bluuebunny/crossref_metadata_2025 huggingface dataset:
Ca2+or Sr2+Partially Rescues Synaptic Transmission in Hippocampal Cultures Treated with Botulinum Toxin A and C, But Not Tetanus Toxin
Authors: Capogna Marco , McKinney R. Anne , O’Connor Vincent , Gähwiler Beat H. , Thompson Scott M.
Published: April 2018

Botulinum (BoNT/A–G) and tetanus toxins (TeNT) are zinc endopeptidases that cleave proteins associated with presynaptic terminals (SNAP-25, syntaxin, or VAMP/synaptobrevin) and block neurotransmitter release. Treatment of hippocampal slice cultures with BoNT/A, BoNT/C, BoNT/E, or TeNT prevented the occurrence of spontaneous or miniature EPSCs (sEPSCs or mEPSCs) as well as the [Ca2+]o-independent increase in their frequency induced by phorbol ester, 0.5 nmα-latrotoxin, or sucrose. [Ca2+]o-independent and -dependent release thus requires that the target proteins of clostridial neurotoxins be uncleaved. In contrast, significant increases in mEPSC frequency were produced in BoNT-treated, but not TeNT-treated, cultures by application of the Ca2+ionophore ionomycin in the presence of 10 mm[Ca2+]o. The frequency of sEPSCs was increased in BoNT-treated, but not TeNT-treated, cultures by increasing [Ca2+]ofrom 2.8 to 5–10 mmor by applying 5 mmSr2+. Large Ca2+and Sr2+influxes thus can rescue release after BoNT treatment, albeit less than in control cultures. The nature of the toxin-induced modification of Ca2+-dependent release was assessed by recordings from monosynaptically coupled CA3 cell pairs. The paired-pulse ratio of unitary EPSCs evoked by two presynaptic action potentials in close succession was 0.5 in control cultures, but it was 1.4 and 1.2 in BoNT/A- or BoNT/C-treated cultures when recorded in 10 mm[Ca2+]o. Log–log plots of unitary EPSC amplitude versus [Ca2+]owere shifted toward higher [Ca2+]oin BoNT/A- or BoNT/C-treated cultures, but their slope was unchanged and the maximal EPSC amplitudes were reduced. We conclude that BoNTs reduce the Ca2+sensitivity of the exocytotic machinery and the number of quanta released.
Evidence That Cysteine String Proteins Regulate an Early Step in the Ca2+-Dependent Secretion of Neurotransmitter atDrosophilaNeuromuscular Junctions
Authors: Umbach Joy A. , Gundersen Cameron B.
Published: April 2018

Previous work indicated that the temperature-dependent block of synaptic transmission in cysteine string protein (csp) mutants ofDrosophilawas attributable to a failure of nerve impulses to trigger transmitter release. The current investigations were undertaken to resolve in more detail the mechanism of this transmission deficit. Our studies reveal that the spider venom toxin α-latrotoxin can trigger a sustained discharge of quanta at neuromuscular junctions ofcspmutant larvae at nonpermissive temperature. The same is true of the calcium ionophore ionomycin. However, solutions with an elevated concentration of K or Ca ions fail to circumvent the block of quantal secretion in these mutants. Likewise, 4-aminopyridine, which augments transmitter release at permissive temperature incspmutants, fails to reverse the inhibition of impulse-evoked transmitter release at elevated temperature. These data are consistent with the hypothesis that there is a deficit either in Ca ion entry or in the ability of Ca ions to trigger exocytosis incspmutants at nonpermissive temperatures. In part, because of previous work showing that csps are important for the functional expression of N-type Ca channels in frog oocytes, we favor the idea that csps participate in a regulatory interaction involving presynaptic Ca channels.
Impact of Cytoplasmic Calcium Buffering on the Spatial and Temporal Characteristics of Intercellular Calcium Signals in Astrocytes
Authors: Wang Zhong , Tymianski Michael , Jones Owen T. , Nedergaard Maiken
Published: April 2018

The impact of calcium buffering on the initiation and propagation of mechanically elicited intercellular Ca2+waves was studied using astrocytes loaded with different exogenous, cell membrane-permeant Ca2+chelators and a laser scanning confocal or video fluorescence microscope. Using an ELISA with a novel antibody to BAPTA, we showed that different cell-permeant chelators, when applied at the same concentrations, accumulate to the same degree inside the cells. Loading cultures with BAPTA, a high Ca2+affinity chelator, almost completely blocked calcium wave occurrence. Chelators having lower Ca2+affinities had lesser affects, as shown in their attenuation of both the radius of spread and propagation velocity of the Ca2+wave. The chelators blocked the process of wave propagation, not initiation, because large [Ca2+]iincreases elicited in the mechanically stimulated cell were insufficient to trigger the wave in the presence of high Ca2+affinity buffers. Wave attenuation was a function of cytoplasmic Ca2+buffering capacity; i.e., loading increasing concentrations of low Ca2+affinity buffers mimicked the effects of lesser quantities of high-affinity chelators. In chelator-treated astrocytes, changes in calcium wave properties were independent of the Ca2+-binding rate constants of the chelators, of chelation of other ions such as Zn2+, and of effects on gap junction function. Slowing of the wave could be completely accounted for by the slowing of Ca2+ion diffusion within the cytoplasm of individual astrocytes. The data obtained suggest that alterations in Ca2+buffering may provide a potent mechanism by which the localized spread of astrocytic Ca2+signals is controlled.
All-or-None Ca2+Release from Intracellular Stores Triggered by Ca2+Influx through Voltage-Gated Ca2+Channels in Rat Sensory Neurons
Authors: Usachev Yuriy M. , Thayer Stanley A.
Published: April 2018

Ca2+-induced Ca2+release (CICR) from intracellular stores amplifies the Ca2+signal that results from depolarization. In neurons, the amplification has been described as a graded process. Here we show that regenerative CICR develops as an all-or-none event in cultured rat dorsal root ganglion neurons in which ryanodine receptors have been sensitized to Ca2+by caffeine. We used indo-1-based microfluorimetry in combination with whole-cell patch-clamp recording to characterize the relationship between Ca2+influx and Ca2+release. Regenerative release of Ca2+was triggered when action potential-induced Ca2+influx increased the intracellular Ca2+concentration ([Ca2+]i) above threshold. The threshold was modulated by caffeine and intraluminal Ca2+. A relative refractory period followed CICR. The pharmacological profile of the response was consistent with Ca2+influx through voltage-gated Ca2+channels triggering release from ryanodine-sensitive stores. The activation of a suprathreshold response increased more than fivefold the amplitude and duration of the [Ca2+]itransient. The switch to a suprathreshold response was regulated very precisely in that addition of a single action potential to the stimulus train was sufficient for this transformation. Confocal imaging experiments showed that CICR facilitated propagation of the Ca2+signal from the plasmalemma to the nucleus. This all-or-none reaction may serve as a switch that determines whether a given electrical signal will be transduced into a local or widespread increase in [Ca2+]i.
Quantitative Three-Dimensional Analysis of the Catecholaminergic Innervation of Identified Neurons in the Macaque Prefrontal Cortex
Authors: Krimer Leonid S. , Jakab Robert L. , Goldman-Rakic Patricia S.
Published: April 2018

The present study provides a complete quantitative three-dimensional analysis of neurons in primate prefrontal cortex targeted by catecholaminergic axons. Individual pyramidal and nonpyramidal cells in fixed slices were filled with Lucifer yellow (LY) and recovered with anti-LY antibody combined with anti-tyrosine hydroxylase (TH) antisera to reveal catecholaminergic axons. The total number of TH contacts and TH apposition density (THAD) was obtained for pyramidal and nonpyramidal cells in different layers. Four TH contacts (two on spines and two on shafts) were selected for correlated electron microscopic examination and serially sectioned; all four were confirmed as membrane appositions. Quantitative analysis revealed 90 TH contacts per pyramidal neuron in layer III, with a density of 0.8 per 100 μm of dendritic length (i.e., averaging one contact per basal dendrite). Remarkably, pyramids of layers III, V, and VI had the same THAD values, with a highly regular distribution of TH terminals on their spiny dendritic trees. In contrast, TH contacts on nonpyramidal neurons in layer III were half as dense and, moreover, were distributed irregularly and showed large variation from cell to cell. Neurons in layers II and superficial III had the highest THAD, as compared with deeper layers (1.4 vs 0.7 per 100 μm of dendritic length for pyramids; 0.53 vs 0.4 for interneurons). The highly organized TH innervation of pyramidal neurons, with at least one contact on virtually every dendrite, indicates that catecholaminergic, presumably dopaminergic, terminals are placed strategically along the entire dendritic tree to modulate most, if not all, of the excitatory input of a neuron. At the same time, the sparsity of contacts per dendrite may explain cortical vulnerability in diseases involving dopamine.
The Effect of Peripherin/rds Haploinsufficiency on Rod and Cone Photoreceptors
Authors: Cheng Tong , Peachey Neal S. , Li Shihong , Goto Yoshinobu , Cao Yun , Naash Muna I.
Published: April 2018

Haploinsufficiency because of a null mutation in the gene encoding peripherin/rds has been thought to be the primary defect associated with the photoreceptor degeneration seen in theretinal degeneration slow(rds) mouse. We have compared the effects of this haploinsufficiency on rod and cone photoreceptors by measuring the levels of rod- and cone-specific gene expression, by determining the relative rates of rod and cone degeneration, and by electroretinography. These analyses were performed at ages before and after the onset of degeneration of the photoreceptor cells. The data were consistent in demonstrating that measures for cone photoreceptors are relatively spared in comparison to comparable measures for rod photoreceptors. Blue cones were retained in higher number than red/green cones for the first 3 months of the degeneration. Our results indicate that the haploinsufficiency present inrds/+mice has a greater impact on the rod than on the cone photoreceptor, a finding that likely reflects the tight regulation of peripherin/rds and the need for two functional alleles to assemble the structure of the rod outer segment and/or differences between the ultrastructure of the rod and cone outer segments.
Nervous System-Specific Expression of a Novel Serine Protease: Regulation in the Adult Rat Spinal Cord by Excitotoxic Injury
Authors: Scarisbrick Isobel A. , Towner Melvin D. , Isackson Paul J.
Published: April 2018

A full-length cDNA clone of a previously unidentified serine protease, myelencephalon-specific protease (MSP), has been isolated by using a PCR cloning strategy and has been shown to be expressed in a nervous system and spinal cord-specific pattern. Sequence analysis demonstrated that MSP is most similar in sequence to neuropsin, trypsin, and tissue kallikrein and is predicted to have trypsin-like substrate specificity. MSP mRNA was found to be ∼10-fold greater in the CNS of the rat and human, as compared with most peripheral tissues, and within the CNS was found to be highest by a factor of four in the medulla oblongata and spinal cord. Levels of mRNA encoding tissue plasminogen activator (tPA) also were elevated in the spinal cord but were more widespread in peripheral tissues as compared with MSP.

In the adult rat lumbosacral spinal cord,in situlocalization of MSP mRNA demonstrated 2-fold higher levels in the white, as compared with the gray, matter. MSP mRNA expression was shown to increase 3-fold in the white matter and 1.5-fold in the gray laminae at 72 hr after intraperitoneal injection of the AMPA/kainate glutamate receptor-specific agonist, kainic acid (KA). MSP mRNA remained elevated in the ventral gray matter, including expression associated with the motor neurons of lamina IX, at 7 d after the initial excitotoxic insult. Together, these observations indicate that MSP is in a position to play a fundamental role in normal homeostasis and in the response of the spinal cord to injury.
Inhibition of Synaptic Transmission by Neuropeptide Y in Rat Hippocampal Area CA1: Modulation of Presynaptic Ca2+Entry
Authors: Qian Jing , Colmers William F. , Saggau Peter
Published: April 2018

Neuropeptide Y (NPY) agonists inhibit glutamate release by a presynaptic action at the CA3–CA1 synapse of rat hippocampus. We have examined the relationship between [Capre]tvia presynaptic, voltage-dependent calcium channels (VDCCs), measured optically by using the fluorescent calcium indicator fura-2, and transmitter release, measured electrophysiologically. Activation of presynaptic NPY Y2receptors reduced [Capre]tand thereby inhibited synaptic transmission. Multiple calcium channels are involved in synaptic transmission at this synapse. Activation of Y2receptors inhibits N-type, P/Q-type, and unidentified presynaptic VDCCs. The inhibition of each of these calcium channel types contributes to the reduction of [Capre]tby Y2receptors. Activation of adenosine receptors fully occluded the inhibition of presynaptic calcium influx by Y2receptors but not the inhibition by GABABreceptors, suggesting a convergent action for Y2and adenosine receptors, probably by coupling to the same G-protein.
Bradykinin-Induced Collapse of Rat Pheochromocytoma (PC12) Cell Growth Cones: A Role for Tyrosine Kinase Activity
Authors: Schindelholz Benno , Reber Bernhard F. X.
Published: April 2018

Pathfinding of growing nerve processes is guided by extracellular guidance cues. Here we report growth cone collapse of NGF-differentiated PC12 cells in culture evoked by the neuropeptide bradykinin. The growth cone response is mediated by B2bradykinin receptors. Two different effects were distinguished. (1) Disappearance of filopodia occurred together with a loss of fibrillar actin (F-actin) in the growth cones at picomolar concentrations of bradykinin. The relative F-actin content was measured by means of rhodamine-phalloidin fluorescence using confocal microscopy. (2) Bradykinin-induced Ca2+release and retraction of the neurite occurred at nanomolar concentrations. Ca2+responses at single growth cones were measured using a 1:1 mixture of fura-red and fluo-3 Ca2+-sensitive dyes. The [Ca2+]irise is not a prerequisite for the observed effects, because F-actin loss and retraction occurred during inhibition of Ca2+responses. In contrast, inhibition by genistein pointed to a tyrosine kinase activity in the bradykinin-evoked cellular events. Subsequent analysis of phosphotyrosine proteins revealed that bradykinin stimulated tyrosine phosphorylation of the cytoskeleton-associated protein paxillin and the nonreceptor protein tyrosine kinase pp60c-src. Paxillin and pp60c-srcco-precipitated after bradykinin treatment. Immunostaining experiments showed punctate distribution of paxillin along PC12 neurites and in growth cones. Taken together, our data suggest that pp60c-srcand paxillin are putative components of the intracellular signaling pathway of bradykinin-mediated neurite retraction and provide evidence for a crosstalk between G-protein- and tyrosine kinase-dependent pathways in these cellular events.
Linearity and Normalization in Simple Cells of the Macaque Primary Visual Cortex
Authors: Carandini Matteo , Heeger David J. , Movshon J. Anthony
Published: April 2018

Simple cells in the primary visual cortex often appear to compute a weighted sum of the light intensity distribution of the visual stimuli that fall on their receptive fields. A linear model of these cells has the advantage of simplicity and captures a number of basic aspects of cell function. It, however, fails to account for important response nonlinearities, such as the decrease in response gain and latency observed at high contrasts and the effects of masking by stimuli that fail to elicit responses when presented alone. To account for these nonlinearities we have proposed a normalization model, which extends the linear model to include mutual shunting inhibition among a large number of cortical cells. Shunting inhibition is divisive, and its effect in the model is to normalize the linear responses by a measure of stimulus energy. To test this model we performed extracellular recordings of simple cells in the primary visual cortex of anesthetized macaques. We presented large stimulus sets consisting of (1) drifting gratings of various orientations and spatiotemporal frequencies; (2) plaids composed of two drifting gratings; and (3) gratings masked by full-screen spatiotemporal white noise. We derived expressions for the model predictions and fitted them to the physiological data. Our results support the normalization model, which accounts for both the linear and the nonlinear properties of the cells. An alternative model, in which the linear responses are subject to a compressive nonlinearity, did not perform nearly as well.
DNA Replication and Postreplication Mismatch Repair in Cell-Free Extracts from Cultured Human Neuroblastoma and Fibroblast Cells
Authors: David Pascale , Efrati Edna , Tocco Georges , Krauss Sharon Wald , Goodman Myron F.
Published: April 2018

DNA synthesis and postreplication mismatch repair were measuredin vitrousing cell-free extracts from cultured human SY5Y neuroblastoma and WI38 fibroblast cells in different growth states. All extracts, including differentiated SY5Y and quiescent WI38 fibroblasts, catalyzed SV40 origin-dependent DNA synthesis, totally dependent on SV40 T-antigen. Thus, although differentiated neuroblastoma and quiescent fibroblasts cells were essentially nondividing, their extracts were competent for DNA replication using DNA polymerases δ, α, and possibly ε, with proliferating cell nuclear antigen. Nonreplicative DNA synthesis and lesion bypass by either α- or β-polymerases were detected independently in extracts using primed or gapped single-stranded DNA templates. Long-patch postreplication mismatch repair was measured for the first time in neuroblastoma cell-free extracts. Extracts from subconfluent and high-density SY5Y cells catalyzed postreplication mismatch repair with efficiencies comparable to those of HeLa cell extracts. No significant differences were observed in repair between SY5Y differentiated and undifferentiated cell extracts. Mismatch repair efficiencies were threefold lower in extracts from subconfluent WI38 cells, and repair in WI38 quiescent cells was fourfold less than in subconfluent cells, suggesting that mismatch repair may be regulated. The spectrum of mismatch repair in SY5Y extracts closely resembled the mismatch removal specificities of HeLa extracts: T · G and G · G mismatches were repaired most efficiently; C · A, A · A, A · G and a five-base loop were repaired with intermediate efficiency; repair of G · A, C · C, and T · T mismatches was extremely inefficient.
Cerebellar Disorganization Characteristic of Reeler in Scrambler Mutant Mice Despite Presence of Reelin
Authors: Goldowitz Dan , Cushing Richard C. , Laywell Eric , D’Arcangelo Gabriella , Sheldon Michael , Sweet Hope O. , Davisson Muriel , Steindler Dennis , Curran Tom
Published: April 2018

Analysis of the molecular basis of neuronal migration in the mammalian CNS relies critically on the discovery and identification of genetic mutations that affect this process. Here, we report the detailed cerebellar phenotype caused by a new autosomal recessive neurological mouse mutation, scrambler (gene symbolscm). The scrambler mutation results in ataxic mice that exhibit several neuroanatomic defects reminiscent of reeler. The most obvious of these lies in the cerebellum, which is small and lacks foliation. Granule cells, although normally placed in an internal granule cell layer, are greatly reduced in number (∼20% of normal). Purkinje cells are also reduced in number, and the majority are located ectopically in deep cerebellar masses. There is a small population of Purkinje cells (∼5% of the total) that occupy a Purkinje cell layer between the molecular and granule cell layers. Despite this apparent disorganization of Purkinje cells, zebrin-positive and zebrin-negative parasagittal zones can be delineated. The ectopic masses of Purkinje cells are bordered by the extracellular matrix protein tenascin and by processes containing glial fibrillary acidic protein. Antibodies specific for these proteins also identify a novel midline raphe structure in both scrambler and reeler cerebellum that is not present in wild-type mice. Thus, in many respects, the scrambler cerebellum is identical to that of reeler. However, the scrambler locus has been mapped to a site distinct from that of reelin (Reln), the gene responsible for the reeler defect. Here we find that there are normal levels ofRelnmRNA in scrambler brain and that reelin protein is secreted normally by scrambler cerebellar cells. These findings imply that the scrambler gene product may function in a molecular pathway critical for neuronal migration that is tightly linked to, but downstream of, reelin.
Inhibitory Interactions between Perigeniculate GABAergic Neurons
Authors: Sanchez-Vives Maria V. , Bal Thierry , McCormick David A.
Published: April 2018

Perigeniculate neurons form an interactive sheet of cells that inhibit one another as well as thalamocortical neurons in the dorsal lateral geniculate nucleus (LGNd). The inhibitory influence of the GABAergic neurons of the perigeniculate nucleus (PGN) onto other PGN neurons was examined with intracellular recordingsin vitro. Intracellular recordings from PGN neurons during the generation of spindle waves revealed barrages of EPSPs and IPSPs. The excitation of local regions of the PGN with the local application of glutamate resulted in activation of IPSPs in neighboring PGN neurons. These IPSPs displayed an average reversal potential of −77 mV and were blocked by application of bicuculline methiodide or picrotoxin, indicating that they are mediated by GABAAreceptors. In the presence of GABAAreceptor blockade, the activation of PGN neurons with glutamate could result in slow IPSPs that were mediated by GABABreceptors in a subset (40%) of cells. Similarly, application of specific agonists muscimol and baclofen demonstrated that PGN neurons possess both functional GABAAand GABABreceptors. Examination of the axon arbors of biocytin-filled PGN neurons often revealed the presence of beaded axon collaterals within the PGN, suggesting that this may be an anatomical substrate for PGN to PGN inhibition.

Functionally, activation of inhibition between PGN neurons could result in a shortening or a complete abolition of the low threshold Ca2+spike or an inhibition of tonic discharge. We suggest that the mutual inhibition between PGN neurons forms a mechanism by which the excitability of these cells is tightly controlled. The activation of a point within the PGN may result in the inhibition of neighboring PGN neurons. This may be reflected in the LGNd as a center of inhibition surrounded by an annulus of disinhibition, thus forming a “center-surround” mechanism for thalamic function.
Short-Term Changes in the Ca2+-Exocytosis Relationship during Repetitive Pulse Protocols in Bovine Adrenal Chromaffin Cells
Authors: Engisch Kathrin L. , Chernevskaya Natalya I. , Nowycky Martha C.
Published: April 2018

Stimulus-secretion coupling was monitored with capacitance detection in bovine chromaffin cells recorded in perforated patch mode and stimulated with trains of depolarizing pulses. A subset of stimulus trains evoked a response with a Ca2+-exocytosis relationship identical to that obtained for single depolarizing pulses (Engisch and Nowycky, 1996). Other trains evoked responses with enhanced or diminished Ca2+efficacy relative to this input–output function. The probability of obtaining a particular Ca2+-exocytosis relationship was correlated with the amount of Ca2+entry per pulse, such that shorter pulses or smaller currents were associated with the greatest efficacy, and longer pulses and larger currents with the lowest efficacy.

Apparent enhancements in Ca2+efficacy were not caused by residual Ca2+summing between pulses, because decreasing the interval between pulses usually reduced efficacy in the same cell; conversely, increasing the interval between pulses did not prevent an enhanced Ca2+-exocytosis relationship. Apparent decreases in Ca2+efficacy were not caused by depletion of an available pool of release-ready vesicles, because an equivalent amount of total Ca2+entry during a single long depolarizing pulse usually evoked a much larger secretory response in the same cell. Finally, there were no striking differences in global Ca2+levels monitored with the fluorescent indicator Fura Red that could account for apparent changes in Ca2+efficacy during repetitive stimulus protocols. It appears that in chromaffin cells, the Ca2+-exocytosis relationship is subject to activity-dependent changes during a stimulus train and can be modulated up or down from a basal state accessed by single pulse stimulations.
Modulation of Rod Photoreceptor Cyclic Nucleotide-Gated Channels by Tyrosine Phosphorylation
Authors: Molokanova Elena , Trivedi Bhavya , Savchenko Alexei , Kramer Richard H.
Published: April 2018

Cyclic nucleotide-gated (CNG) channels in vertebrate photoreceptors are crucial for transducing light-induced changes in cGMP concentration into electrical signals. In this study, we show that both native and exogenously expressed CNG channels from rods are modulated by tyrosine phosphorylation. The cGMP sensitivity of CNG channels, composed of rod α-subunits expressed inXenopusoocytes, gradually increases after excision of inside-out patches from the oocyte membrane. This increase in sensitivity is inhibited by a protein tyrosine phosphatase (PTP) inhibitor and is unaffected by three different Ser/Thr phosphatase inhibitors. Moreover, it is suppressed or reversed by application of ATP but not by a nonhydrolyzable ATP analog. Application of protein tyrosine kinase (PTK) inhibitors causes an increase in cGMP sensitivity, but only in the presence of ATP. Taken together, these results suggest that CNG channels expressed in oocytes are associated with active PTK(s) and PTP(s) that regulate their cGMP sensitivity by changing phosphorylation state. The cGMP sensitivity of native CNG channels from salamander rod outer segments also increases and decreases after incubation with inhibitors of PTP(s) and PTK(s), respectively. These results suggest that rod CNG channels are modulated by tyrosine phosphorylation, which may function as a novel mechanism for regulating the sensitivity of rods to light.
Birthdate and Cell Marker Analysis of Scrambler: A Novel Mutation Affecting Cortical Development with a Reeler-Like Phenotype
Authors: González Jorge L. , Russo Christopher J. , Goldowitz Dan , Sweet Hope O. , Davisson Muriel T. , Walsh Christopher A.
Published: April 2018

The reeler mutation in mice produces an especially well characterized disorder, with systematically abnormal migration of cerebral cortical neurons. The reeler gene encodes a large protein, termed Reelin, that in the cortex is synthesized and secreted exclusively in the Cajal-Retzius neurons of the cortical marginal zone (D’Arcangelo et al., 1995). In reeler mutant mice, loss of Reelin protein is associated with a systematic loss of the normal, “inside-out” sequence of neurogenesis in the cortex: neurons are formed in the normal sequence but become localized in the cortex in a somewhat inverted, although relatively disorganized “outside-in” pattern. Here we show that the scrambler mutant mouse exhibits a loss of lamination in the cortex and hippocampus that is indistinguishable from that seen in the reeler mouse. We use BrdU birthdating studies to show that scrambler cortex shows a somewhat inverted “outside-in” sequence of birthdates for cortical neurons that is similar to that previously described in reeler cortex. Finally, we perform staining with the CR-50 monoclonal antibody (Ogawa et al., 1995), which recognizes the Reelin protein (D’Arcangelo et al., 1997). We show that Reelin immunoreactivity is present in the scrambler cortex in a normal pattern, suggesting that Reelin is synthesized and released normally. Our data suggest that scrambler is a mutation in the same gene pathway as the reeler gene (Relnrl) and is most likely downstream ofRelnrl.
Evidence of Contextual Fear after Lesions of the Hippocampus: A Disruption of Freezing But Not Fear-Potentiated Startle
Authors: McNish Kenneth A. , Gewirtz Jonathan C. , Davis Michael
Published: April 2018

The roles of the dorsal hippocampus and the central nucleus of the amygdala in the expression of contextual fear were assessed using two measures of conditioned fear: freezing and fear-potentiated startle. A discriminable context conditioning paradigm was developed that demonstrated both conditioned freezing and fear-potentiated startle in a context paired previously with foot shock, relative to a context in which foot shock had never been presented. Post-training lesions of the central nucleus of the amygdala completely blocked both contextual freezing and fear-potentiated startle. Post-training lesions of the dorsal hippocampus attenuated contextual freezing, consistent with previous reports in the literature; however, these same lesions had no effect on fear-potentiated startle, suggesting preserved contextual fear. These results suggest that lesions of the hippocampus disrupt the freezing response but not contextual fear itself.
Regulation of Amyloid Precursor Protein Catabolism Involves the Mitogen-Activated Protein Kinase Signal Transduction Pathway
Authors: Mills Julia , Laurent Charest David , Lam Fred , Beyreuther Konrad , Ida Nobuo , Pelech Steven L. , Reiner Peter B.
Published: April 2018

Catabolic processing of the amyloid precursor protein (APP) is subject to regulatory control by protein kinases. We hypothesized that this regulation involves sequential activation of the enzymes mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated protein kinase (ERK). In the present investigation, we provide evidence that MEK is critically involved in regulating APP processing by both nerve growth factor and phorbol esters. Western blot analysis of the soluble N-terminal APP derivative APPsdemonstrated that the synthetic MEK inhibitor PD 98059 antagonized nerve growth factor stimulation of both APPsproduction and ERK activation in PC12 cells. Moreover, PD 98059 inhibited phorbol ester stimulation of APPsproduction and activation of ERK in both human embryonic kidney cells and cortical neurons. Furthermore, overexpression of a kinase-inactive MEK mutant inhibited phorbol ester stimulation of APP secretion and activation of ERK in human embryonic kidney cell lines. Most important, PD 98059 antagonized phorbol ester-mediated inhibition of Aβ secretion from cells overexpressing human APP695carrying the “Swedish mutation.” Taken together, these data indicate that MEK and ERK may be critically involved in protein kinase C and nerve growth factor regulation of APP processing. The mitogen-activated protein kinase cascade may provide a novel target for altering catabolic processing of APP.
Repression of the Calcitonin Gene-Related Peptide Promoter by 5-HT1Receptor Activation
Authors: Durham Paul L. , Sharma Ram V. , Russo Andrew F.
Published: April 2018

We have investigated the control of calcitonin gene-related peptide (CGRP) expression by a serotonergic agonist that is related pharmacologically to currently used antimigraine drugs. During migraines, CGRP levels are elevated but then returned to normal by a 5-HT1receptor agonist, sumatriptan. However, neither the molecular nor cellular targets of this drug are known. Trigeminal neurons are the major source of cerebrovascular CGRP, and thus we have used trigeminal primary cultures and the neuronal-like CA77 thyroid C-cell line as a model. We first demonstrate that sumatriptan and another 5-HT1agonist, CGS 12066A (CGS), cause a robust and prolonged increase with oscillations in intracellular calcium in CA77 cells. CGS caused a similar increase in trigeminal cultures. We then show that CGS treatment leads to a decrease in CGRP mRNA levels in the CA77 cells. This decrease is attributable to the repression of promoter activity through two discrete elements: (1) the cAMP-responsive region, via a cAMP-independent mechanism; and (2) the cell-specific enhancer, which binds the upstream stimulatory factor helix–loop–helix protein and a cell-specific activator. These results demonstrate that activation of the endogenous 5-HT1receptor is coupled to calcium signaling pathways and leads to inhibition of CGRP gene transcription.
Development of Membrane Properties in Taste Cells of Fungiform Papillae: Functional Evidence for Early Presence of Amiloride-Sensitive Sodium Channels
Authors: Kossel A. H. , McPheeters M. , Lin W. , Kinnamon S. C.
Published: April 2018

Behavioral and physiological studies have demonstrated a reduced sensitivity to several taste stimuli early in development. It has been suggested that this reduced sensitivity results from a late maturation of underlying transduction mechanisms. Little is known, however, about maturation of membrane properties of taste cells early in development. We have obtained whole-cell recordings from single fungiform taste cells of rat pups to examine the development of the NaCl transduction system. Although taste buds undergo a considerable increase in size during development, membrane capacitance measurements revealed no change in membrane surface area of individual taste cells, suggesting that the increase in size results from an increase in the total number of cells per bud. Whole-cell recordings showed that taste cells from very young pups [postnatal day 2 (PND2)] already possessed voltage-activated Na+and K+currents with no apparent differences in size or kinetics compared with adults. Surprisingly, amiloride-sensitive Na+responses, important for Na+transduction, were found as early as PND2. The magnitude of responses to amiloride and the percentage of amiloride-sensitive cells remained the same throughout all age groups. Furthermore, the similarity of amiloride inhibition constants suggested that the channel in neonates is the same channel that is expressed in adult taste buds. Our results indicate that taste cells at PND2 already have acquired the transduction elements necessary for signaling NaCl responses to the afferent nerve. We hypothesize that complete functionality of the salt taste transduction system, however, may not be reached until amiloride-sensitive Na+channels become selectively localized at the apical membrane. This would explain previous studies indicating that amiloride sensitivity cannot be detected before PND12 in the intact tongue. Apical clustering of channels along with the opening of the taste pore and an increase in the total number of taste cells per bud likely constitute additional important steps toward a fully functional sensory system.
Sox10, a Novel Transcriptional Modulator in Glial Cells
Authors: Kuhlbrodt Kirsten , Herbarth Beate , Sock Elisabeth , Hermans-Borgmeyer Irm , Wegner Michael
Published: April 2018

Sox proteins are characterized by possession of a DNA-binding domain with similarity to the high-mobility group domain of the sex determining factor SRY. Here, we report on Sox10, a novel protein with predominant expression in glial cells of the nervous system. During development Sox10 first appeared in the forming neural crest and continued to be expressed as these cells contributed to the forming PNS and finally differentiated into Schwann cells. In the CNS, Sox10 transcripts were originally confined to glial precursors and later detected in oligodendrocytes of the adult brain. Functional studies failed to reveal autonomous transcriptional activity for Sox10. Instead, Sox10 functioned synergistically with the POU domain protein Tst-1/Oct6/SCIP with which it is coexpressed during certain stages of Schwann cell development. Synergy depended on binding to adjacent sites in target promoters, was mediated by the N-terminal regions of both proteins, and could not be observed between Sox10 and several other POU domain proteins. Interestingly, Sox10 also modulated the function of Pax3 and Krox-20, two other transcription factors involved in Schwann cell development. We propose a role for Sox10 in conferring cell specificity to the function of other transcription factors in developing and mature glia.
A Model for Encoding Multiple Object Motions and Self-Motion in Area MST of Primate Visual Cortex
Authors: Zemel Richard S. , Sejnowski Terrence J.
Published: April 2018

Many cells in the dorsal part of the medial superior temporal (MST) region of visual cortex respond selectively to specific combinations of expansion/contraction, translation, and rotation motions. Previous investigators have suggested that these cells may respond selectively to the flow fields generated by self-motion of an observer. These patterns can also be generated by the relative motion between an observer and a particular object. We explored a neurally constrained model based on the hypothesis that neurons in MST partially segment the motion fields generated by several independently moving objects. Inputs to the model were generated from sequences of ray-traced images that simulated realistic motion situations, combining observer motion, eye movements, and independent object motions. The input representation was based on the response properties of neurons in the middle temporal area (MT), which provides the primary input to area MST. After applying an unsupervised optimization technique, the units became tuned to patterns signaling coherent motion, matching many of the known properties of MST cells. The results of this model are consistent with recent studies indicating that MST cells primarily encode information concerning the relative three-dimensional motion between objects and the observer.
Stability in Synapse Number and Size at 2 Hr after Long-Term Potentiation in Hippocampal Area CA1
Authors: Sorra Karin E. , Harris Kristen M.
Published: April 2018

Long-term potentiation (LTP) is an important model for examining synaptic mechanisms of learning and memory. A key question is whether the enhanced synaptic transmission occurring with LTP involves the addition of new synapses, the enlargement of existing synapses, or a redistribution in synaptic weight among synapses. Two experimental designs were used to address this question. In the first experimental design three conditions were evaluated across hippocampal slices maintainedin vitro, including slices with LTP analyzed at 2 hr post-tetanus, slices tetanized in the presence of APV, and control slices receiving test stimulation only. In the second experimental design independent LTP and control (low-frequency stimulation) sites were examined. Synapse density was estimated by an unbiased volume sampling procedure. Synapse size was computed by three-dimensional reconstruction from serial electron microscopy (EM). Serial EM also was used to compute synapse number per unit length of dendrite. In both experimental designs there were no significant effects of LTP on total synapse number, on the distribution of different types of synapses (thin, mushroom, stubby, or branched dendritic spines and macular, perforated, or segmented postsynaptic densities), on the frequency of shaft synapses, nor on the relative proportion of single or multiple synapse axonal boutons. There was also no increase in synapse size. These results suggest that LTP does not cause an overall formation of new synapses nor an enlargement of synapses at 2 hr post-tetanus in hippocampal area CA1, and these results support the hypothesis that LTP could involve a redistribution of synaptic weights among existing synapses.
Angiotensin II Type 2 Receptor Stimulation of Neuronal Delayed-Rectifier Potassium Current Involves Phospholipase A2and Arachidonic Acid
Authors: Zhu Mingyan , Gelband Craig H. , Moore Jennifer M. , Posner Philip , Sumners Colin
Published: April 2018

Angiotensin II (Ang II) elicits an Ang II type 2 (AT2) receptor-mediated increase in delayed-rectifier K+current (IK) in neurons cultured from newborn rat hypothalamus and brainstem. This effect involves a pertussis toxin (PTX)-sensitive Giprotein and is abolished by inhibition of serine and threonine phosphatase 2A (PP-2A). Here, we determined that Ang II stimulates [3H]arachidonic acid (AA) release from cultured neurons via AT2receptors. This effect of Ang II was blocked by inhibition of phospholipase A2(PLA2) and by PTX. Because AA and its metabolites are powerful modulators of neuronal K+currents, we investigated the involvement of PLA2and AA in the AT2receptor-mediated stimulation ofIKby Ang II. Single-cell reverse transcriptase (RT)-PCR analyses revealed the presence of PLA2mRNA in neurons that responded to Ang II with an increase inIK. The stimulation of neuronalIKby Ang II was attenuated by selective inhibitors of PLA2and was mimicked by application of AA to neurons. Inhibition of lipoxygenase (LO) enzymes significantly reduced both Ang II- and AA-stimulatedIK, and the 12-LO metabolite of AA 12S-hydroxyeicosatetraenoic acid (12S-HETE) stimulatedIK. These data indicate the involvement of a PLA2, AA, and LO metabolite intracellular pathway in the AT2receptor-mediated stimulation of neuronalIKby Ang II. Furthermore, the demonstration that inhibition of PP-2A abolished the stimulatory effects of Ang II, AA, and 12S-HETE on neuronalIKbut did not alter Ang II-stimulated [3H]-AA release suggests that PP-2A is a distal event in this pathway.
Phosphorylation of c-Jun Is Necessary for Apoptosis Induced by Survival Signal Withdrawal in Cerebellar Granule Neurons
Authors: Watson Andrea , Eilers Andreas , Lallemand Dominique , Kyriakis John , Rubin Lee L. , Ham Jonathan
Published: April 2018

Cerebellar granule neurons die by apoptosis when deprived of survival signals. This death can be blocked by inhibitors of transcription or protein synthesis, suggesting that new gene expression is required. Here we show thatc-junmRNA and protein levels increase rapidly after survival signal withdrawal and that transfection of the neurons with an expression vector for a c-Jun dominant negative mutant protects them against apoptosis. Phosphorylation of serines 63 and 73 in the c-Jun transactivation domain is known to increase c-Jun activity. By using an antibody specific for c-Jun phosphorylated on serine 63, we show that this site is phosphorylated soon after survival signal withdrawal. To determine whether c-Jun phosphorylation is necessary for apoptosis, we have expressed c-Jun phosphorylation site mutants in granule neurons. c-Junasp, a constitutively active c-Jun mutant in which the known and potential serine and threonine phosphoacceptor sites in the transactivation domain have been mutated to aspartic acid, induces apoptosis under all conditions tested. In contrast, c-Junala, which cannot be phosphorylated because the same sites have been mutated to alanine, blocks apoptosis caused by survival signal withdrawal. Finally, we show that cerebellar granule neurons contain high levels of Jun kinase activity and low levels of p38 kinase activity, neither of which increases after survival signal withdrawal. Mitogen-activated protein kinase activity decreases under the same conditions. These results suggest that c-Jun levels and c-Jun phosphorylation may be regulated by novel mechanisms in cerebellar granule neurons.
Slow Synaptic Inhibition in Nucleus HVc of the Adult Zebra Finch
Authors: Schmidt Marc F. , Perkel David J.
Published: April 2018

Nervous systems process information over a broad range of time scales and thus need corresponding cellular mechanisms spanning that range. In the avian song system, long integration times are likely necessary to process auditory feedback of the bird’s own vocalizations. For example, in nucleus HVc, a center that contains both auditory and premotor neurons and that is thought to act as a gateway for auditory information into the song system, slow inhibitory mechanisms appear to play an important role in the processing of auditory information. These long-lasting processes include inhibitory potentials thought to shape auditory selectivity and a vocalization-induced inhibition of auditory responses lasting several seconds. To investigate the possible cellular mechanisms of these long-lasting inhibitory processes, we have made intracellular recordings from HVc neurons in slices of adult zebra finch brains and have stimulated extracellularly within HVc. A brief, high-frequency train of stimuli (50 pulses at 100 Hz) could elicit a hyperpolarizing response that lasted 2–20 sec. The slow hyperpolarization (SH) could still be elicited in the presence of glutamate receptor blockers, suggesting that it does not require polysynaptic excitation. Three major components contribute to this activity-induced SH: a long-lasting GABABreceptor-mediated IPSP, a slow afterhyperpolarization requiring action potentials but not Ca2+influx, and a long-lasting IPSP, the neurotransmitter and receptor of which remain unidentified. These three slow hyperpolarizing events are well placed to contribute to the observed inhibition of HVc neurons after singing and could shape auditory feedback during song learning.
Cysteine String Protein Is Required for Calcium Secretion Coupling of Evoked Neurotransmission inDrosophilaBut Not for Vesicle Recycling
Authors: Ranjan Ravi , Bronk Peter , Zinsmaier Konrad E.
Published: April 2018

The entire deletion of the cysteine string protein (CSP) gene causes a temperature-sensitive (ts) block of evoked neurotransmission inDrosophila. CSP has been found to interactin vitrowith the clathrin-uncoating ATPase HSC70, suggesting a potential role of CSP in vesicle recycling. Using FM1-43 imaging, we analyzed whether the ts block of neurotransmission incspmutants is caused by a defect in vesicle exocytosis or vesicle recycling. We determined that FM1-43-labeled synaptic boutons ofcspmutant neuromuscular junctions fail to destain at 32°C after K+depolarization, and that FM1-43 dye uptake cannot be evoked by K+stimulation at 32°C. However, when we stimulated dye uptake independent of depolarization by using black widow spider venom (BWSV), we observed endocytotic uptake of FM1-43. This suggests that endocytosis exhibits no primary ts defect. In addition, we found no ts defect of vesicle recycling at 32°C that would correlate with the ts block of neurotransmission. We also discovered that BWSV and the calcium ionophore calcimycin stimulate FM1-43 destaining and quantal release incspmutants at 32°C when depolarization fails to evoke any response. The wild-type-like, calcimycin-induced response incspnull mutants indicates that some aspect of the depolarization-dependent calcium signaling pathway must be impaired, either calcium entry, calcium action, or both. Collectively, our results indicate that thecspmutation affects calcium secretion coupling of evoked exocytosis but not vesicle recycling. This supports the hypothesis that CSP links synaptic vesicles to calcium secretion coupling.