bluuebunny/crossref_metadata_2025 huggingface dataset:
Rescue of Hearing, Auditory Hair Cells, and Neurons by CEP-1347/KT7515, an Inhibitor of c-Jun N-Terminal Kinase Activation
Authors: Pirvola Ulla , Xing-Qun Liang , Virkkala Jussi , Saarma Mart , Murakata Chikara , Camoratto Anna Marie , Walton Kevin M. , Ylikoski Jukka
Published: April 2018

We have studied the mechanisms of auditory hair cell death after insultsin vitroandin vivo. We show DNA fragmentation of hair cell nuclei after ototoxic drug and intense noise trauma. By using phospho-specific c-Jun-N-terminal kinase (JNK) and c-Jun antibodies in immunohistochemistry, we show that the JNK pathway, associated with stress, injury, and apoptosis, is activated in hair cells after trauma. CEP-1347, a derivative of the indolocarbazole K252a, is a small molecule that has been shown to attenuate neurodegeneration by blocking the activation of JNK (Maroney et al., 1998). Subcutaneously delivered CEP-1347 attenuated noise-induced hearing loss. The protective effect was demonstrated by functional tests, which showed less hearing threshold shift in CEP-1347-treated than in nontreated guinea pigs, and by morphometric methods showing less hair cell death in CEP-1347-treated cochleas. In organotypic cochlear cultures, CEP-1347 prevented neomycin-induced hair cell death. In addition to hair cells, CEP-1347 promoted survival of dissociated cochlear neurons. These results suggest that therapeutic intervention in the JNK signaling cascade, possibly by using CEP-1347, may offer opportunities to treat inner ear injuries.
Electrophysiological Characterization of Voltage-Gated K+Currents in Cerebellar Basket and Purkinje Cells: Kv1 and Kv3 Channel Subfamilies Are Present in Basket Cell Nerve Terminals
Authors: Southan Andrew P. , Robertson Brian
Published: April 2018

To understand the processes underlying fast synaptic transmission in the mammalian CNS, we must have detailed knowledge of the identity, location, and physiology of the ion channels in the neuronal membrane. From labeling studies we can get clues regarding the distribution of ion channels, but electrophysiological methods are required to determine the importance of each ion channel in CNS transmission. Dendrotoxin-sensitive potassium channel subunits are highly concentrated in cerebellar basket cell nerve terminals, and we have previously shown that they are responsible for a significant fraction of the voltage-gated potassium current in this region. Here, we further investigate the characteristics and pharmacology of the voltage-dependent potassium currents in these inhibitory nerve terminals and compare these observations with those obtained from somatic recordings in basket and Purkinje cell soma regions. We find that α-DTX blocks basket cell nerve terminal currents and not somatic currents, and the IC50for α-DTX in basket cell terminals is 3.2 nm. There are at least two distinct types of potassium currents in the nerve terminal, a DTX-sensitive low-threshold component, and a second component that activates at much more positive voltages. Pharmacological experiments also reveal that nerve terminal potassium currents are also markedly reduced by 4-AP and TEA, with both high-sensitivity (micromolar) and low-sensitivity (millimolar) components present. We suggest that basket cell nerve terminals have potassium channels from both the Kv1 and Kv3 subfamilies, whereas somatic currents in basket cell and Purkinje cell bodies are more homogeneous.
Neuronal α-Bungarotoxin Receptors Are α7 Subunit Homomers
Authors: Drisdel Renaldo C. , Green William N.
Published: April 2018

Nicotinic acetylcholine receptors in the nervous system are heterogeneous with distinct pharmacological and functional properties resulting from differences in post-translational processing and subunit composition. Because of nicotinic receptor diversity, receptor purification and biochemical characterization have been difficult, and the precise subunit composition of each receptor subtype is poorly characterized. Evidence is presented that α-bungarotoxin (Bgt)-binding nicotinic receptors found in pheochromocytoma 12 (PC12) cells are pentamers composed solely of α7 subunits. Metabolically labeled, affinity-purified Bgt receptors (BgtRs) consisted of a single 55 kDa band on SDS gels, which was recognized by anti-α7 antibodies on immunoblots. Isoelectric focusing separated the 55 kDa band into multiple spots, all recognized by anti-α7 antibodies and, therefore, each a differentially processed α7 subunit. Cell-surface BgtR subunits, cross-linked to each other and125I-Bgt, migrated on gels as a ladder of five bands with each band a multiple of an α7 subunit monomer. Similar characteristics of BgtRs from rat brain suggest that they, like PC12 BgtRs, are α7 pentamers containing differentially processed α7 subunits.
Recovery from Open Channel Block by Acetylcholine during Neuromuscular Transmission in Zebrafish
Authors: Legendre Pascal , Ali Declan W. , Drapeau Pierre
Published: April 2018

At larval zebrafish neuromuscular junctions (NMJs), miniature end plate currents (mEPCs) recordedin vivohave an unusually fast time course. We used fast-flow application of acetylcholine (ACh) onto outside-out patches to mimic the effect of synaptic release onto small numbers of ACh receptor channels (AChRs). Positively charged ACh acted at hyperpolarized potentials and at millimolar concentrations as a fast (“flickering”) open channel blocker of AChRs. Because of filtering, the open channel block resulted in reduced amplitude of single channel currents. Immediately after brief (1 msec) application (without significant desensitization) of millimolar ACh at hyperpolarized potentials, a slower, transient current appeared because of delayed reversal of the block. This rebound current depended on the ACh concentration and resembled in time course the mEPC. A simple kinetic model of the AChR that includes an open channel-blocking step accounted for our single channel results, as well as the experimentally observed slowing of the time course of mEPCs recorded at a hyperpolarized compared with a depolarized potential. Recovery from AChR block is a novel mechanism of synaptic transmission that may contribute in part at all NMJs.
Neuronal–Glial Interactions Mediated by Interleukin-1 Enhance Neuronal Acetylcholinesterase Activity and mRNA Expression
Authors: Li Yuekui , Liu Ling , Kang Jinsong , Sheng Jin G. , Barger Steven W. , Mrak Robert E. , Griffin W. Sue T.
Published: April 2018

Cholinergic dysfunction in Alzheimer's disease has been attributed to stress-induced increases in acetylcholinesterase (AChE) activity. Interleukin-1 (IL-1) is overexpressed in Alzheimer's disease, and stress-related changes in long-term potentiation, an ACh-related cerebral function, are triggered by interleukin-1. Microglial cultures (N9) synthesized and released IL-1 in response to conditioned media obtained from glutamate-treated primary neuron cultures or PC12 cells. This conditioned media contained elevated levels of secreted β-amyloid precursor protein (sAPP). Naive PC12 cells cocultured with stimulated N9 cultures showed increased AChE activity and mRNA expression. These effects on AChE expression and activity could be blocked by either preincubating the glutamate-treated PC12 supernatants with anti-sAPP antibodies or preincubating naive PC12 cells with IL-1 receptor antagonist. These findings were confirmedin vivo; IL-1-containing pellets implanted into rat cortex also increased AChE mRNA levels. Neuronal stress in Alzheimer's disease may induce increases in AChE expression and activity through a molecular cascade that is mediated by sAPP-induced microglial activation and consequent overexpression of IL-1.
Presynaptic Ca2+Influx at a Mouse Central Synapse with Ca2+Channel Subunit Mutations
Authors: Qian Jing , Noebels Jeffrey L.
Published: April 2018

Genetic alterations in Ca2+channel subunits can be used to study the interaction among channel subunits and their roles in channel function. P/Q- and N-type Ca2+channels reside at the presynaptic terminal and control the release of neurotransmitter at mammalian central synapses. We used fluorescence imaging techniques to investigate presynaptic Ca2+currents and neurotransmitter release at hippocampal Schaffer collateral synapses in bothtottering(tg, α1Asubunit) andlethargic(lh, β4subunit) mutant mice. Application of selective toxins revealed a large reduction in presynaptic P/Q-type Ca2+transients, from 39% of total in +/+ mice to 6% intg/tgmice, whereas the proportion of N-type increased from 35 to 68%, respectively. Neurotransmitter release in thetg/tgmutant relied almost exclusively on N-type channels, as shown by the complete blockade of synaptic transmission with ω-conotoxin GVIA. Remarkably, loss of β4, a subunit predicted to regulate the subcellular targeting and modulation of both P/Q- and N-type channels, resulted in no significant difference in the ratio of Ca2+channel subtypes or Ca2+dependence of neurotransmitter release in lethargic mice. G-protein-mediated inhibition of Ca2+channels was also unaltered. These results indicate that a profound decrease in presynaptic P/Q-type currents leads to dependence of neurotransmitter release on N-type channels. In contrast, absence of β4appears not to compromise either P/Q- or N-type channel function at this hippocampal synapse, implicating rescue of presynaptic Ca2+currents by other available β subunits. The present study reveals compensatory molecular mechanisms in the regulation of presynaptic Ca2+entry and neurotransmitter release.
Visualization of Cranial Motor Neurons in Live Transgenic Zebrafish Expressing Green Fluorescent Protein Under the Control of theIslet-1Promoter/Enhancer
Authors: Higashijima Shin-ichi , Hotta Yoshiki , Okamoto Hitoshi
Published: April 2018

We generated germ line-transmitting transgenic zebrafish that express green fluorescent protein (GFP) in the cranial motor neurons. This was accomplished by fusing GFP sequences toIslet-1promoter/enhancer sequences that were sufficient for neural-specific expression. The expression of GFP by the motor neurons in the transgenic fish enabled visualization of the cell bodies, main axons, and the peripheral branches within the muscles. GFP-labeled motor neurons could be followed at high resolution for at least up to day four, when most larval neural circuits become functional, and larvae begin to swim and capture prey. Using this line, we analyzed axonal outgrowth by the cranial motor neurons. Furthermore, by selective application of DiI to specific GFP-positive nerve branches, we showed that the two clusters of trigeminal motor neurons in rhombomeres 2 and 3 innervate different peripheral targets. This finding suggests that the trigeminal motor neurons in the two clusters adopt distinct fates. In future experiments, this transgenic line of zebrafish will allow for a genetic analysis of cranial motor neuron development.
AMPA Exposures Induce Mitochondrial Ca2+Overload and ROS Generation in Spinal Motor NeuronsIn Vitro
Authors: Carriedo Sean G. , Sensi Stefano L. , Yin Hong Z. , Weiss John H.
Published: April 2018

The reason for the selective vulnerability of motor neurons in amyotrophic lateral sclerosis (ALS) is primarily unknown. A possible factor is the expression by motor neurons of Ca2+-permeable AMPA/kainate channels, which may permit rapid Ca2+influx in response to synaptic receptor activation. However, other subpopulations of central neurons, most notably forebrain GABAergic interneurons, consistently express large numbers of these channels but do not degenerate in ALS. Indeed, when subjected to identical excitotoxic exposures, motor neurons were more susceptible than GABAergic neurons to AMPA/kainate receptor-mediated neurotoxicity. Microfluorimetric studies were performed to examine the basis for the difference in vulnerability. First, AMPA or kainate exposures appeared to trigger substantial mitochondrial Ca2+loading in motor neurons, as indicated by a sharp increase in intracellular Ca2+after addition of the mitochondrial uncoupler carbonyl cyanidep-(trifluoromethoxy)phenyl hydrazone (FCCP) after the agonist exposure. The same exposures caused little mitochondrial Ca2+accumulation in GABAergic cortical neurons. Subsequent experiments examined other measures of mitochondrial function to compare sequelae of AMPA/kainate receptor activation between these populations. Brief exposure to either AMPA or kainate caused mitochondrial depolarization, assessed using tetramethylrhodamine ethylester, and reactive oxygen species (ROS) generation, assessed using hydroethidine, in motor neurons. However, these effects were only seen in the GABAergic neurons after exposure to the nondesensitizing AMPA receptor agonist kainate. Finally, addition of either antioxidants or toxins (FCCP or CN−) that block mitochondrial Ca2+uptake attenuated AMPA/kainate receptor-mediated motor neuron injury, suggesting that the mitochondrial Ca2+uptake and consequent ROS generation are central to the injury process.
AMPA–Kainate Subtypes of Glutamate Receptor in Rat Cerebral Microglia
Authors: Noda Mami , Nakanishi Hiroshi , Nabekura Junichi , Akaike Norio
Published: April 2018

Microglial cells were isolated from rat cerebral cortex, and kainate (KA)-induced inward current was measured at a holding potential of −40 or −60 mV. 6-Cyano-7-nitroquinoxaline-2, 3-dione-sensitive KA-induced currents increased with increasing KA concentration. The half-activation concentration and Hill coefficient were 3.3 × 10−4mand 1.4, respectively. Although glutamate (Glu) and AMPA-induced currents were much smaller than that induced by KA, all KA-, Glu-, and AMPA-induced currents were greatly and consistently enhanced in the presence of cyclothiazide (CTZ). On the other hand, KA-induced currents were much less sensitive to potentiation by concanavain A, suggesting that the KA-induced response in rat microglia is predominantly mediated by AMPA-preferring receptors (subunits GluR1–GluR4). The current–voltage relationships of KA- and AMPA–CTZ-induced currents were almost linear or slightly outward rectifying. The reversal potential of KA-induced current shifted to negative potentials (from +4 to −40 mV) on switching from high Na+to high Ca2+external solution, indicating the low Ca2+permeability through the AMPA–KA receptor channel complexes. AMPA–KA receptor expression was studied with immunohistochemistry and reverse transcription-PCR, from which GluR2, GluR3, GluR4, and GluR5 were identified. Lower levels of mRNAs for GluR7 and KA-1–KA-2 were also indicated. Finally, activation of these receptors with KA or Glu significantly enhanced the production of tumor necrosis factor-α. These results suggest that primary cultured rat microglia possesses functional Glu receptor, which may mediate neuron to microglia communication in the physiological and pathological states.
Critical Dependence of cAMP Response Element-Binding Protein Phosphorylation on L-Type Calcium Channels Supports a Selective Response to EPSPs in Preference to Action Potentials
Authors: Mermelstein Paul G. , Bito Haruhiko , Deisseroth Karl , Tsien Richard W.
Published: April 2018

Activity-dependent gene expression in neurons shows a remarkable ability to differentiate between different types of stimulation: orthodromic inputs that engage synaptic transmission are much more effective than antidromic stimuli that do not. We have studied the basis of such selectivity in cultured hippocampal neurons in which nuclear cAMP response element-binding protein (CREB) phosphorylation is induced by synaptic activity but not by action potential (AP) stimulation in the absence of EPSPs, although spikes by themselves generate large elevations in intracellular Ca2+. Previous work has shown that Ca2+entry through L-type Ca2+channels plays a dominant role in triggering calmodulin mobilization and activation of calmodulin-dependent kinases that phosphorylate CREB, raising the possibility that L-type channels contribute to the selective response to EPSPs rather than APs. Accordingly, we performed voltage-clamp experiments to compare the currents carried by L-type channels during depolarizing waveforms that approximated APs or dendritic EPSPs. The integrated current generated by L-type channels was significantly less after mock APs than with EPSP-like depolarizations. The difference was traced to two distinct factors. Compared with other channels, L-type channels activated at relatively negative potentials, favoring their opening with EPSP stimulation; they also exhibited relatively slow activation kinetics, weighing against their contribution during an AP. The relative ineffectiveness of APs as a stimulus for CREB phosphorylation could be overcome by exposure to the agonist Bay K8644, which potentiated the AP-induced influx through L-type channels by ∼10-fold. Under normal conditions, the unique biophysical properties of L-type channels allow them to act as a kinetic filter to support spike–EPSP discrimination.
The bHLH GeneHes1as a Repressor of the Neuronal Commitment of CNS Stem Cells
Authors: Nakamura Yuki , Sakakibara Shin-ichi , Miyata Takaki , Ogawa Masaharu , Shimazaki Takuya , Weiss Samuel , Kageyama Ryoichiro , Okano Hideyuki
Published: April 2018

Hes1is one of the basic helix-loop-helix transcription factors that regulate mammalian CNS development, and its loss- and gain-of-function phenotypes indicate that it negatively regulates neuronal differentiation.

Here we report thatHes1−/−mice expressed both early (TuJ1 and Hu) and late (MAP2 and Neurofilament) neuronal markers prematurely, and that there were approximately twice the normal number of neurons in theHes1−/−brain during early neural development. However, immunochemical analyses of sections and dissociated cells using neural progenitor markers, including nestin, failed to detect any changes inHes1−/−progenitor population. Therefore, further characterization of neural progenitor cells that discriminated between multipotent and monopotent cells was performed using two culture methods, low-density culture, and a neurosphere assay. We demonstrate that the self-renewal activity of multipotent progenitor cells was reduced in theHes1−/−brain, and that their subsequent commitment to the neuronal lineage was accelerated. TheHes1−/−neuronal progenitor cells were functionally abnormal, in that they divided, on average, only once, and then generated two neurons, (instead of one progenitor cell and one neuron), whereas wild-type progenitor cells divided more. In addition, someHes1−/−progenitors followed an apoptotic fate. The overproduction of neurons in the earlyHes1−/−brains may reflect this premature and immediate generation of neurons as well as a net increase in the number of neuronal progenitor cells.

Taken together, we conclude thatHes1is important for maintaining the self-renewing ability of progenitors and for repressing the commitment of multipotent progenitor cells to a neuronal fate, which is critical for the correct number of neurons to be produced and for the establishment of normal neuronal function.
Serotonin and the 5-HT2BReceptor in the Development of Enteric Neurons
Authors: Fiorica-Howells Elena , Maroteaux Luc , Gershon Michael D.
Published: April 2018

We tested the hypothesis that 5-HT promotes the differentiation of enteric neurons by stimulating a developmentally regulated receptor expressed by crest-derived neuronal progenitors. 5-HT and the 5-HT2agonist (±)-2,5-dimethoxy-4-iodoamphetamine.HCl (DOI) enhancedin vitrodifferentiation of enteric neurons, both in dissociated cultures of mixed cells and in cultures of crest-derived cells isolated from the gut by immunoselection with antibodies to p75NTR. The promotion ofin vitroneuronal differentiation by 5-HT and DOI was blocked by the 5-HT1/2antagonist methysergide, the pan-5-HT2antagonist ritanserin, and the 5-HT2B/2C-selective antagonist SB206553. The 5-HT2A-selective antagonist ketanserin did not completely block the developmental effects of 5-HT. 5-HT induced the nuclear translocation of mitogen-activated protein kinase. This effect was blocked by ritanserin. mRNA encoding 5-HT2Aand 5-HT2Breceptors was detected in the fetal bowel (stomach and small and large intestine), but that encoding the 5-HT2Creceptor was not. mRNA encoding the 5-HT2Breceptor and 5-HT2Bimmunoreactivity were found to be abundant in primordial [embryonic day 15 (E15)–E16] but not in mature myenteric ganglia. 5-HT2B-immunoreactive cells were found to be a subset of cells that expressed the neuronal marker PGP9.5. These data demonstrate for the first time that the 5-HT2Breceptor is expressed in the small intestine as well as the stomach and that it is expressed by enteric neurons as well as by muscle. It is possible that by stimulating 5-HT2Breceptors, 5-HT affects the fate of the large subset of enteric neurons that arises after the development of endogenous sources of 5-HT.
Hepatocyte Growth Factor/Scatter Factor Is a Neurotrophic Survival Factor for Lumbar But Not for Other Somatic Motoneurons in the Chick Embryo
Authors: Novak Kristine D. , Prevette David , Wang Siwei , Gould Tom W. , Oppenheim Ronald W.
Published: April 2018

Hepatocyte growth factor/scatter factor (HGF/SF) is expressed in the developing limb muscles of the chick embryo during the period of spinal motoneuron (MN) programmed cell death, and its receptor c-met is expressed in lumbar MNs during this same period. Although cultured motoneurons from brachial, thoracic, and lumbar segments are all rescued from cell death by chick embryo muscle extract (CMX) as well as by other specific trophic agents, HGF/SF only promotes the survival of lumbar MNs. Similarly, treatment of embryosin ovowith exogenous HGF/SF rescues lumbar but not other somatic MNs from cell death. Blocking antibodies to HGF/SF (anti-HGF) reduce the effects of CMX on MN survivalin vitroand decrease the number of lumbar MNsin vivo. The expression of c-met on MNsin vivois regulated by a limb-derived trophic signal distinct from HGF/SF. HGF/SF is a potent, select, and physiologically relevant survival factor for a subpopulation of developing spinal MNs in the lumbar segments of the chick embryo.
Increased Spontaneous Unit Activity and Appearance of Spontaneous Negative Potentials in the Goldfish Tectum during Refinement of the Optic Projection
Authors: Kolls Bradley J. , Meyer Ronald L.
Published: April 2018

Spontaneous (not retinally driven) postsynaptic activity was examined during activity-dependent refinement of optic fibers in the goldfish tectum. Unit recordingsin vivoandin vitrodemonstrated that spontaneous tectal activity increased to 150% of normal during refinement at 1–2 months after optic nerve crush and subsequently returned to baseline over the next month. This increase was not mimicked by long-term denervation indicating an effect specifically influenced by regenerating fibers. Loss of optic input was also found to induce spontaneous negative potentials (SNPs) rapidly in the tectum. SNPs were negative, monophasic potentials of 70–120 msec duration and −0.15 to −1.5 mV amplitude. SNPs occurred with no apparent periodicity at a frequency of ∼0.3–0.6 Hz. Multiple electrode recordings and depth analysis showed that SNPs were localized events occurring in columnar domains of tectum a few hundred micrometers wide. Cross-correlation analysis revealed that SNPs were strongly correlated with local unit bursting, suggesting SNPs are generated by the summed synaptic and spike currents of coactive cells in small regions of the tectum. SNPs were suppressed by a low concentration of APV indicating they were regulated by NMDA receptors. During regeneration, the number and size of SNPs reached a peak during refinement and subsequently decreased, eventually disappearing. This temporal association with refinement suggests that these patterns of postsynaptic activity may have functional relevance. It is hypothesized that SNPs or the underlying activity that produces them increases the excitability of target cells, allowing the weak, less-convergent input from regenerating axons to drive target groups of cells in the tectum during refinement.
Developmental Changes in the Neurotransmitter Regulation of Correlated Spontaneous Retinal Activity
Authors: Wong Wai T. , Myhr Karen L. , Miller Ethan D. , Wong Rachel O. L.
Published: April 2018

Synchronized spontaneous rhythmic activity is a feature common to many parts of the developing nervous system. In the early visual system, before vision, developing circuits in the retina generate synchronized patterns of bursting activity that contain information useful for patterning connections between retinal ganglion cells and their central targets. However, how developing retinal circuits generate and regulate these spontaneous activity patterns is still incompletely understood. Here we show that in developing retinal circuits, the nature of excitatory neurotransmission driving correlated bursting activity in ganglion cells is not fixed but undergoes a developmental shift from cholinergic to glutamatergic transmission. In addition, we show that this shift occurs as presynaptic glutamatergic bipolar cells form functional connections onto the ganglion cells, implicating the role of bipolar cells in providing endogenous drive to bursting activity later in development. This transition coincides with the period when subsets of ganglion cells (On and Off cells) develop distinct activity patterns that are thought to underlie the refinement of their connectivity with their central targets. Here, our results suggest that the differences in activity patterns of On and Off ganglion cells may be conferred by differential synaptic drive from On and Off bipolar cells, respectively. Taken together, our results suggest that the regulation of patterned spontaneous activity by neurotransmitters undergoes systematic change as new cellular elements are added to developing circuits and also that these new elements can help specify distinct activity patterns appropriate for shaping connectivity patterns at later ages.
Rapid Correction of Aimed Movements by Summation of Force-Field Primitives
Authors: Kargo William J. , Giszter Simon F.
Published: April 2018

Spinal circuits form building blocks for movement construction. In the frog, such building blocks have been described as isometric force fields. Microstimulation studies showed that individual force fields can be combined by vector summation. Summation and scaling of a few force-field types can, in theory, produce a large range of dynamic force-field structures associated with limb behaviors. We tested for the first time whether force-field summation underlies the construction of real limb behavior in the frog. We examined the organization of correction responses that circumvent path obstacles during hindlimb wiping trajectories. Correction responses were triggered on-line during wiping by cutaneous feedback signaling obstacle collision. The correction response activated a force field that summed with an ongoing sequence of force fields activated during wiping. Both impact force and time of impact within the wiping motor pattern scaled the evoked correction response amplitude. However, the duration of the correction response was constant and similar to the duration of other muscles activated in different phases of wiping. Thus, our results confirm that both force-field summation and scaling occur during real limb behavior, that force fields represent fixed-timing motor elements, and that these motor elements are combined in chains and in combination contingent on the interaction of feedback and central motor programs.
Spatial and Temporal Structure of Receptive Fields in Primate Somatosensory Area 3b: Effects of Stimulus Scanning Direction and Orientation
Authors: DiCarlo James J. , Johnson Kenneth O.
Published: April 2018

This is the third in a series of studies of the neural representation of tactile spatial form in somatosensory cortical area 3b of the alert monkey. We previously studied the spatial structure of >350 fingerpad receptive fields (RFs) with random-dot patterns scanned in one direction (DiCarlo et al., 1998) and at varying velocities (DiCarlo and Johnson, 1999). Those studies showed that area 3b RFs have a wide range of spatial structures that are virtually unaffected by changes in scanning velocity. In this study, 62 area 3b neurons were studied with three to eight scanning directions (58 with four or more directions). The data from all three studies are described accurately by an RF model with three components: (1) a single, central excitatory region of short duration, (2) one or more inhibitory regions, also of short duration, that are adjacent to and nearly synchronous with the excitation, and (3) a region of inhibition that overlaps the excitation partially or totally and is temporally delayed with respect to the first two components. The mean correlation between the observed RFs and the RFs predicted by this three-component model was 0.81. The three-component RFs also predicted orientation sensitivity and preferred orientation to a scanned bar accurately. The orientation sensitivity was determined most strongly by the intensity of the coincident RF inhibition in relation to the excitation. Both orientation sensitivity and this ratio were stronger in the supragranular and infragranular layers than in layer IV.
Axonal Transport of Microtubule-Associated Protein 1B (MAP1B) in the Sciatic Nerve of Adult Rat: Distinct Transport Rates of Different Isoforms
Authors: Ma Dongling , Himes B. Timothy , Shea Thomas B. , Fischer Itzhak
Published: April 2018

Cytoskeletal proteins are axonally transported with slow components a and b (SCa and SCb). In peripheral nerves, the transport velocity of SCa, which includes neurofilaments and tubulin, is 1–2 mm/d, whereas SCb, which includes actin, tubulin, and numerous soluble proteins, moves as a heterogeneous wave at 2–4 mm/d. We have shown that two isoforms of microtubule-associated protein 1B (MAP1B), which can be separated on SDS polyacrylamide gels on the basis of differences in their phosphorylation states (band I and band II), were transported at two different rates. All of band I MAP1B moved as a coherent wave at a velocity of 7–9 mm/d, distinct from slow axonal transport components SCa and SCb. Several other proteins were detected within the component that moved at the velocity of 7–9 mm/d, including the leading wave of tubulin and actin. The properties of this component define a distinct fraction of the slow axonal transport that we suggest to term slow component c (SCc). The relatively fast transport of the phosphorylated MAP1B isoform at 7–9 mm/d may account for the high concentration of phosphorylated MAP1B in the distal end of growing axons. In contrast to band I MAP1B, the transport profile of band II was complex and contained components moving with SCa and SCb and a leading edge at SCc. Thus, MAP1B isoforms in different phosphorylation states move with distinct components of slow axonal transport, possibly because of differences in their abilities to associate with other proteins.
The Neuronal Monoamine Transporter VMAT2 Is Regulated by the Trimeric GTPase Go2
Authors: Höltje Markus , von Jagow Burkhard , Pahner Ingrid , Lautenschlager Marion , Hörtnagl Heide , Nürnberg Bernd , Jahn Reinhard , Ahnert-Hilger Gudrun
Published: April 2018

Monoamines such as noradrenaline and serotonin are stored in secretory vesicles and released by exocytosis. Two related monoamine transporters, VMAT1 and VMAT2, mediate vesicular transmitter uptake. Previously we have reported that in the rat pheochromocytoma cell line PC 12 VMAT1, localized to peptide-containing secretory granules, is controlled by the heterotrimeric G-protein Go2. We now show that in BON cells, a human serotonergic neuroendocrine cell line derived from a pancreatic tumor expressing both transporters on large, dense-core vesicles, VMAT2 is even more sensitive to G-protein regulation than VMAT1. The activity of both transporters is only downregulated by Gαo2, whereas comparable concentrations of Gαo1are without effect. In serotonergic raphe neurons in primary culture VMAT2 is also downregulated by pertussis toxin-sensitive Go2. By electron microscopic analysis from prefrontal cortex we show that VMAT2 and Gαo2associate preferentially to locally recycling small synaptic vesicles in serotonergic terminals. In addition, Go2-dependent modulation of VMAT2 also works when using a crude synaptic vesicle preparation from this brain area. We conclude that regulation of monoamine uptake by the heterotrimeric G proteins is a general feature of monoaminergic neurons that controls the content of both large, dense-core and small synaptic vesicles.
Distinct Roles for Ionotropic and Metabotropic Glutamate Receptors in the Maturation of Excitatory Synapses
Authors: Gomperts Stephen N. , Carroll Reed , Malenka Robert C. , Nicoll Roger A.
Published: April 2018

We used the single-cell culture preparation to study the role of activity in the development of glutamatergic synapsesin vitro. Rat hippocampal cells grown in isolation on glial islands formed functional autaptic connections and continued to elaborate new synapses throughout the 2 week investigation, resulting in increases in both the evoked AMPA receptor (AMPAR) and NMDA receptor (NMDAR) components of the EPSC. Synaptogenesis was not prevented by chronic blockade of sodium channels or all of the known glutamate receptors. Analysis of miniature EPSCs revealed that AMPAR quantal size doubled over timein vitrowhereas NMDAR quantal size remained constant. However, the proportion of synaptic responses mediated only by NMDARs increased over timein vitro. The increase in AMPAR quantal size was prevented by TTX and ionotropic glutamate receptor antagonists, whereas the increase in the proportion of NMDAR-only synapses was prevented by metabotropic glutamate receptor antagonists. Notably, chronic NMDAR blockade incubation did not block the formation of the AMPAR EPSC, indicating that NMDAR-dependent plasticity is not necessary for the onset of AMPAR synaptic transmission in this system. We conclude that action potentials and ionotropic glutamate receptor activation are necessary for the developmental increase in AMPAR quantal size and that metabotropic glutamate receptor activation is required for the production of NMDAR-only synapses, but none of these is essential for synapse formation.
Growth Cones Are Not Required for Initial Establishment of Polarity or Differential Axon Branch Growth in Cultured Hippocampal Neurons
Authors: Ruthel Gordon , Hollenbeck Peter J.
Published: April 2018

Hippocampal neurons developing in culture exhibit two types of differential, seemingly competitive, process outgrowth in the absence of external cues. During the initial acquisition of polarity, one of several equivalent undifferentiated minor neurites preferentially grows to become the axon. Once the axon has formed, it typically branches, and the branches grow differentially rather than concurrently. In axons with only two branches, growth alternates between branches. In both axon establishment and branch growth alternation, growth among sibling processes or branches must be differentially regulated. We found that elaborate and dynamic growth cones were associated with growth, whereas diminished growth cones were associated with nongrowing processes or branches. To test whether growth cones were necessary for differential growth, growth cone motility was eliminated by application of cytochalasin E. Although cytochalasin treatment before axon formation yielded longer processes overall, a similar percentage of both treated and untreated neurons had one process that grew more rapidly and became much longer than its sibling processes. Immunostaining to visualize dephospho-tau, an axonal marker, demonstrated that these single dominant processes were axons. Axons that formed in cytochalasin were thicker and showed more intense anti-tubulin staining than their sibling processes. Branched axons deprived of growth cones retained a pattern of differential growth and often included alternation. These results indicate that neither formation of a single axon nor differential growth of branches are dependent on growth cone motility and suggest that the neuron can regulate neurite elongation at sites other than at the growth cone.
Transformations of an Auditory Temporal Code in the Medulla of a Sound-Producing Fish
Authors: Kozloski James , Crawford John D.
Published: April 2018

The fish auditory system provides important insights into the evolution and mechanisms of vertebrate hearing. Fish have relatively simple auditory systems, without a cochlea for mechanical frequency analysis. However, as in all vertebrates, the primary auditory afferents of fish represent sounds as stimulus-entrained spike trains. Thus, fish provide important models for studying how temporal spiking patterns are used in higher level neural computations. In this paper we demonstrate that one of the fundamental transformations of information in the auditory system of a sound-producing fish,Pollimyrus,takes place in the auditory medulla. We discovered a class of neurons in which evoked spiking patterns were relatively independent of the stimulus fine structure and appeared to reflect intrinsic properties of the neurons. These neurons generated sustained responses but were poorly phase-locked to tones compared with the primary afferents. The interval histograms showed that spike timing was regular. However, in contrast to primary afferents, the mode of the interspike interval distribution was independent of the period of tonal stimuli. The tuning of the neurons was broad, with best sensitivity in the same spectral region where these animals concentrate energy in their communication sounds. The physiology of these neurons was similar to that of the chopper neurons known in the auditory brainstem of mammals. Our findings suggest that this medullary transformation, from phase-locked afferent input to chopper-like physiology, is basic to vertebrate auditory processing, even within lineages that have not evolved a cochlea.
The Plasmin System Is Induced by and Degrades Amyloid-β Aggregates
Authors: Tucker H. Michael , Kihiko Muthoni , Caldwell Joseph N. , Wright Sarah , Kawarabayashi Takeshi , Price Douglas , Walker Donald , Scheff Stephen , McGillis Joseph P. , Rydel Russell E. , Estus Steven
Published: April 2018

Amyloid-β (Aβ) appears critical to Alzheimer's disease. To clarify possible mechanisms of Aβ action, we have quantified Aβ-induced gene expressionin vitroby using Aβ-treated primary cortical neuronal cultures andin vivoby using mice transgenic for the Aβ precursor (AβP). Here, we report that aggregated, but not nonaggregated, Aβ increases the level of the mRNAs encoding tissue plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Moreover, tPA and uPA were also upregulated in aged AβP overexpressing mice. Because others have reported that Aβ aggregates can substitute for fibrin aggregates in activating tPA post-translationally, the result oftPAinduction by Aβ would be cleavage of plasminogen to the active protease plasmin. To gain insights into the possible actions of plasmin, we evaluated the hypotheses that tPA and plasmin may mediate Aβin vitrotoxicity or, alternatively, that plasmin activation may lead to Aβ degradation. In evaluating these conflicting hypotheses, we found that purified plasmin degrades Aβ with physiologically relevant efficiency, i.e., ∼1/10th the rate of plasmin on fibrin. Mass spectral analyses show that plasmin cleaves Aβ at multiple sites. Electron microscopy confirms indirect assays suggesting that plasmin degrades Aβ fibrils. Moreover, exogenously added plasmin blocks Aβ neurotoxicity. In summation, we interpret these results as consistent with the possibility that the plasmin pathway is induced by aggregated Aβ, which can lead to Aβ degradation and inhibition of Aβ actions.
Neuromodulatory Effects of Gonadotropin Releasing Hormone on Olfactory Receptor Neurons
Authors: Eisthen Heather L. , Delay Rona J. , Wirsig-Wiechmann Celeste R. , Dionne Vincent E.
Published: April 2018

The terminal nerve is an anterior cranial nerve that innervates the lamina propria of the chemosensory epithelia of the nasal cavity. The function of the terminal nerve is ambiguous, but it has been suggested to serve a neuromodulatory role. We tested this hypothesis by exposing olfactory receptor neurons from mudpuppies (Necturus maculosus) to a peptide, gonadotropin releasing hormone (GnRH), that is found in cells and fibers of the terminal nerve. We used voltage-clamped whole-cell recordings to examine the effects of 0.5–50 μmGnRH on voltage-activated currents in olfactory receptor neurons from epithelial slices. We found that GnRH increases the magnitude, but does not alter the kinetics, of a tetrodotoxin-sensitive inward current. This increase in magnitude generally begins 5–10 min after initial exposure to GnRH, is sustained for at least 60 min during GnRH exposure, and recovers to baseline within 5 min after GnRH is washed off. This effect occurred in almost 60% of the total number of olfactory receptor neurons examined and appeared to be seasonal: ∼67% of neurons responded to GnRH during the courtship and mating season, compared with ∼33% during the summer, when the sexes separate. GnRH also appears to alter an outward current in the same cells. Taken together, these data suggest that GnRH increases the excitability of olfactory receptor neurons and that the terminal nerve functions to modulate the odorant sensitivity of olfactory receptor neurons.
Motoneuron Activity Patterns Related to the Earliest Behavior of the Zebrafish Embryo
Authors: Saint-Amant Louis , Drapeau Pierre
Published: April 2018

As a first step in the study of the developing motor circuitry of the embryonic zebrafish spinal cord, we obtained patch-clamp recordingsin vivofrom identified motoneurons in curarized embryos from the onset of the first motor behavior. At an early developmental stage in which embryos showed slow and repetitive spontaneous contractions of the trunk, motoneurons showed periodic depolarizations that triggered rhythmic bursts of action potentials with a frequency and duration that were consistent with those of the spontaneous contractions. The periodic depolarizations were blocked by tetrodotoxin or Cd2+. Surprisingly, the contractions and periodic depolarizations were insensitive to general blockade of synaptic transmission (by elevated Mg2+and reduced Ca2+, or by Co2+) and to selective blockade of the major neurotransmitter receptors of the mature spinal cord (acetylcholine, GABAA, NMDA, AMPA/kainate, and glycine). The periodic depolarizations were suppressed by heptanol or by intracellular acidification, treatments that are known to uncouple gap junctions, indicating that electrotonic synapses could underlie the earliest motor behavior. A few hours later, most motoneurons already showed a new pattern of repetitive activity consisting of bursts of glycinergic synaptic events, but these were not necessary for the spontaneous contractions. Transecting the spinal cord at the hindbrain border did not affect the rhythmic activity patterns of the motoneurons. We suggest that spontaneous contractions of the zebrafish embryo are mediated by an early spinal circuit that is independent of the main neurotransmitter systems and descending hindbrain projections that are required for locomotion in the mature vertebrate spinal cord.
Intracellular Modulation of NMDA Receptor Function by Antipsychotic Drugs
Authors: Leveque Jean-Christophe , Macı́as Wendy , Rajadhyaksha Anjali , Carlson Richard R. , Barczak Amy , Kang Stanley , Li Xin-Min , Coyle Joseph T. , Huganir Richard L. , Heckers Stephan , Konradi Christine
Published: April 2018

The present study deals with the functional interaction of antipsychotic drugs and NMDA receptors. We show that both the conventional antipsychotic drug haloperidol and the atypical antipsychotic drug clozapine mediate gene expression via intracellular regulation of NMDA receptors, albeit to different extents. Data obtained in primary striatal culture demonstrate that the intraneuronal signal transduction pathway activated by haloperidol, the cAMP pathway, leads to phosphorylation of the NR1 subtype of the NMDA receptor at897Ser. Haloperidol treatment is likewise shown to increase897Ser–NR1 phosphorylation in ratsin vivo. Mutation of896Ser and897Ser to alanine, which prevents phosphorylation at both sites, inhibits cAMP-mediated gene expression. We conclude that antipsychotic drugs have the ability to modulate NMDA receptor function by an intraneuronal signal transduction mechanism. This facilitation of NMDA activity is necessary for antipsychotic drug-mediated gene expression and may contribute to the therapeutic benefits as well as side effects of antipsychotic drug treatment.
Cytoskeletal Links of Neuronal Acetylcholine Receptors Containing α7 Subunits
Authors: Shoop Richard D. , Yamada Naoko , Berg Darwin K.
Published: April 2018

Nicotinic acetylcholine receptors serve a variety of signaling functions in the nervous system depending on cellular location, but little is known about mechanisms responsible for tethering them at specific sites. Among the most interesting are receptors containing the α7 gene product, because of their abundance and high relative permeability to calcium. On chick ciliary ganglion neurons α7-containing receptors are highly concentrated on somatic spines folded into discrete patches on the cell. We show that the spines contain filamentous actin and drebrin. After cell dissociation, the actin slowly redistributes, the spines retract, and the α7-containing receptors disperse and are subsequently lost from the surface. Latrunculin A, a drug that depolymerizes filamentous actin, accelerates receptor dispersal, whereas jasplikinolide, a drug that stabilizes the actin cytoskeleton, preserves large receptor clusters and prevents receptor loss from the surface. The receptors are resistant to extraction by nonionic detergent even after latrunculin A treatment. Other, less abundant, nicotinic receptors on the neurons are readily solubilized by the detergent even though these receptors are located in part on the spines. The results demonstrate that the actin cytoskeleton is important for retaining receptor-rich spines and indicate that additional cytoskeletal elements or molecular interactions specific for α7-containing receptors influence their fate in the membrane. The cytoskeletal elements involved are not dependent on the architecture of the postsynaptic density because α7-containing receptors are excluded from such sites on ciliary ganglion neurons.
cAMP Response Element-Mediated Gene Transcription Is Upregulated by Chronic Antidepressant Treatment
Authors: Thome J. , Sakai N. , Shin K.-H. , Steffen C. , Zhang Y.-J. , Impey S. , Storm D. , Duman R. S.
Published: April 2018

Regulation of gene transcription via the cAMP-mediated second messenger pathway has been implicated in the actions of antidepressant drugs, but studies to date have not demonstrated such an effectin vivo. To directly study the regulation of cAMP response element (CRE)-mediated gene transcription by antidepressants, transgenic mice with a CRE–LacZ reporter gene construct were administered one of three different classes of antidepressants: a norepinephrine selective reuptake inhibitor (desipramine), a serotonin selective reuptake inhibitor (fluoxetine), or a monoamine oxidase inhibitor (tranylcypromine). Chronic, but not acute, administration of these antidepressants significantly increased CRE-mediated gene transcription, as well as the phosphorylation of CRE binding protein (CREB), in several limbic brain regions thought to mediate the action of antidepressants, including the cerebral cortex, hippocampus, amygdala, and hypothalamus. These results demonstrate that chronic antidepressant treatment induces CRE-mediated gene expression in a neuroanatomically differentiated pattern and further elucidate the molecular mechanisms underlying the actions of these widely used therapeutic agents.