bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid Resetting of the Mammalian Circadian Clock
Authors: Best Jonathan D. , Maywood Elizabeth S. , Smith Karen L. , Hastings Michael H.
Published: April 2018

The suprachiasmatic nuclei (SCN) contain the principal circadian clock governing overt daily rhythms of physiology and behavior. The endogenous circadian cycle is entrained to the light/dark via direct glutamatergic retinal afferents to the SCN. To understand the molecular basis of entrainment, it is first necessary to define how rapidly the clock is reset by a light pulse. We used a two-pulse paradigm, in combination with cellular and behavioral analyses of SCN function, to explore the speed of resetting of the circadian oscillator in Syrian hamster and mouse. Analysis ofc-fosinduction and cAMP response element-binding protein phosphorylation in the retinorecipient SCN demonstrated that the SCN are able to resolve and respond to light pulses presented 1 or 2 hr apart. Analysis of the phase shifts of the circadian wheel-running activity rhythm of hamsters presented with single or double pulses demonstrated that resetting of the oscillator occurred within 2 hr. This was the case for both delaying and advancing phase shifts. Examination of delaying shifts in the mouse showed resetting within 2 hr and in addition showed that resetting is not completed within 1 hr of a light pulse. These results establish the temporal window within which to define the primary molecular mechanisms of circadian resetting in the mammal.
Ca2+-Induced Deprotonation of Peptide Hormones Inside Secretory Vesicles in Preparation for Release
Authors: Han Weiping , Li Danqing , Stout Amy K. , Takimoto Koichi , Levitan Edwin S.
Published: April 2018

The acidic environment inside secretory vesicles ensures that neuropeptides and peptide hormones are packaged in a concentrated condensed form. Although this is optimal for storage, decondensation limits release. Thus, it would be advantageous to alter the physical state of peptides in preparation for exocytosis. Here, we report that depolarization of the plasma membrane rapidly increases enhanced green fluorescent protein (EGFP)-tagged hormone fluorescence inside secretory vesicles. This effect requires Ca2+influx and persists when exocytosis is inhibited byN-ethylmaleimide. Peptide deprotonation appears to produce this response, because it is not seen when the vesicle pH gradient is collapsed or when a pH-insensitive GFP variant is used. These data demonstrate that Ca2+evokes alkalinization of the inside of secretory vesicles before exocytosis. Thus, Ca2+influx into the cytoplasm alters the physical state of intravesicular contents in preparation for release.
Differential Modulation of Synaptic Transmission by Calcium Chelators in Young and Aged Hippocampal CA1 Neurons: Evidence for Altered Calcium Homeostasis in Aging
Authors: Ouanounou Aviv , Zhang Liang , Charlton Milton P. , Carlen Peter L.
Published: April 2018

The effects of membrane-permeant Ca2+chelators on field EPSPs (fEPSPs) were measured in the hippocampal CA1 region of brain slices from young (2–4 months) and old (24–27 months) Fischer 344 rats. BAPTA-AM depressed fEPSPs in young slices by up to 70% but enhanced fEPSPs by 30% in aged slices. EGTA-AM, with slower binding kinetics, did not affect fEPSPs from young slices but enhanced fEPSPs in aged slices. BAPTA derivatives with calcium dissociation constants (Kd) of 0.2–3.5 μmreduced or enhanced fEPSPs in young and aged slices, respectively, but 5′,5′-dinitro BAPTA-AM (Kdof ∼7000 μm) had no effect. Frequency facilitation of the fEPSPs occurred in young, but not in aged, slices, except when BAPTA-AM or EGTA-AM was perfused onto aged slices. The differential effects of BAPTA-AM in young and old slices were eliminated by perfusing with a low Ca2+–high Mg2+saline or with the calcium blocker Co2+. These data suggest that intracellular Ca2+regulation is altered and raised in aged neurons. Cell-permeant calcium buffers may be able to “ameliorate” deficits in synaptic transmission in the aged brain.
Mutant Huntingtin Expression in Clonal Striatal Cells: Dissociation of Inclusion Formation and Neuronal Survival by Caspase Inhibition
Authors: Kim Manho , Lee H-S , LaForet Genevieve , McIntyre Charmian , Martin Eileen J. , Chang Patrick , Kim Tae Wan , Williams M. , Reddy P. H. , Tagle Dan , Boyce Frederick M. , Won Lisa , Heller Alfred , Aronin Neil , DiFiglia Marian
Published: April 2018

Neuronal intranuclear inclusions are found in the brains of patients with Huntington’s disease and form from the polyglutamine-expanded N-terminal region of mutant huntingtin. To explore the properties of inclusions and their involvement in cell death, mouse clonal striatal cells were transiently transfected with truncated and full-length human wild-type and mutant huntingtin cDNAs. Both normal and mutant proteins localized in the cytoplasm, and infrequently, in dispersed and perinuclear vacuoles. Only mutant huntingtin formed nuclear and cytoplasmic inclusions, which increased with polyglutamine expansion and with time after transfection. Nuclear inclusions contained primarily cleaved N-terminal products, whereas cytoplasmic inclusions contained cleaved and larger intact proteins. Cells with wild-type or mutant protein had distinct apoptotic features (membrane blebbing, shrinkage, cellular fragmentation), but those with mutant huntingtin generated the most cell fragments (apoptotic bodies). The caspase inhibitor Z-VAD-FMK significantly increased cell survival but did not diminish nuclear and cytoplasmic inclusions. In contrast, Z-DEVD-FMK significantly reduced nuclear and cytoplasmic inclusions but did not increase survival. A series of N-terminal products was formed from truncated normal and mutant proteins and from full-length mutant huntingtin but not from full-length wild-type huntingtin. One prominent N-terminal product was blocked by Z-VAD-FMK. In summary, the formation of inclusions in clonal striatal cells corresponds to that seen in the HD brain and is separable from events that regulate cell death. N-terminal cleavage may be linked to mutant huntingtin’s role in cell death.
Regulation of Neurotrophin Receptor Expression by Retinoic Acid in Mouse Sympathetic Neuroblasts
Authors: Wyatt Sean , Andres Rosa , Rohrer Hermann , Davies Alun M.
Published: April 2018

We have studied the effect of retinoic acid on the expression of the neurotrophin receptors trkA, trkC, and p75 by neuroblasts and neurons at different axial levels along the embryonic mouse paravertebral sympathetic chain. In dissociated cultures of sympathetic neuroblasts, retinoic acid inhibited the developmental increase in trkA mRNA expression and the developmental decrease in trkC mRNA expression that normally occurs in these cells but did not affect p75 mRNA expression. At higher concentrations, retinoic acid also increased the proliferation of sympathetic neuroblasts. After sympathetic neuroblasts became postmitotic, retinoic acid no longer affected receptor expression. Studies with retinoic acid receptor agonists and antagonists indicated that the effects of retinoic acid on neurotrophin receptor expression were mediated mainly by α retinoic acid receptors, not β or γ receptors. The observation that α-antagonists increased trkA mRNA expression in intact sympathetic ganglion explants suggests that endogenous retinoic acid is a physiological regulator of trkA receptor expression.
Inhibition of Light- or Glutamate-InducedmPer1Expression Represses the Phase Shifts into the Mouse Circadian Locomotor and Suprachiasmatic Firing Rhythms
Authors: Akiyama Masashi , Kouzu Yasuko , Takahashi Satomi , Wakamatsu Hisanori , Moriya Takahiro , Maetani Miyuki , Watanabe Shigenori , Tei Hajime , Sakaki Yoshiyuki , Shibata Shigenobu
Published: April 2018

mPer1,a mouse gene, is a homolog of theDrosophilaclock geneperiodand has been shown to be closely associated with the light-induced resetting of a mammalian circadian clock. To investigate whether the rapid induction ofmPer1after light exposure is necessary for light-induced phase shifting, we injected an antisense phosphotioate oligonucleotide (ODN) tomPer1mRNA into the cerebral ventricle. Light-induced phase delay of locomotor activity at CT16 was significantly inhibited when the mice were pretreated withmPer1antisense ODN 1 hr before light exposure.mPer1sense ODN or random ODN treatment had little effect on phase delay induced by light pulses. In addition, glutamate-induced phase delay of suprachiasmatic nucleus (SCN) firing rhythm was attenuated by pretreatment withmPer1antisense ODN, but not by random ODN. The present results demonstrate that induction ofmPer1mRNA is required for light- or glutamate-induced phase shifting, suggesting that the acute induction ofmPer1mRNA in the SCN after light exposure is involved in light-induced phase shifting of the overt rhythm.
Low Resting Potential and Postnatal Upregulation of NMDA Receptors May Cause Cajal–Retzius Cell Death
Authors: Mienville Jean-Marc , Pesold Christine
Published: April 2018

Usingin situpatch-clamp techniques in rat telencephalic slices, we have followed resting potential (RP) properties and the functional expression of NMDA receptors in neocortical Cajal–Retzius (CR) cells from embryonic day 18 to postnatal day 13, the time around which these cells normally disappear. We find that throughout their lives CR cells have a relatively depolarized RP (approximately −50 mV), which can be made more hyperpolarized (approximately −70 mV) by stimulation of the Na/K pump with intracellular ATP. The NMDA receptors of CR cells are subjected to intense postnatal upregulation, but their similar properties (EC50, Hill number, sensitivity to antagonists, conductance, and kinetics) throughout development suggest that their subunit composition remains relatively homogeneous. The low RP of CR cells is within a range that allows for the relief of NMDA channels from Mg2+blockade. Our findings are consistent with the hypothesis that CR cells may degenerate and die subsequent to uncontrolled overload of intracellular Ca2+via NMDA receptor activation by ambient glutamate. In support of this hypothesis we have obtained evidence showing the protection of CR cells viain vivoblockade of NMDA receptors with dizocilpine.
Neural Changes after Operant Conditioning of the Aerial Respiratory Behavior inLymnaea stagnalis
Authors: Spencer Gaynor E. , Syed Naweed I. , Lukowiak Ken
Published: April 2018

In this study, we demonstrate neural changes that occurred during operant conditioning of the aerial respiratory behavior ofLymnaea stagnalis. Aerial respiration inLymnaeaoccurs at the water interface and is achieved by opening and closing movements of its respiratory orifice, the pneumostome. This behavior is controlled by a central pattern generator (CPG), the neurons of which, as well as the motoneurons innervating the pneumostome, have previously been identified and their synaptic connections well characterized.

The respiratory behavior was operantly conditioned by applying a mechanical stimulus to the open pneumostome whenever the animal attempted to breathe. This negative reinforcement to the open pneumostome resulted in its immediate closure and a significant reduction in the overall respiratory activity. Electrophysiological recordings from the isolated CNSs after operant conditioning showed that the spontaneous patterned respiratory activity of the CPG neurons was significantly reduced. This included reduced spontaneous activity of the CPG interneuron involved in pneumostome opening (input 3 interneuron) and a reduced frequency of spontaneous tonic activity of the CPG interneuron [right pedal dorsal 1 (RPeD1)]. The ability to trigger the patterned respiratory activity by electrical stimulation of RPeD1 was also significantly reduced after operant conditioning. This study therefore demonstrates significant changes within a CPG that are associated with changes in a rhythmic homeostatic behavior after operant conditioning.
Modification of Postsynaptic Densities after Transient Cerebral Ischemia: A Quantitative and Three-Dimensional Ultrastructural Study
Authors: Martone Maryann E. , Jones Ying Z. , Young Steve J. , Ellisman Mark H. , Zivin Justin A. , Hu Bing-Ren
Published: April 2018

Abnormal synaptic transmission has been hypothesized to be a cause of neuronal death resulting from transient ischemia, although the mechanisms are not fully understood. Here, we present evidence that synapses are markedly modified in the hippocampus after transient cerebral ischemia. Using both conventional and high-voltage electron microscopy, we performed two- and three-dimensional analyses of synapses selectively stained with ethanolic phosphotungstic acid in the hippocampus of rats subjected to 15 min of ischemia followed by various periods of reperfusion. Postsynaptic densities (PSDs) from both area CA1 and the dentate gyrus were thicker and fluffier in postischemic hippocampus than in controls. Three-dimensional reconstructions of selectively stained PSDs created using electron tomography indicated that postsynaptic densities became more irregular and loosely configured in postischemic brains compared with those in controls. A quantitative study based on thin sections of the time course of PSD modification indicated that the increase in thickness was both greater and more long-lived in area CA1 than in dentate gyrus. Whereas the magnitude of morphological change in dentate gyrus peaked at 4 hr of reperfusion (140% of control values) and declined thereafter, changes in area CA1 persisted and increased at 24 hr of reperfusion (191% of control values). We hypothesize that the degenerative ultrastructural alteration of PSDs may produce a toxic signal such as a greater calcium influx, which is integrated from the thousands of excitatory synapses onto dendrites, and is propagated to the neuronal somata where it causes or contributes to neuronal damage during the postischemic phase.
Glial Cell Line-Derived Neurotrophic Factor Rescues Target-Deprived Sympathetic Spinal Cord Neurons But Requires Transforming Growth Factor-β as CofactorIn Vivo
Authors: Schober Andreas , Hertel Richard , Arumäe Urmas , Farkas Lilla , Jaszai Jozsef , Krieglstein Kerstin , Saarma Mart , Unsicker Klaus
Published: April 2018

Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for several populations of CNS and peripheral neurons. Synthesis and storage of GDNF by the neuron-like adrenal medullary cells suggest roles in adrenal functions and/or in the maintenance of spinal cord neurons that innervate the adrenal medulla. We show that unilateral adrenomedullectomy causes degeneration of all sympathetic preganglionic neurons within the intermediolateral column (IML) of spinal cord segments T7–T10 that project to the adrenal medulla.In situhybridization revealed that IML neurons express the glycosylphosphatidylinositol-linked α receptor 1 and c-Ret receptors, which are essential for GDNF signaling. IML neurons also display immunoreactivity for transforming growth factor-β (TGF-β) receptor II. Administration of GDNF (recombinant human, 1 μg) in Gelfoam implanted into the medullectomized adrenal gland rescued all Fluoro-Gold-labeled preganglionic neurons projecting to the adrenal medulla after four weeks. Cytochromecapplied as a control protein was not effective. The protective effect of GDNF was prevented by co-administration to the Gelfoam of neutralizing antibodies recognizing all three TGF-β isoforms but not GDNF. This suggests that the presence of endogenous TGF-β was essential for permitting a neurotrophic effect of GDNF. Our data indicate that GDNF has a capacity to protect a population of autonomic spinal cord neurons from target-deprived cell death. Furthermore, our results demonstrate for the first time that the previously reported requirement of TGF-β for permitting trophic actions of GDNFin vitro(Krieglstein et al., 1998) also applies to thein vivosituation.
Cloning and Characterization of RGS9-2: A Striatal-Enriched Alternatively Spliced Product of the RGS9 Gene
Authors: Rahman Z. , Gold S. J. , Potenza M. N. , Cowan C. W. , Ni Y. G. , He W. , Wensel T. G. , Nestler E. J.
Published: April 2018

Regulators of G-protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) for α subunits of heterotrimeric G-proteins. Previousin situhybridization analysis of mRNAs encoding RGS3–RGS11 revealed region-specific expression patterns in rat brain. RGS9 showed a particularly striking pattern of almost exclusive enrichment in striatum. In a parallel study, RGS9 cDNA, here referred to as RGS9-1, was cloned from retinal cDNA libraries, and the encoded protein was identified as a GAP for transducin (Gαt) in rod outer segments. In the present study we identify a novel splice variant of RGS9, RGS9-2, cloned from a mouse forebrain cDNA library, which encodes a striatal-specific isoform of the protein. RGS9-2 is 191 amino acids longer than the retinal isoform, has a unique 3′ untranslated region, and is highly enriched in striatum, with much lower levels seen in other brain regions and no expression detectable in retina. Immunohistochemistry showed that RGS9-2 protein is restricted to striatal neuropil and absent in striatal terminal fields. The functional activity of RGS9-2 is supported by the finding that it, but not RGS9-1, dampens the Gi/o-coupled μ-opioid receptor responsein vitro. Characterization of a bacterial artificial chromosome genomic clone of ∼200 kb indicates that these isoforms represent alternatively spliced mRNAs from a single gene and that the RGS domain, conserved among all known RGS members, is encoded over three distinct exons. The distinct C-terminal domains of RGS9-2 and RGS9-1 presumably contribute to unique regulatory properties in the neural and retinal cells in which these proteins are selectively expressed.
BDNF Is a Target-Derived Survival Factor for Arterial Baroreceptor and Chemoafferent Primary Sensory Neurons
Authors: Brady Roseann , Zaidi Syed Ishrat Ali , Mayer Catherine , Katz David M.
Published: April 2018

Brain-derived neurotrophic factor (BDNF) supports survival of 50% of visceral afferent neurons in the nodose/petrosal sensory ganglion complex (NPG; Ernfors et al., 1994a; Jones et al., 1994; Conover et al., 1995; Liu et al., 1995; Erickson et al., 1996), including arterial chemoafferents that innervate the carotid body and are required for development of normal breathing (Erickson et al., 1996). However, the relationship between BDNF dependence of visceral afferents and the location and timing of BDNF expression in visceral tissues is unknown. The present study demonstrates that BDNF mRNA and protein are transiently expressed in NPG targets in the fetal cardiac outflow tract, including baroreceptor regions in the aortic arch, carotid sinus, and right subclavian artery, as well as in the carotid body. The period of BDNF expression corresponds to the onset of sensory innervation and to the time at which fetal NPG neurons are BDNF-dependentin vitro. Moreover, baroreceptor innervation is absent in newborn mice lacking BDNF. In addition to vascular targets, vascular afferents themselves express high levels of BDNF, both during and after the time they are BDNF-dependent. However, endogenous BDNF supports survival of fetal NPG neuronsin vitroonly under depolarizing conditions. Together, these data indicate two roles for BDNF during vascular afferent pathway development; initially, as a target-derived survival factor, and subsequently, as a signaling molecule produced by the afferents themselves. Furthermore, the fact that BDNF is required for survival of functionally distinct populations of vascular afferents demonstrates that trophic requirements of NPG neurons are not modality-specific but may instead be associated with innervation of particular organ systems.
Proteolipid Protein Gene Product Can Be Secreted and Exhibit Biological Activity during Early Development
Authors: Yamada Masahisa , Ivanova Anna , Yamaguchi Yoshihide , Lees Marjorie B. , Ikenaka Kazuhiro
Published: April 2018

A gene encoding myelin proteolipid protein (PLP) and its smaller isoform DM20 is expressed at least 1 week before myelination. Mutations within the gene cause abnormalities in the development of premyelinating oligodendrocytes, resulting in hypomyelinating disorders. These findings suggest a premyelinating function of the PLP gene products. We previously demonstrated that PLP gene expression is directly associated with secretion of a factor that increases the number of oligodendrocytes. Here we show that this activity is mediated by a secreted fragment containing the C-terminal portion of PLP. This factor increased the bromodeoxyuridine incorporation rate in both oligodendrocyte and astrocyte lineage cells; a synthetic peptide (PLP 215–232) exhibited a similar activity. Dose–response curves of PLP and PLP peptide showed maximum activities at a concentration in the picomolar range, which decreased at higher concentrations. These observations demonstrate that a secreted PLP gene product exerts biological activity at a premyelinating stage before the major induction of the gene.
Role of Protein Kinase A in the Maintenance of Inflammatory Pain
Authors: Aley Kochuvelikakam O. , Levine Jon D.
Published: April 2018

Although the initiation of inflammatory pain (hyperalgesia) has been demonstrated to require the cAMP second messenger signaling cascade, whether this mechanism and/or other mechanisms underlie the continued maintenance of the induced hyperalgesia is unknown. We report that injection of adenylyl cyclase inhibitors before but not after injection of direct-acting hyperalgesic agents (prostaglandin E2and purine and serotonin receptor agonists) resulted in reduction in hyperalgesia, evaluated by the Randall–Selitto paw-withdrawal test. In contrast, injection of protein kinase A (PKA) inhibitors either before or after these hyperalgesic agents resulted in reduced hyperalgesia, suggesting that hyperalgesia after its activation was maintained by persistent PKA activity but not by adenylyl cyclase activity. To evaluate further the role of PKA activity in the maintenance of hyperalgesia, we injected the catalytic subunit of PKA (PKACS) that resulted in hyperalgesia similar in magnitude to that induced by the direct-acting hyperalgesic agents but much longer in duration (>48 vs 2 hr). Injection of WIPTIDE (a PKA inhibitor) at 24 hr after PKACS reduced hyperalgesia, suggesting that PKACS hyperalgesia is not independently maintained by steps downstream from PKA. In summary, our results indicate that, once established, inflammatory mediator-induced hyperalgesia is no longer maintained by adenylyl cyclase activity but rather is dependent on ongoing PKA activity. An understanding of the mechanism maintaining hyperalgesia may provide important insight into targets for the treatment of persistent pain.
Transient Potassium Currents Regulate the Discharge Patterns of Dorsal Cochlear Nucleus Pyramidal Cells
Authors: Kanold Patrick O. , Manis Paul B.
Published: April 2018

Pyramidal cells in the dorsal cochlear nucleus (DCN) show three distinct temporal discharge patterns in response to sound: “pauser,” “buildup,” and “chopper.” Similar discharge patterns are seenin vitroand depend on the voltage from which the cell is depolarized. It has been proposed that an inactivating A-type K+current (IKI) might play a critical role in generating the three different patterns. In this study we examined the characteristics of transient currents in DCN pyramidal cells to evaluate this hypothesis. Morphologically identified pyramidal cells in rat brain slices (P11–P17) exhibited the three voltage-dependent discharge patterns. Two inactivating currents were present in outside-out patches from pyramidal cells: a rapidly inactivating (IKIF, τ ∼11 msec) current insensitive to block by tetraethylammonium (TEA) and variably blocked by 4-aminopyridine (4-AP) with half-inactivation near −85 mV, and a slowly inactivating TEA- and 4-AP-sensitive current (IKIS, τ ∼145 msec) with half-inactivation near −35 mV. Recovery from inactivation at 34°C was described by a single exponential with a time constant of 10–30 msec, similar to the rate at which first spike latency increases with the duration of a hyperpolarizing prepulse. Acutely isolated cells also possessed a rapidly activating (<1 msec at 22°C) transient current that activated near −45 mV and showed half-inactivation near −80 mV. A model demonstrated that the deinactivation ofIKIFwas correlated with the discharge patterns. Overall, the properties of the fast inactivating K+current were consistent with their proposed role in shaping the discharge pattern of DCN pyramidal cells.
Role of the CNS Melanocortin System in the Response to Overfeeding
Authors: Hagan Mary M. , Rushing Paul A. , Schwartz Michael W. , Yagaloff Keith A. , Burn Paul , Woods Stephen C. , Seeley Randy J.
Published: April 2018

The voluntary suppression of food intake that accompanies involuntary overfeeding is an effective regulatory response to positive energy balance. Because the pro-opiomelanocortin (POMC)-derived melanocortin system in the hypothalamus promotes anorexia and weight loss and is an important mediator of energy regulation, we hypothesized that it may contribute to the hypophagic response to overfeeding. Two groups of rats were overfed to 105 and 116% of control body weight via a gastric catheter. In the first group,in situhybridization was used to measure POMC gene expression in the rostral arcuate (ARC). Overfeeding increased POMC mRNA in the ARC by 180% relative to levels in control rats. For rats in the second group, the overfeeding was stopped, and they were infused intracerebroventricularly with SHU9119 (SHU), a melanocortin (MC) antagonist at the MC3 and MC4 receptor, or vehicle. Although SHU (0.1 nmol) had no effect on food intake of control rats, intake of overfed rats increased by 265% relative to CSF-treated controls. This complete reversal of regulatory hypophagia not only maintained but actually increased the already elevated weight of overfed rats, whereas CSF-treated overfed rats lost weight. These results indicate that CNS MCs mediate hypophagic signaling in response to involuntary overfeeding and support the hypothesis that MCs are important in the central control of energy homeostasis.
Alteration of Descending Modulation of Nociception during the Course of Monoarthritis in the Rat
Authors: Danziger Nicolas , Weil-Fugazza Jeanne , Le Bars Daniel , Bouhassira Didier
Published: April 2018

Diffuse noxious inhibitory controls (DNIC), which involve supraspinal structures and modulate the transmission of nociceptive signals, were investigated at different stages during the development of adjuvant-induced monoarthritis in the rat. After behavioral evaluation, recordings of trigeminal convergent neurons were performed in anesthetized animals with acute (24–48 hr) or chronic (3–4 weeks) monoarthritis of the ankle. Inhibitions of C-fiber-evoked neuronal responses during and after the application of noxious conditioning stimuli to the ankle were measured to evaluate DNIC. The conditioning stimuli consisted of mechanical (maximal flexion and graded pressures) and graded thermal stimuli and were applied alternately to normal and arthritic ankles. Behaviorally, the two groups of animals exhibited a similar increased sensitivity to mechanical stimuli applied to the arthritic joint (i.e., an increased ankle-bend score and a decreased vocalization threshold to pressure stimuli). However, they showed different electrophysiological profiles. In the animals with acute monoarthritis, the DNIC-induced inhibitions produced by mechanical or thermal stimulation of the arthritic joint were significantly increased at all intensities compared with the normal joint. In contrast, in the chronic stage of monoarthritis, the DNIC-induced inhibitions triggered by thermal or pressure stimuli were similar for both ankles, except with the most intense mechanical stimuli. This discrepancy between the behavioral and electrophysiological findings suggests that inputs activated during chronic monoarthritis may fail to recruit DNIC and may thus be functionally different from those activated in the acute stage of inflammation.
Functional Analysis of a Mouse Brain Elk-Type K+Channel
Authors: Trudeau Matthew C. , Titus Steven A. , Branchaw Janet L. , Ganetzky Barry , Robertson Gail A.
Published: April 2018

Members of the Ether à go-go (Eag) K+channel subfamilies Eag, Erg, and Elk are widely expressed in the nervous system, but their neural functionsin vivoremain largely unknown. The biophysical properties of channels from the Eag and Erg subfamilies have been described, and based on their characteristic features and expression patterns, Erg channels have been associated with native currents in the heart. Little is known about the properties of channels from the Elk subfamily. We have identified a mouse gene,Melk2, that encodes a predicted polypeptide with 48% amino acid identity toDrosophilaElk but only 40 and 36% identity with mouse Erg (Merg) and Eag (Meag), respectively.Melk2RNA appears to be expressed at high levels only in brain tissue. Functional expression ofMelk2inXenopusoocytes reveals large, transient peaks of current at the onset of depolarization. Like Meag currents, Melk2 currents activate relatively quickly, but they lack the nonsuperimposable Cole–Moore shift characteristic of the Eag subfamily. Melk2 currents are insensitive to E-4031, a class III antiarrhythmic compound that blocks the Human Ether-à-go-go-Related Gene (HERG) channel and its counterpart in native tissues, IKr. Melk2 channels exhibit inward rectification because of a fast C-type inactivation mechanism, but the slower rate of inactivation and the faster rate of activation results in less inward rectification than that observed in HERG channels. This characterization of Melk currents should aid in identification of native counterparts to the Elk subfamily of channels in the nervous system.
A 29 kDa Intracellular Chloride Channel p64H1 Is Associated with Large Dense-Core Vesicles in Rat Hippocampal Neurons
Authors: Chuang Jen-Zen , Milner Teresa A. , Zhu Meicai , Sung Ching-Hwa
Published: April 2018

A novel class of intracellular chloride channels, the p64 family, has been found on several types of vesicles. These channels, acting in concert with the electrogenic proton pump, regulate the pH of the vesicle interior, which is critical for vesicular function. Here we describe the molecular cloning of p64H1, a p64 homolog, from both human and cow. Northern blot analysis showed that p64H1 is expressed abundantly in brain and retina, whereas the other members of this family (e.g., p64 and NCC27) are expressed only at low levels in these tissues. Immunohistochemical analysis of p64H1 in rat brain, using an affinity-purified antibody, revealed a high level of expression in the limbic system—the hippocampal formation, the amygdala, the hypothalamus, and the septum. Immunoelectron microscopic analysis of p64H1 in hippocampal neurons demonstrated a striking association between p64H1 and large dense-core vesicles (LDCVs) and microtubules. In contrast, very low p64H1 labeling was found in perikarya or associated with small synaptic vesicles (SSVs) in axonal profiles. Immunoblot analysis confirmed that p64H1 is colocalized with heavy membrane fractions containing LDCVs rather than the fractions containing SSVs. These results suggest that p64H1-mediated Cl−permeability may be involved in the maintenance of low internal pH in LDCVs and in the maturation of LDCVs and the biogenesis of functional neuropeptides.
AMPA Receptor Activates a G-Protein that Suppresses a cGMP-Gated Current
Authors: Kawai Fusao , Sterling Peter
Published: April 2018

The AMPA receptor, ubiquitous in brain, is termed “ionotropic” because it gates an ion channel directly. We found that an AMPA receptor can also modulate a G-protein to gate an ion channel indirectly. Glutamate applied to a retinal ganglion cell briefly suppresses the inward current through a cGMP-gated channel. AMPA and kainate also suppress the current, an effect that is blocked both by their general antagonist CNQX and also by the relatively specific AMPA receptor antagonist GYKI-52466. Neither NMDA nor agonists of metabotropic glutamate receptors are effective. The AMPA-induced suppression of the cGMP-gated current is blocked when the patch pipette includes GDP-β-S, whereas the suppression is irreversible when the pipette contains GTP-γ-S. This suggests a G-protein mediator, and, consistent with this, pertussis toxin blocks the current suppression. Nitric oxide (NO) donors induce the current suppressed by AMPA, and phosphodiesterase inhibitors prevent the suppression. Apparently, the AMPA receptor can exhibit a “metabotropic” activity that allows it to antagonize excitation evoked by NO.
Single-Channel Properties of Synaptic and Extrasynaptic GABAAReceptors Suggest Differential Targeting of Receptor Subtypes
Authors: Brickley Stephen G. , Cull-Candy Stuart G. , Farrant Mark
Published: April 2018

Many neurons express a multiplicity of GABAAreceptor subunit isoforms. Despite having only a single source of inhibitory input, the cerebellar granule cell displays, at various stages of development, more than 10 different GABAAsubunit types. This subunit diversity would be expected to result in significant receptor heterogeneity, yet the functional consequences of such heterogeneity remain poorly understood. Here we have used single-channel properties to characterize GABAAreceptor types in the synaptic and extrasynaptic membrane of granule cells. In the presence of high concentrations of GABA, which induced receptor desensitization, extrasynaptic receptors in outside-out patches from the soma entered long-lived closed states interrupted by infrequent clusters of openings. Each cluster of openings, which is assumed to result from the repeated activation of a single channel, was to one of three main conductance states (28, 17, or 12 pS), the relative frequency of which differed between patches. Such behavior indicates the presence of at least three different receptor types. This heterogeneity was not replicated by individual recombinant receptors (α1β2γ2Sor α1β3γ2S), which gave rise to clusters of a single type only. By contrast, the conductance of synaptic receptors, determined by fluctuation analysis of the synaptic current or direct resolution of channel events, was remarkably uniform and similar to the highest conductance value seen in extrasynaptic patches. These results suggest that granule cells express multiple GABAAreceptor types, but only those with a high conductance, most likely containing a γ subunit, are activated at the synapse.
Cannabinoids and Neuroprotection in Global and Focal Cerebral Ischemia and in Neuronal Cultures
Authors: Nagayama Tetsuya , Sinor Amy D. , Simon Roger P. , Chen Jun , Graham Steven H. , Jin Kunlin , Greenberg David A.
Published: April 2018

Marijuana and related drugs (cannabinoids) have been proposed as treatments for a widening spectrum of medical disorders.R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate(R(+)-WIN 55212-2), a synthetic cannabinoid agonist, decreased hippocampal neuronal loss after transient global cerebral ischemia and reduced infarct volume after permanent focal cerebral ischemia induced by middle cerebral artery occlusion in rats. The less active enantiomerS(−)-WIN 55212-3 was ineffective, and the protective effect ofR(+)-WIN 55212-2 was blocked by the specific central cannabinoid (CB1) cannabinoid receptor antagonistN-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide-hydrochloride.R(+)-WIN 55212-2 also protected cultured cerebral cortical neurons fromin vitrohypoxia and glucose deprivation, but in contrast to the receptor-mediated neuroprotection observedin vivo, thisin vitroeffect was not stereoselective and was insensitive to CB1and CB2receptor antagonists. Cannabinoids may have therapeutic potential in disorders resulting from cerebral ischemia, including stroke, and may protect neurons from injury through a variety of mechanisms.
Spatiotemporal Patterns of Activity in an Intact Mammalian Network with Single-Cell Resolution: Optical Studies of Nicotinic Activity in an Enteric Plexus
Authors: Obaid Ana Lía , Koyano Takeshi , Lindstrom Jon , Sakai Tetsuro , Salzberg B. M.
Published: April 2018

Multiple Site Optical Recording of Transmembrane Voltage (MSORTV) has been used to measure, continuously and simultaneously, the spontaneous electrical activity from all of the neurons in individual ganglia or up to five interconnected ganglia of the submucous plexus of the guinea pig small intestine. These are the first optical recordings of electrical activity with single-cell resolution from a mammalian nervous system. They are used to investigate the effects of acute and chronic application of nicotine on the firing patterns of this neural network containing important cholinergic components. After washout of acutely applied nicotine, the firing rates of selected neurons were dramatically elevated. These results suggest that nAChRs that reversibly desensitize after exposure to nicotine may be responsible for the enhancement of activity that is observed after a brief application of this agonist. In addition, immunostaining with monoclonal antibodies was used to localize α3/α5, α7, and β2 nAChR subunits, and the results demonstrate the prevalence of α3/α5. It is this α3-containing nAChR subtype that probably accounts for most of the excess activity elicited by nicotine application.
Glossopharyngeal Nerve Transection Eliminates Quinine-Stimulated Fos-Like Immunoreactivity in the Nucleus of the Solitary Tract: Implications for a Functional Topography of Gustatory Nerve Input in Rats
Authors: King Camille T. , Travers Susan P. , Rowland Neil E. , Garcea Mircea , Spector Alan C.
Published: April 2018

The relationship between specific gustatory nerve activity and central patterns of taste-evoked neuronal activation is poorly understood. To address this issue within the first central synaptic relay in the gustatory system, we examined the distribution of neurons in the nucleus of the solitary tract (NST) activated by the intraoral infusion of quinine using Fos immunohistochemistry in rats with bilateral transection of the chorda tympani (CTX), bilateral transection of the glossopharyngeal nerve (GLX), or combined neurotomy (DBLX). Compared with nonstimulated and water-stimulated controls, quinine evoked significantly more Fos-like-immunoreactive (FLI) neurons across the rostrocaudal extent of the gustatory NST (gNST), especially within its dorsomedial portion (subfield 5). Although the somatosensory aspects of fluid stimulation contributed to the observed increase in FLI neurons, the elevated number and spatial distribution of FLI neurons in response to quinine were remarkably distinguishable from those in response to water. GLX and DBLX produced a dramatic attenuation of quinine-evoked FLI neurons and a shift in their spatial distribution such that their number and pattern were indiscernable from those observed in water-stimulated controls. Although CTX had no effect on the number of quinine-evoked FLI neurons within subfield 5 at intermediate levels of the gNST, it produced intermediate effects elsewhere; yet, the spatial distribution of the quinine-evoked FLI neurons was not altered by CTX. These findings suggest that the GL provides input to all FLI neurons responsive to quinine, however, some degree of convergence with CT input apparently occurs in this subpopulation of neurons. Although the role of these FLI neurons in taste-guided behavioral responses to quinine remains speculative, their possible function in oromotor reflex control is considered.
Axons from Anteroventral Cochlear Nucleus that Terminate in Medial Superior Olive of Cat: Observations Related to Delay Lines
Authors: Beckius Gretchen E. , Batra Ranjan , Oliver Douglas L.
Published: April 2018

The differences in path length of axons from the anteroventral cochlear nuclei (AVCN) to the medial superior olive (MSO) are thought to provide the anatomical substrate for the computation of interaural time differences (ITD). We made small injections of biotinylated dextran into the AVCN that produced intracellular-like filling of axons. This permitted three-dimensional reconstructions of individual axons and measurements of axonal length to individual terminals in MSO. Some axons that innervated the contralateral MSO had collaterals with lengths that were graded in the rostrocaudal direction with shorter collaterals innervating more rostral parts of MSO and longer collaterals innervating more caudal parts of MSO. These could innervate all or part of the length of the MSO. Other axons had restricted terminal fields comparable to the size of a single dendritic tree in the MSO. In the ipsilateral MSO, some axons had a reverse, but less steep, gradient in axonal length with greater axonal length associated with more rostral locations; others had restricted terminal fields. Thus, the computation of ITDs is based on gradients of axonal length in both the contralateral and ipsilateral MSO, and these gradients may account for a large part of the range of ITDs encoded by the MSO. Other factors may be involved in the computation of ITDs to compensate for differences between axons.
Characterization of Progressive Motor Deficits in Mice Transgenic for the Human Huntington’s Disease Mutation
Authors: Carter Rebecca J. , Lione Lisa A. , Humby Trevor , Mangiarini Laura , Mahal Amarbirpal , Bates Gillian P. , Dunnett Stephen B. , Morton A. Jennifer
Published: April 2018

Transgenic mice expressing exon 1 of the human Huntington’s disease (HD) gene carrying a 141–157 CAG repeat (line R6/2) develop a progressive neurological phenotype with motor symptoms resembling those seen in HD. We have characterized the motor deficits in R6/2 mice using a battery of behavioral tests selected to measure motor aspects of swimming, fore- and hindlimb coordination, balance, and sensorimotor gating [swimming tank, rotarod, raised beam, fore- and hindpaw footprinting, and acoustic startle/prepulse inhibition (PPI)]. Behavioral testing was performed on female hemizygotic R6/2 transgenic mice (n= 9) and female wild-type littermates (n= 22) between 5 and 14 weeks of age. Transgenic mice did not show an overt behavioral phenotype until around 8 weeks of age. However, as early as 5–6 weeks of age they had significant difficulty swimming, traversing the narrowest square (5 mm) raised beam, and maintaining balance on the rotarod at rotation speeds of 33–44 rpm. Furthermore, they showed significant impairment in prepulse inhibition (an impairment also seen in patients with HD). Between 8 and 15 weeks, R6/2 transgenic mice showed a progressive deterioration in performance on all of the motor tests. Thus R6/2 mice show measurable deficits in motor behavior that begin subtly and increase progressively until death. Our data support the use of R6/2 mice as a model of HD and indicate that they may be useful for evaluating therapeutic strategies for HD, particularly those aimed at reducing the severity of motor symptoms or slowing the course of the disease.
Human Cone Pigment Expressed in Transgenic Mice Yields Altered Vision
Authors: Jacobs Gerald H. , Fenwick John C. , Calderone Jack B. , Deeb Samir S.
Published: April 2018

Genetically driven alterations in the complement of retinal photopigments are fundamental steps in the evolution of vision. We sought to determine how a newly added photopigment might impact vision by studying a transgenic mouse that expresses a human cone photopigment. Electroretinogram (ERG) measurements indicate that the added pigment works well, significantly changing spectral sensitivity without deleteriously affecting the operation of the native cone pigments. Visual capacities of the transgenic mice were established in behavioral tests. The new pigment was found to provide a significant expansion of the spectral range over which mice can perceive light, thus underlining the immediate utility of acquiring a new photopigment. The transgenic mouse also has the receptor basis for a novel color vision capacity, but tests show that potential was not realized. This failure likely reflects limitations in the organizational arrangement of the mouse retina.
Electrotonic Coupling Interacts with Intrinsic Properties to Generate Synchronized Activity in Cerebellar Networks of Inhibitory Interneurons
Authors: Mann-Metzer Puah , Yarom Yosef
Published: April 2018

Exploring the organization and function of local inhibitory networks is an essential step on the way to understand the principles of brain operation. We show here that molecular layer inhibitory interneurons of the guinea pig cerebellar cortex are organized as local networks, generating synchronous activity. Simultaneous recording from two adjacent interneurons revealed a direct current flow between synchronized pairs of neurons. Blocking inhibitory or excitatory synaptic transmission did not alter the synchronization. The electrotonic coupling coefficient (average 0.1) depended mainly on the input resistance of the postsynaptic cell, indicating a homogenous coupling resistance between different pairs. A presynaptic action potential generated a short, attenuated spikelet in the postsynaptic cell. The passive current flow was amplified by voltage-dependent intrinsic currents to create a reciprocal interplay between the presynaptic and postsynaptic cells. This interplay results in a time window for synchronization that is wider than expected from the duration of the spikelet. Intracellular staining with biocytin revealed high incidence of dye coupling. Furthermore, the interneurons located superficially in the molecular layer tend to form larger networks compared with the inner interneurons. We propose that weakly coupled inhibitory networks can generate loosely synchronous activity, which results from the interaction of electrical coupling and intrinsic currents.
Genetic Dissection of Behavior: Modulation of Locomotion by Light in theDrosophila melanogasterLarva Requires Genetically Distinct Visual System Functions
Authors: Busto Macarena , Iyengar Balaji , Campos Ana Regina
Published: April 2018

TheDrosophilalarva modulates its pattern of locomotion when exposed to light. Modulation of locomotion can be measured as a reduction in the distance traveled and by a sharp change of direction when the light is turned on. When the light is turned off this change of direction, albeit significantly smaller than when the light is turned on, is still significantly larger than in the absence of light transition. Mutations that disrupt adult phototransduction disrupt a subset of these responses. In larvae carrying these mutations the magnitude of change of direction when the light is turned on is reduced to levels indistinguishable from that recorded when the light is turned off, but it is still significantly higher than in the absence of any light transition. Similar results were obtained when these responses were measured in strains where the larval photoreceptor neurons were ablated by mutations in theglass(gl) gene or by the targeted expression of the cell death genehead involution defective(hid). A mutation in the homeobox genesine oculis(so) that ablates the larval visual system, or the targeted expression of thereaper(rpr) cell death gene, abolishes all responses to light detected as a change of direction. We propose the existence of an extraocular light perception that does not use the same phototransduction cascade as the adult photoreceptors. Our results indicate that this novel visual function depends on the blue-absorbing rhodopsin Rh1 and is specified by thesogene.