bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and Characterization ofAplysiaAdducin, anAplysiaCytoskeletal Protein Homologous to Mammalian Adducins: Increased Phosphorylation at a Protein Kinase C Consensus Site during Long-Term Synaptic Facilitation
Authors: Gruenbaum Lore M. , Gilligan Diana M. , Picciotto Marina R. , Marinesco Stéphane , Carew Thomas J.
Published: April 2018

Structural changes at synapses are associated with long-term facilitation (LTF) of synaptic transmission between sensory and motor neurons inAplysia. We have cloned a cDNA encodingAplysiaadducin (ApADD), theAplysiahomolog of mammalian adducins that are regulatory components of the membrane cytoskeleton. ApADD is recovered in the particulate fraction of nervous system extracts and is localized predominantly in the submembraneous region ofAplysianeurons. ApADD is phosphorylatedin vitroby protein kinase C (PKC) at a site homologous to thein vivoPKC phosphorylation site in mammalian adducins. Phosphorylation of ApADD at this site is also detectedin vivoin the intactAplysianervous system and is increased 18 hr after serotonin-induced LTF. In contrast, there is no change in phosphorylation during short-term facilitation or 1 hr after initial LTF induction. Thus, ApADD is modulated specifically with later phases of LTF and provides an attractive candidate protein that contributes to structural changes accompanying long-lasting synaptic alteration.
Caenorhabditis elegansUNC-103 ERG-Like Potassium Channel Regulates Contractile Behaviors of Sex Muscles in Males before and during Mating
Authors: Garcia L. Rene , Sternberg Paul W.
Published: April 2018

During mating behavior theCaenorhabditis elegansmale must regulate periodic and prolonged protractor muscle contractions to insert his copulatory spicules into his mate. The protractors undergo periodic contractions to allow the spicules to reattempt insertion if a previous thrust failed to breach the vulva. When the spicule tips penetrate the vulva, the protractors undergo prolonged contraction to keep the spicules inside the hermaphrodite until sperm transfer is complete. To understand how these contractions are regulated, we isolated EMS-induced mutations that cause males to execute prolonged contraction inappropriately. Loss-of-function mutations in theunc-103ERG-like K+channel gene cause the protractor muscles to contract in the absence of mating stimulation.unc-103-induced spicule protraction can be suppressed by killing the SPC motor neurons and the anal depressor muscle: cells that directly contact the protractors. Also, reduction in acetylcholine suppressesunc-103-induced protraction, suggesting that UNC-103 keeps cholinergic neurons from stimulating the protractors before mating behavior. UNC-103 also regulates the timing of spicule protraction during mating behavior.unc-103males that do not display mating-independent spicule protraction show abnormal spicule insertion behavior during sex. In contrast to wild-type males,unc-103mutants execute prolonged contractions spontaneously within sequences of periodic protractor contractions. The premature prolonged contractions cause the spicules to extend from the male tail before the spicule tips penetrate the vulva. These observations demonstrate thatunc-103controls various aspects of spicule function.
Partial Rescue of the p35−/− Brain Phenotype by Low Expression of a Neuronal-Specific Enolase p25 Transgene
Authors: Patzke Holger , Maddineni Upendra , Ayala Ramses , Morabito Maria , Volker Janet , Dikkes Pieter , Ahlijanian Michael K. , Tsai Li-Huei
Published: April 2018

Cyclin-dependent kinase 5 (Cdk5) is activated on binding of activator proteins p35 and p39. A N-terminally truncated p35, termed p25, is generated through cleavage by the Ca2+-dependent protease calpain after induction of ischemia in rat brain. p25 has been shown to accumulate in brains of patients with Alzheimer's disease and may contribute to A-β peptide-mediated toxicity. Studies from transfected neurons as well as p35 and p25 transgenic mice have indicated that Cdk5, when activated by p25, gains some toxic function compared with p35/Cdk5. It remains unclear, however, whether p25/Cdk5 signaling additionally channels into pathways usually used by p35/Cdk5 and whether p25 is associated with a loss of p35 function. To clarify these issues, we have generated p25-transgenic mice in a p35-null background. We find that low levels of p25 during development induce a partial rescue of the p35−/− phenotype in several brain regions analyzed, including a rescue of cell positioning of a subset of neurons in the neocortex. In accordance with the partial rescue of brain anatomy, phosphorylation of the Cdk5 substrate mouse disabled 1 is partially restored during development. Besides this, p25/Cdk5 fails to phosphorylate other substrates that are normally phosphorylated by p35/Cdk5. Our results show that p25 can substitute for p35/Cdk5 under certain circumstances during development. In addition, they suggest that p25 may have lost some functions of p35.
Visualizing Changes in Circuit Activity Resulting from Denervation and Reinnervation Using Immediate Early Gene Expression
Authors: Temple Meredith D. , Worley Paul F. , Steward Oswald
Published: April 2018

We describe a novel strategy to evaluate circuit function after brain injury that takes advantage of experience-dependent immediate early gene (IEG) expression. When normal rats undergo training or are exposed to a novel environment, there is a strong induction of IEG expression in forebrain regions, including the hippocampus. This gene induction identifies the neurons that are engaged during the experience. Here, we demonstrate that experience-dependent IEG induction is diminished after brain injury in young adult rats (120–200 gm), specifically after unilateral lesions of the entorhinal cortex (EC), and then recovers with a time course consistent with reinnervation.In situhybridization techniques were used to assess the expression of the activity-regulated cytoskeleton-associated protein Arc at various times after the lesion (4, 8, 12, 16, or 30 d). One group of rats was allowed to explore a complex novel environment for 1 hr; control operated animals remained in their home cage. In unoperated animals, exposure to the novel environment induced Arc mRNA levels in most pyramidal neurons in CA1, in many pyramidal neurons in CA3, and in a small number of dentate granule cells. This characteristic pattern of induction was absent at early time points after unilateral EC lesions (4 and 8 d) but recovered progressively at later time points. The recovery of Arc expression occurred with approximately the same time course as the reinnervation of the dentate gyrus as a result of postlesion sprouting. These results document a novel approach for quantitatively assessing activity-regulated gene expression in polysynaptic circuits after trauma.
A Chondroitin Sulfate Proteoglycan PTPζ/RPTPβ Regulates the Morphogenesis of Purkinje Cell Dendrites in the Developing Cerebellum
Authors: Tanaka Masahiko , Maeda Nobuaki , Noda Masaharu , Marunouchi Tohru
Published: April 2018

PTPζ/RPTPβ, a receptor-type protein tyrosine phosphatase synthesized as a chondroitin sulfate (CS) proteoglycan, uses a heparin-binding growth factor pleiotrophin (PTN) as a ligand, in which the CS portion plays an essential role in ligand binding. Using an organotypic slice culture system, we tested the hypothesis that PTN-PTPζ signaling is involved in the morphogenesis of Purkinje cell dendrites. An aberrant morphology of Purkinje cell dendrites such as multiple and disoriented primary dendrites was induced in slice cultures by (1) addition of a polyclonal antibody against the extracellular domain of PTPζ, (2) inhibition of protein tyrosine phosphatase activity, (3) enzymatic removal of the CS chains, (4) addition of exogenous CS chains, and (5) addition of exogenous PTN, all of which disturb PTN-PTPζ signaling. These treatments also reduced the immunoreactivity to GLAST, a glial glutamate transporter, on Bergmann glial processes. Furthermore, a glutamate transporter inhibitor also induced the abnormal morphogenesis of Purkinje cell dendrites. Altogether, these findings suggest that PTN-PTPζ signaling regulates the morphogenesis of Purkinje cell dendrites and that the mechanisms underlying that regulation involve the GLAST activity in Bergmann glial processes.
Regulation of Spike Initiation and Propagation in anAplysiaSensory Neuron: Gating-In via Central Depolarization
Authors: Evans Colin G. , Jing Jian , Rosen Steven C. , Cropper Elizabeth C.
Published: April 2018

Afferent transmission can be regulated (or gated) so that responses to peripheral stimuli are adjusted to make them appropriate for the ongoing phase of a motor program. Here, we characterize a gating mechanism that involves regulation of spike propagation inAplysiamechanoafferent B21. B21 is striking in that afferent transmission to the motor neuron B8 does not occur when B21 is at resting membrane potential. Our data suggest that this results from the fact that spikes are not actively propagated to the lateral process of B21 (the primary contact with B8). When B21 is peripherally activated at its resting potential, electrotonic potentials in the lateral process are on average 11 mV. In contrast, mechanoafferent activity is transmitted to B8 when B21 is centrally depolarized via current injection. Our data suggest that central depolarization relieves propagation failure. Full-size spikes are recorded in the lateral process when B21 is depolarized and then peripherally activated. Moreover, changes in membrane potential in the lateral process affect spike amplitude, even when the somatic membrane potential is virtually unchanged. During motor programs, both the lateral process and the soma of B21 are phasically depolarized via synaptic input. These depolarizations are sufficient to convert subthreshold potentials to full-size spikes in the lateral process. Thus, our data strongly suggest that afferent transmission from B21 to B8 is, at least in part, regulated via synaptic control of spike initiation in the lateral process. Consequences of this control for compartmentalization in B21 are discussed, as are specific consequences for feeding behavior.
Synaptic Pathways to Phrenic Motoneurons Are Enhanced by Chronic Intermittent Hypoxia after Cervical Spinal Cord Injury
Authors: Fuller David D. , Johnson Stephen M. , Olson E. Burdette , Mitchell Gordon S.
Published: April 2018

Spinal hemisection at C2 reveals caudal synaptic pathways that cross the spinal midline (crossed phrenic pathways) and can restore inspiratory activity in ipsilateral phrenic motoneurons. Intermittent hypoxia induces plasticity in the cervical spinal cord, resulting in enhanced inspiratory phrenic motor output. We hypothesized that chronic intermittent hypoxia (CIH) (alternating 11% O2and air; 5 min periods; 12 hr per night; 7 nights) would strengthen crossed phrenic pathways. Experiments were performed on anesthetized, vagotomized, paralyzed, ventilated, and spinally injured (C2 hemisection) rats that were exposed to either normoxia or CIH before acute injury (preconditioning) or after chronic injury (postconditioning). Spontaneous inspiratory bursts or compound action potentials evoked via stimulation of the ventrolateral funiculus (contralateral to injury) were recorded in both phrenic nerves. Spontaneous or evoked activity in crossed phrenic pathways were minimal or absent in all acutely injured rats regardless of preconditioning. In rats postconditioned with normoxia, crossed phrenic inspiratory bursts were observed occasionally during baseline conditions and always during chemoreceptor stimulation (hypoxia and hypercapnia). However, CIH postconditioned rats had substantially larger crossed phrenic inspiratory bursts during baseline, hypoxia, and hypercapnia (allp< 0.05 vs normoxic group). Short-latency (0.7 msec) evoked crossed phrenic potentials were also enhanced by CIH conditioning in chronically injured rats (p< 0.05). We conclude that CIH induced spinal cord plasticity-enhanced phrenic motor output. This plasticity required preconditions established by chronic spinal injury.
Directional Avoidance Turns Encoded by Single Interneurons and Sustained by Multifunctional Serotonergic Cells
Authors: Jing Jian , Gillette Rhanor
Published: April 2018

Avoidance turns in the sea slugPleurobranchaeaare responses to noxious stimuli and replace orienting turns to food stimuli after avoidance conditioning or satiation. Avoidance turns proved to be centrally patterned behaviors, the fictive expression of which could be elicited in reduced preparations and the isolated CNS. Activity in one of a bilateral interneuron pair, the A4 cells, was necessary and sufficient to drive the avoidance turn toward the contralateral side. Single A4 cells appeared to encode both turn direction and angle, in contrast to directional behaviors of other animals in which displacement angle is usually encoded by multiple units.

The As1–4 cells, bilateral serotonergic cell clusters, excited the prolonged A4 burst during the turn through electrical and chemical coupling. However, during the escape swim, As1–4 became integral elements of the swim motor network, and A4 activity was entrained to the swim rhythm by alternating excitatory–inhibitory inputs, with only weak spiking. This provides a likely mechanism for the previously observed suppression of the avoidance turn by escape swimming. These observations add significant new aspects to the multiplying known functions of As1–4 and their homologs in other molluscs and point to a pivotal role of these neurons in the organization of gastropod behavior.

Simple functional models predict (1) the essential actions of inhibitor neurons in the directionality of the turning network motor output and (2) an integrating role for As1–4 in the behavioral switch between turning avoidance and swimming escape, on the basis of their response to increasing stimulus intensity.
Neuroprotection by the Endogenous Cannabinoid Anandamide and Arvanil againstIn VivoExcitotoxicity in the Rat: Role of Vanilloid Receptors and Lipoxygenases
Authors: Veldhuis W. B. , van der Stelt M. , Wadman M. W. , van Zadelhoff G. , Maccarrone M. , Fezza F. , Veldink G. A. , Vliegenthart J. F. G. , Bär P. R. , Nicolay K. , Di Marzo V.
Published: April 2018

Type 1 vanilloid receptors (VR1) have been identified recently in the brain, in which they serve as yet primarily undetermined purposes. The endocannabinoid anandamide (AEA) and some of its oxidative metabolites are ligands for VR1, and AEA has been shown to afford protection against ouabain-inducedin vivoexcitotoxicity, in a manner that is only in part dependent on the type 1 cannabinoid (CB1) receptor. In the present study, we assessed whether VR1is involved in neuroprotection by AEA and by arvanil, a hydrolysis-stable AEA analog that is a ligand for both VR1and CB1. Furthermore, we assessed the putative involvement of lipoxygenase metabolites of AEA in conveying neuroprotection. Using HPLC and gas chromatography/mass spectroscopy, we demonstrated that rat brain and blood cells converted AEA into 12-hydroxy-N-arachidoylethanolamine (12-HAEA) and 15-hydroxy-N-arachidonoylethanolamine (15-HAEA) and that this conversion was blocked by addition of the lipoxygenase inhibitor nordihydroguaiaretic acid. Using magnetic resonance imaging we show the following: (1) pretreatment with the reduced 12-lipoxygenase metabolite of AEA, 12-HAEA, attenuated cytotoxic edema formation in a CB1receptor-independent manner in the acute phase after intracranial injection of the Na+/K+-ATPase inhibitor ouabain; (2) the reduced 15-lipoxygenase metabolite, 15-HAEA, enhanced the neuroprotective effect of AEA in the acute phase; (3) modulation of VR1, as tested using arvanil, the VR1agonist capsaicin, and the antagonist capsazepine, leads to neuroprotective effects in this model, and arvanil is a potent neuroprotectant, acting at both CB1and VR1; and (4) thein vivoneuroprotective effects of AEA are mediated by CB1but not by lipoxygenase metabolites or VR1.
Serotonin 1A Receptor Agonists Reverse Respiratory Abnormalities in Spinal Cord-Injured Rats
Authors: Teng Yang Dong , Bingaman Marian , Taveira-DaSilva Angelo M. , Pace Peter P. , Gillis Richard A. , Wrathall Jean R.
Published: April 2018

Contusion spinal cord injury (SCI) at T8 produces respiratory abnormalities in conscious rats breathing room air and challenged with CO2. In seeking ways to improve respiration after SCI, we tested drugs that stimulate serotonin 1A (5-HT1A) receptors, based on our previous findings that these agents can counteract respiratory depression produced by morphine overdose. Respiratory function was measured with a head-out plethysmograph system in conscious rats. T8 SCI rats (n= 5) showed decreased tidal volume (Vt; 0.90 ± 0.02-0.66 ± 0.03 ml;p< 0.05) and increased respiratory rate (f;91 ± 3.7-132 ± 5.7 breaths/min;p< 0.05) with room air ventilation at 24 hr after injury. They also exhibited a diminished response to the respiratory stimulating effect of 7% CO2; minute ventilation increased to 250 ± 17 ml/min before, but only to 162 ± 15 ml/min at 24 hr after SCI (p< 0.05). Respiratory deficits during room air ventilation were also observed at 7 d after injury (n= 3). Treatment with the 5-HT1Areceptor agonist 8-hydroxy-2-(di-n-propylmino)tetralin (8-OH-DPAT; 250μg/kg, i.p.) at 24 hr (n= 5) or 7 d (n= 3) after injury normalized Vt,f, and the respiratory response to 7% CO2. Identical results were obtained with another 5-HT1Areceptor agonist, buspirone (1.5 mg/kg, i.p.;n= 3). In contrast, intraperitoneal saline vehicle administration (n= 5) showed no beneficial effects on SCI-impaired respiration. Finally, pretreatment with a specific antagonist of 5-HT1Areceptors, 4-iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-benzamide (3 mg/kg, i.p.;n= 3) given 20 min before 8-OH-DPAT, prevented 8-OH-DPAT from restoring respiration to normal. Our results demonstrate that drugs that stimulate 5-HT1Areceptors counteract respiratory abnormalities in conscious rats after SCI.
TheGrowth Arrest-SpecificGene Product Gas6 Promotes the Survival of Human Oligodendrocytes via a Phosphatidylinositol 3-Kinase-Dependent Pathway
Authors: Shankar Sai Latha , O'Guin Kathleen , Cammer Michael , McMorris F. Arthur , Stitt Trevor N. , Basch Ross S. , Varnum Brian , Shafit-Zagardo Bridget
Published: April 2018

Microarray analysis revealed that transcripts for the Axl and Mer receptor tyrosine kinases are expressed at high levels in O4+-immunopanned oligodendrocytes isolated from second trimester human fetal spinal cord. In humans the sole known ligand for the Axl/Rse/Mer kinases isgrowth arrest-specificgene 6 (Gas6), which in the CNS is secreted by neurons and endothelial cells. We hypothesized that Gas6 is a survival factor for oligodendrocytes and receptor activation signals downstream to the phosphatidylinositol 3 (PI3)-kinase/Akt pathway to increase cell survival in the absence of cell proliferation. To test this hypothesis, we grew enriched human oligodendrocytes for 6 d on a monolayer of NIH3T3 cells stably expressing Gas6. CNP+oligodendrocytes on Gas6-secreting 3T3 cells had more primary processes and arborizations than those plated solely on 3T3 cells. Also, a twofold increase in CNP+and MBP+oligodendrocytes was observed when they were plated on the Gas6-secreting cells. The effect was abolished in the presence of Axl-Fc but remained unchanged in the presence of the irrelevant receptor fusion molecule TrkA-Fc. A significant decrease in CNP+/TUNEL+oligodendrocytes was observed when recombinant human Gas6 (rhGas6) was administered to oligodendrocytes plated on poly-l-lysine, supporting a role for Gas6 signaling in oligodendrocyte survival during a period of active myelination in human fetal spinal cord development. PI3-kinase inhibitors blocked the anti-apoptotic effect of rhGas6, whereas a MEK/ERK inhibitor had no effect. Thus Gas6 sustains human fetal oligodendrocyte viability by receptor activation and downstream signaling via the PI3-kinase/Akt pathway.
Osmotic Regulation of Estrogen Receptor-β in Rat Vasopressin and Oxytocin Neurons
Authors: Somponpun Suwit J. , Sladek Celia D.
Published: April 2018

The vasopressin (VP) magnocellular neurosecretory cells (MNCs) in the supraoptic and paraventricular (PVN) nuclei are regulated by estrogen and exhibit robust expression of estrogen receptor (ER)-β. In contrast, only ∼7.5% of oxytocin (OT) MNCs express ER-β. We examined the osmotic regulation of ER-β mRNA expression in MNCs using quantitativein situhybridization histochemistry. Hyper-osmolality induced via 2% hypertonic saline ingestion significantly decreased, whereas sustained hypo-osmolality induced via d-d-arginine VP and liquid diet increased ER-β mRNA expression in MNCs (p< 0.05). Thus, the expression of ER-β mRNA correlated inversely with changes in plasma osmolality. Because hyper-osmolality is a potent stimulus for VP and OT release, this suggests an inhibitory role for ER-β in MNCs. Immunocytochemistry demonstrated that the decrease in ER-β mRNA was translated into depletion of receptor protein content in hyper-osmotic animals. Numerous MNCs were positive for ER-β in control animals, but they were virtually devoid of ER-β-immunoreactivity (IR) in hyper-osmotic animals. The osmotically induced decrease in ER-β expression was selective for MNCs because ER-β-IR remained unaltered in PVN parvocellular neurons. Plasma estradiol and testosterone were not correlated with ER-β mRNA expression after osmotic manipulation, suggesting that ER-β expression was not driven by ligand availability. Expression of FOS-IR in MNCs with attenuated ER-β-IR, and the absence of FOS-IR in parvocellular neurons that retain ER-β-IR suggest a role for neuronal activation in the regulation of ER-β expression in MNCs. Thus, osmotic modulation of ER-β expression in MNCs may augment or attenuate an inhibitory effect of gonadal steroids on VP release.
Rapid Motor Learning in the Translational Vestibulo-Ocular Reflex
Authors: Zhou Wu , Weldon Patrick , Tang Bingfeng , King W. M.
Published: April 2018

Motor learning was induced in the translational vestibulo-ocular reflex (TVOR) when monkeys were repeatedly subjected to a brief (0.5 sec) head translation while they tried to maintain binocular fixation on a visual target for juice rewards. If the target was world-fixed, the initial eye speed of the TVOR gradually increased; if the target was head-fixed, the initial eye speed of the TVOR gradually decreased. The rate of learning acquisition was very rapid, with a time constant of ∼100 trials, which was equivalent to <1 min of accumulated stimulation. These learned changes were consolidated over ≥1 d without any reinforcement, indicating induction of long-term synaptic plasticity. Although the learning generalized to targets with different viewing distances and to head translations with different accelerations, it was highly specific for the particular combination of head motion and evoked eye movement associated with the training. For example, it was specific to the modality of the stimulus (translation vs rotation) and the direction of the evoked eye movement in the training. Furthermore, when one eye was aligned with the heading direction so that it remained motionless during training, learning was not expressed in this eye, but only in the other nonaligned eye. These specificities show that the learning sites are neither in the sensory nor the motor limb of the reflex but in the sensory-motor transformation stage of the reflex. The dependence of the learning on both head motion and evoked eye movement suggests that Hebbian learning may be one of the underlying cellular mechanisms.
Nitric Oxide-Mediated Cortical Activation: A Diffuse Wake-Up System
Authors: Mariño Jorge , Cudeiro Javier
Published: April 2018

Nitric oxide (NO) has been implicated in some of the central pathways engaged in the regulation of the sleep–wake cycle. The existence of nitric oxide synthase (NOS) in the cholinergic basal forebrain (BF) cells projecting to the cortex suggests a role for NO in the activation induced by the BF during arousal. We tested, in the anesthetized cat, the hypothesis that inhibition of NOS would decrease the ability of BF cholinergic fibers to induce cortical activation. In control conditions, BF stimulation evoked an awake-like EEG pattern (i.e., a decrease in the low-frequency–high-amplitude oscillatory activity and an increase in the high-frequency–low-amplitude activity). After blocking NOS activity, the capacity of BF stimulation to induce cortical activation was strongly impaired. Furthermore, voltammetric measurements of NO levels revealed an increase in cortical NO after BF stimulation, also blocked by systemic NOS inhibition. These results indicate that the blockade of NOS activity significantly reduces the ability of BF stimulation to induce changes in the EEG pattern and suggest a role for NO in the BF–cholinergic system implicated in arousal mechanisms.
Neuronal Activity in the Rostral Superior Colliculus Related to the Initiation of Pursuit and Saccadic Eye Movements
Authors: Krauzlis Richard J.
Published: April 2018

The extinction of the central fixated stimulus before the appearance of a new target stimulus reduces the latency of saccades and pursuit, a phenomenon known as the “gap effect.” The superior colliculus (SC) plays a prominent role in the gap effect for saccades, and recent data indicate that this structure also plays some role in the control of pursuit. We now show that the firing rate of buildup neurons in the rostral SC exhibits a gap effect during the initiation of both pursuit and saccadic eye movements to parafoveal targets. Most neurons exhibited an increase in tonic activity starting ∼100 msec after the offset of the fixation spot, regardless of whether the target later appeared inside or outside of the response field of the neuron. The subsequent appearance of the target in the response field evoked phasic increases in activity that were approximately twice as large as the effects on tonic activity. For both pursuit and saccades, the levels of tonic and phasic activity were inversely correlated with latency on a trial-by-trial basis. These changes in activity provide a neuronal correlate for the shared effects on latency observed previously with the gap paradigm for pursuit and saccades. Finally, the phasic activity at pursuit onset exhibited a gap effect just like the target-evoked response, whereas the burst activity at saccade onset was fixed in amplitude. These results suggest how SC neurons may coordinate the initiation of pursuit and saccades: buildup activity may gate the initiation of pursuit, whereas it indirectly triggers saccades by recruiting a saccade-related burst.
Math1Gene Transfer Generates New Cochlear Hair Cells in Mature Guinea PigsIn Vivo
Authors: Kawamoto Kohei , Ishimoto Shin-Ichi , Minoda Ryosei , Brough Douglas E. , Raphael Yehoash
Published: April 2018

Hair cell loss in the mammalian cochlea is irreversible and results in permanent hearing loss.Math1, the basic helix-loop-helix transcription factor homolog of theDrosophila atonalgene, is a positive regulator of hair cell differentiation during cochlear development. Developing hair cells expressMath1, and nonsensory cells do not. We set out to determine the outcome of overexpression ofMath1in nonsensory cells of the cochlea on the phenotype of these cells. We demonstrate thatin vivoinoculation of adenovirus with theMath1gene insert into the endolymph of the mature guinea pig cochlea results inMath1overexpression in nonsensory cochlear cells, as evident from the presence of Math1 protein in supporting cells of the organ of Corti and in adjacent nonsensory epithelial cells.Math1overexpression leads to the appearance of immature hair cells in the organ of Corti and new hair cells adjacent to the organ of Corti in the interdental cell, inner sulcus, and Hensen cell regions. Axons are extended from the bundle of auditory nerve toward some of the new hair cells, suggesting that the new cells attract auditory neurons. We conclude that nonsensory cells in the mature cochlea retain the competence to generate new hair cells after overexpression ofMath1 in vivoand thatMath1is necessary and sufficient to direct hair cell differentiation in these mature nonsensory cells.
Alzheimer's Presenilin 1 Mutations Impair Kinesin-Based Axonal Transport
Authors: Pigino Gustavo , Morfini Gerardo , Pelsman Alejandra , Mattson Mark P. , Brady Scott T. , Busciglio Jorge
Published: April 2018

Several lines of evidence indicate that alterations in axonal transport play a critical role in Alzheimer's disease (AD) neuropathology, but the molecular mechanisms that control this process are not understood fully. Recent work indicates that presenilin 1 (PS1) interacts with glycogen synthase kinase 3β (GSK3β).In vivo, GSK3β phosphorylates kinesin light chains (KLC) and causes the release of kinesin-I from membrane-bound organelles (MBOs), leading to a reduction in kinesin-I driven motility (Morfini et al., 2002b). To characterize a potential role for PS1 in the regulation of kinesin-based axonal transport, we used PS1-/-and PS1 knock-inM146V(KIM146V) mice and cultured cells. We show that relative levels of GSK3β activity were increased in cells either in the presence of mutant PS1 or in the absence of PS1 (PS1-/-). Concomitant with increased GSK3β activity, relative levels of KLC phosphorylation were increased, and the amount of kinesin-I bound to MBOs was reduced. Consistent with a deficit in kinesin-I-mediated fast axonal transport, densities of synaptophysin- and syntaxin-I-containing vesicles and mitochondria were reduced in neuritic processes of KIM146Vhippocampal neurons. Similarly, we found reduced levels of PS1, amyloid precursor protein, and synaptophysin in sciatic nerves of KIM146Vmice. Thus PS1 appears to modulate GSK3β activity and the release of kinesin-I from MBOs at sites of vesicle delivery and membrane insertion. These findings suggest that mutations in PS1 may compromise neuronal function by affecting GSK-3 activity and kinesin-I-based motility.
Inducible cAMP Early Repressor, an Endogenous Antagonist of cAMP Responsive Element-Binding Protein, Evokes Neuronal ApoptosisIn Vitro
Authors: Jaworski Jacek , Mioduszewska Barbara , Sánchez-Capelo Amelia , Figiel Izabela , Habas Agata , Gozdz Agata , Proszynski Tomasz , Hetman Michal , Mallet Jacques , Kaczmarek Leszek
Published: April 2018

Active CREB (cAMP responsive element-binding protein) transcription factor is crucial for neuronal survival. Several members of the CREM/ICER (cAMP responsive element modulator/inducible cAMP early repressor) protein family may act as endogenous CREB antagonists. However, their involvement in a process of programmed cell death remains unexplored. Here we report that ICER may play such a role in neuronal apoptosis because it is upregulated in apoptotic neuronsin vitro, and overexpression of ICER, delivered in adenoviral vector, evokes programmed cell death of three different kinds of cultured neurons, namely those derived from hippocampal dentate gyrus, cerebral cortex, and superior cervical ganglion. Reporter gene assay with a promoter containing a CREB-responsive sequence revealed a decrease in both basal and induced CRE-dependent gene expression in neurons overexpressing ICER. Finally, the level of expression of the anti-apoptotic protein Bcl-2, a well known CREB target, was markedly diminished in ICER-treated neurons. We suggest that the naturally occurring CREB functional antagonist ICER may have a specific function in programmed cell death of neurons, probably by silencing the expression of anti-apoptotic genes.
Synapse-Associated Protein-97 Isoform-Specific Regulation of Surface AMPA Receptors and Synaptic Function in Cultured Neurons
Authors: Rumbaugh Gavin , Sia Gek-Ming , Garner Craig C. , Huganir Richard L.
Published: April 2018

Members of the synapse-associated protein-97 (SAP97) family of scaffold proteins have been implicated as central organizers of synaptic junctions to build macromolecular signaling complexes around specific postsynaptic neurotransmitter receptors. In this regard, SAP97 has been suggested to regulate the synaptic localization of glutamate receptor type 1 subunits of the AMPA-type glutamate receptors. To test this hypothesis directly, we assessed the effects of SAP97 overexpression on surface expression of synaptic AMPA receptors. We find that recombinant SAP97 not only becomes concentrated at synaptic junctions but also leads to an increase in synaptic AMPA receptors, spine enlargement, and an increase in miniature EPSC (mEPSC) frequency, indicating that SAP97 has both postsynaptic and presynaptic effects on synaptic transmission. Synaptic targeting of SAP97, increased surface AMPA receptors, and increased mEPSC frequency are dependent on the presence of specific alternatively spliced sequences in SAP97 that encode a protein 4.1 binding site. These results suggest that SAP97 can affect the synaptic recruitment of AMPA receptors and spine morphology and that these effects may be regulated by alternative splicing.
The Distribution of Calcium Buffering Proteins in the Turtle Cochlea
Authors: Hackney Carole M. , Mahendrasingam Shanthini , Jones Eugenia M. C. , Fettiplace Robert
Published: April 2018

Hair cells of the inner ear contain high concentrations of calcium-binding proteins that limit calcium signals and prevent cross talk between different signaling pathways during auditory transduction. Using light microscope immunofluorescence and post-embedding immunogold labeling in the electron microscope, we characterized the distribution of three calcium-buffering proteins in the turtle cochlea. Both calbindin-D28k and parvalbumin-β were confined to hair cells in which they showed a similar distribution, whereas calretinin was present mainly in hair-cell nuclei but also occurred in supporting cells and nerve fibers. The hair-cell concentration of calbindin-D28k but not of parvalbumin-β increased from the low- to high-frequency end of the cochlea. Calibration against standards containing known amounts of calcium-buffering protein processed in the same fluid drop as the cochlear sections gave cytoplasmic concentrations of calbindin-D28k as 0.13–0.63 mmand parvalbumin-β as ∼0.25 mm, but calretinin was an order of magnitude less. Total amount of Ca2+-binding sites on the proteins is at least 1.0 mmin low-frequency hair cells and 3.0 mmin high-frequency cells. Reverse transcription-PCR showed that mRNA for all three proteins was expressed in turtle hair cells. We suggest that calbindin-D28k and parvalbumin-β may serve as endogenous mobile calcium buffers, but the predominantly nuclear location of calretinin argues for another role in calcium signaling. The results support conclusions from electrophysiological measurements that millimolar concentrations of endogenous calcium buffers are present in turtle hair cells. Parvalbumin-β was also found in both inner and outer hair cells of the guinea pig cochlea.
Regulated Expression of ATF5 Is Required for the Progression of Neural Progenitor Cells to Neurons
Authors: Angelastro James M. , Ignatova Tatyana N. , Kukekov Valery G. , Steindler Dennis A. , Stengren George B. , Mendelsohn Cathy , Greene Lloyd A.
Published: April 2018

An important milestone in brain development is the transition of neuroprogenitor cells to postmitotic neurons. We report that the bZIP transcription factor ATF5 plays a major regulatory role in this process. In developing brain ATF5 expression is high within ventricular zones containing neural stem and progenitor cells and is undetectable in postmitotic neurons. In attached clonal neurosphere cultures ATF5 is expressed by neural stem/progenitor cells and is undetectable in tau-positive neurons. In PC12 cell cultures nerve growth factor (NGF) dramatically downregulates endogenous ATF5 protein and transcripts, whereas exogenous ATF5 suppresses NGF-promoted neurite outgrowth. Such inhibition requires the repression of CRE sites. In contrast, loss of function conferred by dominant-negative ATF5 accelerates NGF-promoted neuritogenesis. Exogenous ATF5 also suppresses, and dominant-negative ATF5 and a small-interfering RNA targeted to ATF5 promote, neurogenesis by cultured nestin-positive telencephalic cells. These findings indicate that ATF5 blocks the differentiation of neuroprogenitor cells into neurons and must be downregulated to permit this process to occur.
Inhibition of the Proteasome by Lactacystin Enhances Oligodendroglial Cell Differentiation
Authors: Pasquini Laura A. , Paez Pablo M. , Moreno Marcos A. N. Besio , Pasquini Juana M. , Soto Eduardo F.
Published: April 2018

We have used lactacystin, a specific inhibitor of the 26S proteasome, in oligodendroglial cell (OLGc) primary cultures to explore the possible participation of the proteasome–ubiquitin-dependent pathway in the decision of the OLGcs to arrest their proliferation and start differentiation. Addition of lactacystin at various concentrations to cultures containing a majority of OLGc was found to produce their withdrawal from the cell cycle and to induce their biochemical and morphological differentiation, with the appearance of extensive myelin-like sheets. The three classic proteolytic activities of the proteasome were significantly decreased in the lactacystin-treated cultures, and the immunocytochemical analysis showed an increase in the number of O4-, O1-, myelin basic protein-, and myelin proteolipid protein-positive cells and a decrease in A2B5-reacting cells. Quantitative immunochemical evaluation of the expression of certain proteins controlling the cell cycle showed an increase in p27kip1-, cyclin D-, and cdk4-positive cells, with a decrease in cyclin E- and cdk2-positive cells. In the lactacystin-treated OLGcs, there was a dose-dependent decrease in the number of cells incorporating bromodeoxyuridine and in the activity of the complexes cyclin D–cdk4 and cyclin E–cdk2. Furthermore, increased levels of expression of several STAT factors were found, suggesting that proteasome inhibition in OLGcs could stabilize signals of survival and differentiation that might be processed through the JAK/STAT signaling cascade.
Voltage-Dependent Switching of Sensorimotor Integration by a Lobster Central Pattern Generator
Authors: Nargeot Romuald
Published: April 2018

Behavioral adaptations and the underlying neural plasticity may not simply result from peripheral information conveyed by sensory inputs. Central neuronal networks often spontaneously generate neuronal activity patterns that may also contribute to sensorimotor integration and behavioral adaptations. The present study explored a novel form of sensory-induced plasticity by which the resulting changes in motor output depend essentially on the preexisting functional state of an identified neuron of an endogenously active central network. In the isolated lobster stomatogastric nervous system, electrical stimulation of a mechanosensory nerve transiently inactivated rhythmic spike bursting in the lateral pyloric (LP) neuron of the pyloric motor pattern-generating network. Repeated sensory nerve stimulation gradually and long-lastingly strengthened the bursting of the LP neuron to the detriment of sensory-elicited inactivation. This strengthening of pyloric-timed rhythmic activity was enhanced by experimental depolarization of the neuron. Conversely, when the LP neuron was hyperpolarized, the same sensory stimulation paradigm now gradually increased the susceptibility of the pyloric-timed bursting of the network neuron to sensory-elicited inactivation. Modulation of depolarization-activated and hyperpolarization-activated ionic conductances that underlie the intrinsic bursting properties of the LP neuron may contribute via differential voltage-dependent recruitment and effects to the respective adaptive processes. These data therefore suggest a novel state-dependent mechanism by which an endogenously active central network can decrease or increase its responsiveness to the same sensory input.
Experiments to Test the Role of Phosphatidylinositol 4,5-Bisphosphate in Neurotransmitter-Induced M-Channel Closure in Bullfrog Sympathetic Neurons
Authors: Ford Christopher P. , Stemkowski Patrick L. , Light Peter E. , Smith Peter A.
Published: April 2018

Various neurotransmitters excite neurons by suppressing a ubiquitous, voltage-dependent, noninactivating K+conductance called the M-conductance (gM). In bullfrog sympathetic ganglion neurons the suppression of gMby the P2Y agonist ATP involves phospholipase C (PLC). The present results are consistent with the involvement of the lipid and inositol phosphate cycles in the effects of both P2Y and muscarinic cholinergic agonists on gM. Impairment of resynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2) with the phosphatidylinositol 4-kinase inhibitor wortmannin (10 μm) slowed or blocked the recovery of agonist-induced gMsuppression. This effect could not be attributed to an action of wortmannin on myosin light chain kinase or on phosphatidylinositol 3-kinase. Inhibition of PIP2synthesis at an earlier point in the lipid cycle by the use of R59022 (40 μm) to inhibit diacylglycerol kinase also slowed the rate of recovery of successive ATP responses. This effect required several applications of agonist to deplete levels of various phospholipid intermediates in the lipid cycle. PIP2antibodies attenuated the suppression of gMby agonists. Intracellular application of 20 μmPIP2slowed the rundown of KCNQ2/3 currents expressed in COS-1 or tsA-201 cells, and 100 μmPIP2produced a small potentiation of native M-current bullfrog sympathetic neurons. These are the results that might be expected if agonist-induced activation of PLC and the concomitant depletion of PIP2contribute to the excitatory action of neurotransmitters that suppress gM.
Oligodendrocytes Promote Neuronal Survival and Axonal Length by Distinct Intracellular Mechanisms: A Novel Role for Oligodendrocyte-Derived Glial Cell Line-Derived Neurotrophic Factor
Authors: Wilkins Alastair , Majed Henry , Layfield Robert , Compston Alastair , Chandran Siddharthan
Published: April 2018

Interactions of CNS cells lead to the establishment of complex neural systems. Specifically, oligodendrocytes form myelin sheaths around axons that enable rapid electrical conduction of impulses. Recent evidence has emerged that oligodendrocytes may also release trophic factors promoting neuronal survival. We therefore studied the effects of factors released from cells of the oligodendrocyte lineage on neuronal survival and also on the morphology of neurons. Neurons derived from rat embryonic cortices were cultured and exposed to media conditioned by oligodendrocyte precursor cells (OPCs) or differentiated oligodendrocytes. In line with previous studies, exposure of OPC and oligodendrocyte-conditioned media (OCM) increased survival, a phosphatidylinositol 3′-kinase (PI3kinase)/Akt-dependent phenomenon. In addition, exposure of neurons to OCM but not OPC conditioned media resulted in increased axonal length per neuron, as detected by antibodies to phosphorylated neurofilaments. OCM exposure resulted in activation of the MAPkinase/extracellular signal-regulated kinase pathway, inhibition of which significantly reduced oligodendrocyte-mediated enhancement of axonal length but, unlike PI3kinase inhibition, had no effect on neuronal survival. Furthermore, we identify glial cell line-derived neurotrophic factor (GDNF) production by differentiated oligodendrocytes and provide evidence that implicates GDNF in OCM-mediated axonal effects, independent of its effect on neuronal survival. Therefore, we have shown that factors released by OPCs and oligodendrocytes induce the activation of distinct intracellular pathways within neurons, which have different functional effects on the cell.
Estrogens and ICI182,780 (Faslodex) Modulate Mitosis and Cell Death in Immature Cerebellar Neurons via Rapid Activation of p44/p42 Mitogen-Activated Protein Kinase
Authors: Wong Jeremy K. , Le Hoa H. , Zsarnovszky Attila , Belcher Scott M.
Published: April 2018

Estrogen influences the development and function of the nervous system through estrogen receptor-dependent changes in gene expression and by rapidly influencing diverse intracellular signaling pathways. We have investigated the influence of estradiol on developing neonatal rat cerebellar neurons in primary culture and found that low concentrations of 17β-estradiol (17β-E2), 17α-E2, 17β-E2-BSA, and ICI182,780 stimulated phosphorylation of the extracellular signal-regulated kinases 1/2 (ERK1/2) mitogen-activated protein kinases (MAPK). Neither testosterone nor progesterone increased ERK1/2 phosphorylation. The effects of the estrogens were specific to the ERK1/2 MAPK pathway and were blocked by U0126, an inhibitor of the ERK1/2 MAPK kinase (MEK1/2). Compared with control cultures, significant MAPK-dependent decreases in viable granule cell numbers were observed in dissociated explant cultures of developing cerebellar neurons 24–96 hr after pulse treatment with 10 pm17β-E2or 10 nmICI182,780. In contrast, continuous exposure to 10 pm17β-E2significantly increased granule cell numbers. Analysis of bromodeoxyuridine incorporation revealed that a 15 min pulsed treatment with 10 pm17β-E2increased mitogenesis, whereas continuous exposure to the same concentration of 17β-E2was anti-mitotic. Estradiol did not increase caspase activity; however, significant increases in cellular permeability and lysis were observed. Cell lysis and death were independent of the pan-caspase inhibitor zVAD-fmk but were blocked fully by the irreversible calpain inhibitor PD150606. These results indicate that rapid activation of the ERK1/2 MAPK pathway by low concentrations of 17β-E2induces oncotic/necrotic, but not apoptotic, programmed cell death in a subpopulation of developing granule cells and increased mitogenesis of the granule cell neuroblasts refractory to estrogen-induced neurotoxicity.
Calpain Is a Major Cell Death Effector in Selective Striatal Degeneration InducedIn Vivoby 3-Nitropropionate: Implications for Huntington's Disease
Authors: Bizat Nicolas , Hermel Jean-Michel , Boyer Frédéric , Jacquard Carine , Créminon Christophe , Ouary Stéphane , Escartin Carole , Hantraye Philippe , Krajewski Stan , Brouillet Emmanuel
Published: April 2018

Striatal cell death in Huntington's Disease (HD) may involve mitochondrial defects, NMDA-mediated excitotoxicity, and activation of death effector proteases such as caspases and calpain. However, the precise contribution of mitochondrial defects in the activation of these proteases in HD is unknown. Here, we addressed this question by studying the mechanism of striatal cell death in rat models of HD using the mitochondrial complex II inhibitor 3-nitropropionic acid (3-NP). The neurotoxin was either given by intraperitoneal injections (acute model) or over 5 d by constant systemic infusion using osmotic pumps (chronic model) to produce either transient or sustained mitochondrial deficits. Caspase-9 activation preceded neurodegeneration in both cases. However, caspase-8 and caspase-3 were activated in the acute model, but not in the chronic model, showing that 3-NP does not require activation of these caspases to produce striatal degeneration. In contrast, activation of calpain was specifically detected in the striatum in both models and this was associated with a calpain-dependent cleavage of huntingtin. Finally, in the chronic model, which mimics a steady blockade of complex II activity reminiscent of HD, selective calpain inhibition prevented the abnormal calpain-dependent processing of huntingtin, reduced the size of the striatal lesions, and almost completely abolished the 3-NP-induced DNA fragmentation in striatal cells. The present results demonstrate that calpain is a predominant effector of striatal cell death associated with mitochondrial defectsin vivo. This suggests that calpain may play an important role in HD pathogenesis and could be a potential therapeutic target to slow disease progression.