bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitochondrial Manganese Superoxide Dismutase Prevents Neural Apoptosis and Reduces Ischemic Brain Injury: Suppression of Peroxynitrite Production, Lipid Peroxidation, and Mitochondrial Dysfunction
Authors: Keller Jeffrey N. , Kindy Mark S. , Holtsberg Fredrick W. , St. Clair Daret K. , Yen Hsiu-Chuan , Germeyer Arriane , Steiner Sheldon M. , Bruce-Keller Annadora J. , Hutchins James B. , Mattson Mark P.
Published: April 2018

Oxidative stress is implicated in neuronal apoptosis that occurs in physiological settings and in neurodegenerative disorders. Superoxide anion radical, produced during mitochondrial respiration, is involved in the generation of several potentially damaging reactive oxygen species including peroxynitrite. To examine directly the role of superoxide and peroxynitrite in neuronal apoptosis, we generated neural cell lines and transgenic mice that overexpress human mitochondrial manganese superoxide dismutase (MnSOD). In cultured pheochromocytoma PC6 cells, overexpression of mitochondria-localized MnSOD prevented apoptosis induced by Fe2+, amyloid β-peptide (Aβ), and nitric oxide-generating agents. Accumulations of peroxynitrite, nitrated proteins, and the membrane lipid peroxidation product 4-hydroxynonenal (HNE) after exposure to the apoptotic insults were markedly attenuated in cells expressing MnSOD. Glutathione peroxidase activity levels were increased in cells overexpressing MnSOD, suggesting a compensatory response to increased H2O2levels. The peroxynitrite scavenger uric acid and the antioxidants propyl gallate and glutathione prevented apoptosis induced by each apoptotic insult, suggesting central roles for peroxynitrite and membrane lipid peroxidation in oxidative stress-induced apoptosis. Apoptotic insults decreased mitochondrial transmembrane potential and energy charge in control cells but not in cells overexpressing MnSOD, and cyclosporin A and caspase inhibitors protected cells against apoptosis, demonstrating roles for mitochondrial alterations and caspase activation in the apoptotic process. Membrane lipid peroxidation, protein nitration, and neuronal death after focal cerebral ischemia were significantly reduced in transgenic mice overexpressing human MnSOD. The data suggest that mitochondrial superoxide accumulation and consequent peroxynitrite production and mitochondrial dysfunction play pivotal roles in neuronal apoptosis induced by diverse insults in cell culture andin vivo.
Temporal Patterns of Gonadotropin-Releasing Hormone (GnRH), c-fos, and Galanin Gene Expression in GnRH Neurons Relative to the Luteinizing Hormone Surge in the Rat
Authors: Finn Patricia D. , Steiner Robert A. , Clifton Donald K.
Published: April 2018

Gonadotropin-releasing hormone (GnRH) neurons increase their expression of Fos and galanin coincident with the luteinizing hormone (LH) surge in the female rat. To define the temporal relationships between the expression of these genes and the GnRH gene itself and to gain insight about the possible functional interactions of these processes, we compared levels of c-fos, galanin, and GnRH mRNA in GnRH neurons and plasma levels of LH in the rat, beginning 6 hr before and continuing for 24 hr after a sex steroid-induced LH surge. LH levels were increased significantly by 1600 hr. They increased twofold further by 1800 hr and then returned to baseline by 2400 hr. Usingin situhybridization, we determined that levels of c-fosmRNA in GnRH neurons were elevated significantly at 1600 hr only, whereas levels of galanin mRNA in GnRH neurons first increased twofold by 1800 hr, increased an additional twofold by 2400 hr, and remained elevated at all time points sampled thereafter. There were no significant changes in cellular levels of GnRH mRNA over the time points sampled. These results are consistent with the hypothesis that the induction of c-fosgene expression in GnRH neurons leads to an increase in galanin gene expression, and that the sustained increase in galanin mRNA levels in GnRH neurons reflects either the need to replenish galanin stores that are depleted at the time of the LH surge or the involvement of galanin with physiological events that occur on the day of estrus.
DrosophilaPhotoreceptors Contain an Autonomous Circadian Oscillator That Can Function withoutperiodmRNA Cycling
Authors: Cheng Yuzhong , Hardin Paul E.
Published: April 2018

Circadian oscillations inperiod(per) mRNA andperprotein (PER) constitute, in part, a feedback loop that is required for circadian pacemaker function inDrosophila melanogaster. Oscillations in PER are required for oscillations inpermRNA, but the converse has not been rigorously tested because of a lack of measurable quantities ofpermRNA and protein in the same cells. This circadian feedback loop operates synchronously in many neuronal and non-neuronal tissues, including a set of lateral brain neurons (LNs) that mediate rhythms in locomotor activity, but whether a hierarchy among these tissues maintains this synchrony is not known. To determine whetherpermRNA cycling is necessary for PER cycling and whether cyclicpergene expression is tissue autonomous, we have generatedper01flies carrying a transgene that constitutively expressespermRNA specifically in photoreceptors, a cell type that supports feedback loop function. These transformants were tested for different aspects of feedback loop function includingpermRNA cycling, PER cycling, and PER nuclear localization. Under both light/dark (LD) cycling and constant dark (DD) conditions, PER abundance cycles in the absence of circadian cycling ofpermRNA. These results show thatpermRNA cycling is not required for PER cycling and indicate thatDrosophilaphotoreceptors R1–R6 contain a tissue autonomous circadian oscillator.
Memory for Spatial Location: Role of the Hippocampus in Mediating Spatial Pattern Separation
Authors: Gilbert Paul E. , Kesner Raymond P. , DeCoteau William E.
Published: April 2018

A paradigm based on measuring short-term memory for spatial location information as a function of spatial similarity between distal cues was developed to examine the role of pattern separation in the modulation of short-term memory for spatial information. A delayed-match-to-sample for spatial location task using a dryland version of the Morris water maze was used to assess spatial pattern separation in male Long–Evans rats. In the sample phase, animals were trained to displace an object that covered a baited food well in one of 15 spatial locations along a row of food wells perpendicular to a start box. In the ensuing choice phase, the animal was allowed to choose between two objects identical to the sample phase object. One covered the same baited food well as did the object in the study phase (correct choice), and another foil object (incorrect choice) covered a different unbaited food well along the row of wells. Five spatial separations were randomly used to separate the correct object from the foil object. After reaching a criterion before the operation, animals were given either hippocampal or cortical control lesions. In trials after the operation, control animals matched their performance before the operation across all spatial separations. In contrast, hippocampal-lesioned animals displayed impairments across all spatial separations with the exception of the longest (105 cm) spatial separation. The results suggest that the hippocampus may serve to separate incoming spatial information by temporarily storing one place separate from another. It is proposed that hippocampal lesions decrease efficiency in pattern separation, resulting in impairments in trials with increased spatial similarity among working-memory representations.
Free Radical Scavenging and Inhibition of Nitric Oxide Synthase Potentiates the Neurotrophic Effects of Brain-Derived Neurotrophic Factor on Axotomized Retinal Ganglion CellsIn Vivo
Authors: Klöcker Nikolaj , Cellerino Alessandro , Bähr Mathias
Published: April 2018

Brain-derived neurotrophic factor (BDNF) partially promotes the survival of axotomized retinal ganglion cells (RGCs). In analogy within vitroexperiments (Koh et al., 1995; Samdami et al., 1996), we tested whether neuroprotection by BDNF is limited by adverse effects as a consequence of excessive free radical formation. First, we investigated whether BDNF and the free radical scavengerN-tert-butyl-(2-sulfophenyl)-nitrone (S-PBN) cooperate in protecting RGCs from axotomy-induced death. Although systemic S-PBN treatment alone did not influence RGC survival after axotomy, it potentiated the neuroprotective effects of BDNF significantly. Single BDNF treatment rescued 27% of the RGCs, which otherwise would have died 14 d after optic nerve transection, whereas a combined treatment of BDNF and S-PBN improved this rescue rate up to 68%. We then investigated whether the adverse effects of BDNF could be ascribed to activation of nitric oxide synthase (NOS). We found colocalization of NOS and the BDNF receptor TrkB in the retina. NADPH-diaphorase reactivity, a reliable marker for NOS in the rat retina, increased after chronic BDNF treatmentin vivo. Systemic application of the NOS-inhibitorN-ω-nitro-l-arginine-methylester (l-NAME) potentiated the neuroprotective action of BDNF (55% rescue rate). We conclude that activation of NOS is a pathological consequence of BDNF application, which reduces its neuroprotective potential. The observation that this adverse effect can be antagonized by systemic application of free radical scavengers could be of relevance for clinical applications of neurotrophins in human neurodegenerative diseases.
Cross-Modal Transfer of Information between the Tactile and the Visual Representations in the Human Brain: A Positron Emission Tomographic Study
Authors: Hadjikhani Nouchine , Roland Per E.
Published: April 2018

Positron emission tomography in three-dimensional acquisition mode was used to identify the neural populations involved in tactile–visual cross-modal transfer of shape. Eight young male volunteers went through three runs of three different matching conditions: tactile–tactile (TT), tactile–visual (TV), and visual–visual (VV), and a motor control condition. Fifteen spherical ellipsoids were used as stimuli.

By subtracting the different matching conditions and calculating the intersections of statistically significant activations, we could identify cortical functional fields involved in the formation of visual and tactile representation of the objects alone and those involved in cross-modal transfer of the shapes of the objects.

Fields engaged in representation of visual shape, revealed in VV–control, TV–control and TV–TT, were found bilaterally in the lingual, fusiform, and middle occipital gyri and the cuneus. Fields engaged in the formation of the tactile representation of shape, appearing in TT–control, TV–control and TV–VV, were found in the left postcentral gyrus, left superior parietal lobule, and right cerebellum.

Finally, fields active in both TV–VV and TV–TT were considered as those involved in cross-modal transfer of information. One field was found, situated in the right insula–claustrum. This region has been shown to be activated in other studies involving cross-modal transfer of information. The claustrum may play an important role in cross-modal matching, because it receives and gives rise to multimodal cortical projections. We propose here that modality-specific areas can communicate, exchange information, and interact via the claustrum.
Modulation of Plasticity in Human Motor Cortex after Forearm Ischemic Nerve Block
Authors: Ziemann Ulf , Corwell Brian , Cohen Leonardo G.
Published: April 2018

Deafferentation leads to cortical reorganization that may be functionally beneficial or maladaptive. Therefore, we were interested in learning whether it is possible to purposely modulate deafferentation-induced reorganization. Transient forearm deafferentation was induced by ischemic nerve block (INB) in healthy volunteers. The following five interventions were tested: INB alone; INB plus low-frequency (0.1 Hz) repetitive transcranial magnetic stimulation of the motor cortex ipsilateral to INB (INB+rTMSi); rTMSialone; INB plus rTMS of the motor cortex contralateral to INB (INB+rTMSc); and rTMScalone. Plastic changes in the motor cortex contralateral to deafferentation were probed with TMS, measuring motor threshold (MT), motor evoked-potential (MEP) size, and intracortical inhibition (ICI) and facilitation (ICF) to the biceps brachii muscle proximal to the level of deafferentation. INB alone induced a moderate increase in MEP size, which was significantly enhanced by INB+rTMScbut blocked by INB+rTMSi. INB alone had no effect on ICI or ICF, whereas INB+rTMScreduced ICI and increased ICF, and conversely, INB+rTMSideepened ICI and suppressed ICF. rTMSiand rTMScalone were ineffective in changing any of these parameters. These findings indicate that the deafferented motor cortex becomes modifiable by inputs that are normally subthreshold for inducing changes in excitability. The deafferentation-induced plastic changes can be up-regulated by direct stimulation of the “plastic” cortex and likely via inhibitory projections down-regulated by stimulation of the opposite cortex. This modulation of cortical plasticity by noninvasive means might be used to facilitate plasticity when it is primarily beneficial or to suppress it when it is predominately maladaptive.
Differential Effects of Neurotrophic Factors on Motoneuron Retrograde Labeling in a Murine Model of Motoneuron Disease
Authors: Sagot Yves , Rossé Thierry , Vejsada Richard , Perrelet Daniel , Kato Ann C.
Published: April 2018

It has been shown that abnormalities in axonal transport occur in several mouse models with motoneuron degeneration and also in the human disease amyotrophic lateral sclerosis. In this report, we have examined the potential of neurotrophic factors to act on axonal transport properties in a mouse mutant, progressive motor neuronopathy (pmn). This mouse mutant has been characterized as a “dying-back” motoneuronopathy, with a loss of motoneuron cell bodies and motor fibers. Retrograde transport to the spinal cord motoneurons was determined using fluorescent tracers either injected into the gastrocnemius muscle or applied directly onto the cut sciatic nerve. Because the rate of retrograde labeling was significantly reduced in the pmn, we examined the potential of neurotrophic factors to compensate for the impairment. Ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) but not glial-derived neurotrophic factor (GDNF) or nerve growth factor (NGF) were capable of significantly improving the rate of labeling. The differential effects of these factors agree with previous studies showing that molecules that promote cell survival do not necessarily compensate for axonal deficiency. Because impairment of axonal properties appears as an early event in motoneuron pathology, our results may have important clinical implications in the treatment of motoneuron diseases.
Heterogeneity in the Molecular Composition of Excitatory Postsynaptic Sites during Development of Hippocampal Neurons in Culture
Authors: Rao Anuradha , Kim Eunjoon , Sheng Morgan , Craig Ann Marie
Published: April 2018

To determine their roles in the assembly of glutamatergic postsynaptic sites, we studied the distributions of NMDA- and AMPA-type glutamate receptors; the NMDA receptor-interacting proteins α-actinin-2, PSD-95, and chapsyn; and the PSD-95-associated protein GKAP during the development of hippocampal neurons in culture. NMDA receptors first formed nonsynaptic proximal dendrite shaft clusters within 2–5 d. AMPA receptors were diffuse at this stage and began to cluster on spines at 9–10 d. NMDA receptor clusters remained partially nonsynaptic and mainly distinct from AMPA receptor clusters until after 3 weeks in culture, when the two began to colocalize at spiny synaptic sites. Thus, the localization of NMDA and AMPA receptors must be regulated by different mechanisms. α-Actinin-2 colocalized with the NMDA receptor only at spiny synaptic clusters, but not at shaft nonsynaptic or synaptic clusters, suggesting a modulatory role in the anchoring of NMDA receptor at spines. PSD-95, chapsyn, and GKAP were present at some, but not all, nonsynaptic NMDA receptor clusters during the first 2 weeks, indicating that none is essential for NMDA receptor cluster formation. When NMDA receptor clusters became synaptic, PSD-95 and GKAP were always present, consistent with an essential function in synaptic localization of NMDA receptors. Furthermore, PSD-95 and GKAP clustered opposite presynaptic terminals several days before either NMDA or AMPA receptors clustered at these presumptive postsynaptic sites. These results suggest that synapse development proceeds by formation of a postsynaptic scaffold containing PSD-95 and GKAP in concert with presynaptic vesicle clustering, followed by regulated attachment of glutamate receptor subtypes to this scaffold.
Laminin and α-Dystroglycan Mediate Acetylcholine Receptor Aggregation via a MuSK-Independent Pathway
Authors: Montanaro Federica , Gee Stephen H. , Jacobson Christian , Lindenbaum Michael H. , Froehner Stanley C. , Carbonetto Salvatore
Published: April 2018

Specific isoforms of laminin (LN) are concentrated at neuromuscular junctions (NMJs) where they may participate in synaptic organization or function. In myotubes from C2 cells, LN is concentrated within the majority of spontaneous acetylcholine receptor (AChR) aggregates. Neural agrin substantially increases this colocalization, suggesting that agrin can recruit LN into AChR aggregates. Addition of LN to C2 myotubes induces a more than twofold increase in the number of AChR aggregates. These aggregates have a larger size and are more dense than are those induced by agrin, suggesting that LN is involved in the growth and/or stabilization of AChR aggregates. Consistent with this hypothesis, an antiserum to LN reduces the size of individual AChR aggregates but increases their number. In C2 myotubes, extracellular matrix receptors containing the integrin β1 subunit are poorly colocalized with AChR aggregates, suggesting that integrins may not be involved in LN-induced aggregation. In contrast, almost all AChR aggregates are associated with dystroglycan immunoreactivity, and monoclonal antibody (mAb) IIH6 against α-dystroglycan (α-DG), a LN and agrin receptor, causes a concentration-dependent inhibition of LN-induced aggregation. Moreover, S27 cells, which lack a functional α-DG, and two C2-derived cell lines expressing antisense DG mRNA fail to aggregate AChRs in response to LN. Finally, LN-induced AChR aggregation does not involve the phosphorylation of the muscle-specific tyrosine kinase receptor (MuSK) or the AChR β subunit. We hypothesize that the interaction of LN with α-DG contributes to the growth and/or stabilization of AChR microaggregates into macroaggregates at the developing NMJ via a MuSK-independent mechanism.
Interaction of Huntingtin-Associated Protein with Dynactin P150Glued
Authors: Li Shi-Hua , Gutekunst Claire-Anne , Hersch Steven M. , Li Xiao-Jiang
Published: April 2018

Huntingtin is the protein product of the gene for Huntington’s disease (HD) and carries a polyglutamine repeat that is expanded in HD (>36 units). Huntingtin-associated protein (HAP1) is a neuronal protein and binds to huntingtin in association with the polyglutamine repeat. Like huntingtin, HAP1 has been found to be a cytoplasmic protein associated with membranous organelles, suggesting the existence of a protein complex including HAP1, huntingtin, and other proteins. Using the yeast two-hybrid system, we found that HAP1 also binds to dynactin P150Glued(P150), an accessory protein for cytoplasmic dynein that participates in microtubule-dependent retrograde transport of membranous organelles. Anin vitrobinding assay showed that both huntingtin and P150 selectively bound to a glutathione transferase (GST)–HAP1 fusion protein. An immunoprecipitation assay demonstrated that P150 and huntingtin coprecipitated with HAP1 from rat brain cytosol. Western blot analysis revealed that HAP1 was enriched in rat brain microtubules and comigrated with P150 and huntingtin in sucrose gradients. Immunofluorescence showed that transfected HAP1 colocalized with P150 and huntingtin in human embryonic kidney (HEK) 293 cells. We propose that HAP1, P150, and huntingtin are present in a protein complex that may participate in dynein–dynactin-associated intracellular transport.
Neuronal Expression of Zinc Finger Transcription Factor REST/NRSF/XBR Gene
Authors: Palm Kaia , Belluardo Natale , Metsis Madis , Timmusk T õnis
Published: April 2018

The identification of a commoncis-acting silencer element, a neuron-restrictive silencer element (NRSE), in multiple neuron-specific genes, together with the finding that zinc finger transcription factor REST/NRSF/XBR could confer NRSE-mediated silencing in non-neuronal cells, suggested that REST/NRSF/XBR is a master negative regulator of neurogenesis. Here we show that, although REST/NRSF/XBR expression decreases during neuronal development, it proceeds in the adult nervous system.In situhybridization analysis revealed neuronal expression of rat REST/NRSF/XBR mRNA in adult brain, with the highest levels in the neurons of hippocampus, pons/medulla, and midbrain. The glutamate analog kainic acid increased REST/NRSF/XBR mRNA levels in various hippocampal and cortical neuronsin vivo, suggesting that REST/NRSF/XBR has a role in neuronal activity-implied processes. Several alternatively spliced REST/NRSF/XBR mRNAs encoding proteins with nine, five, or four zinc finger motifs are transcribed from REST/NRSF/XBR gene. Two of these transcripts are generated by neuron-specific splicing of a 28-bp-long exon. Rat REST/NRSF/XBR protein isoforms differ in their DNA binding specificities; however, all mediate repression in transient expression assays. Our data suggest that REST/NRSF/XBR is a negative regulator rather than a transcriptional silencer of neuronal gene expression and counteracts with positive regulators to modulate target gene expression quantitatively in different cell types, including neurons.
p75 Neurotrophin Receptor Expression on Adult Human Oligodendrocytes: Signaling without Cell Death in Response to NGF
Authors: Ladiwala Uma , Lachance Christian , Simoneau Steve J. J. , Bhakar Asha , Barker Philip A. , Antel Jack P.
Published: April 2018

Oligodendrocytes (OLs) are the primary targets in the autoimmune disease multiple sclerosis (MS). Cell receptors belonging to the tumor necrosis factor receptor (TNF-R) superfamily, such as TNF receptors and fas, are implicated in signaling the injury response of OLs. The p75 neurotrophin receptor (p75NTR), another member of the TNF-R superfamily, has been reported to mediate nerve growth factor (NGF)-induced apoptosis in some neural systems. To address the potential relationship between p75NTRsignaling and OL injury, we assayed adult human OLs cultured under several different conditions for p75NTRand tyrosine kinase receptor trkA expression, for NGF-mediated apoptosis, and for NGF-mediated jun kinase (JNK) or nuclear factor (NF) κB activation. In the current study, we have found expression of p75NTRon cultured adult CNS-derived human OLs but not on other glial cells. TrkA was not detected on these OLs in any of the culture conditions tested. Treatment of the OLs with varying concentrations of NGF over a range of time periods resulted in no significant increase in numbers of terminal transferase (TdT)-mediated d-uridine triphosphate (UTP)-biotin nick-end labeling positive OLs, whereas significant cell death was observed using TNF-α. Furthermore, unlike TNF-α treatment, NGF treatment did not significantly activate JNK, although both TNF-α and NGF induced nuclear translocation of NF-κB. These findings contrast with the recent report of NGF-mediated apoptosis in the OLs of neonatal rats maturedin vitro, which express p75NTRbut not trkA (Casaccia-Bonnefil et al., 1996), and suggest that, at least in humans, p75NTRsignaling may mediate responses other than apoptosis of OLs.
Synaptic Structure and Transmitter Release in Crustacean Phasic and Tonic Motor Neurons
Authors: Msghina M. , Govind C. K. , Atwood H. L.
Published: April 2018

The paired phasic and tonic motor neurons supplying the extensor muscle in the crayfish leg were investigated to establish whether differences in synaptic structure could account for large differences in transmitter release at the neuromuscular junctions. Nerve terminals with transmitter release that had been assessed from recordings made with a focal “macro-patch” electrode were subsequently labeled, processed for electron microscopy, and reconstructed from serial sections.

At a frequency of 1 Hz, quantal contents of phasic terminals were 90–1300 times greater than those of tonic terminals when both were recorded at the same location. At higher frequencies, facilitation was pronounced at tonic, but not phasic, terminals.

Reconstructions of recording sites showed that both phasic and tonic terminals possessed many small synapses, usually with one or more structurally defined active zones. Mean synaptic contact area was larger for tonic terminals, and the number of individual synapses per length of nerve terminal was also larger. Active zones were not different in size for the two terminals.

At low frequencies, quantal emission per synapse is much greater for phasic terminals. The higher quantal content of phasic terminals and their synapses cannot reasonably be accounted for by more or larger synapses or active zones at the recording sites. Because structural features alone are not likely to produce the very large differences in quantal content of phasic and tonic terminals observed at low stimulation frequencies, it is likely that other properties of the nerve terminal are largely responsible for these differences.
Outward Currents inDrosophilaLarval Neurons:dunceLacks a Maintained Outward Current Component Downregulated by cAMP
Authors: Delgado Ricardo , Davis Ronald , Bono Maria Rosa , Latorre Ramon , Labarca Pedro
Published: April 2018

Outward current modulation by cAMP was investigated in wild type (wt) anddunce(dnc)Drosophilalarval neurons.dncis deficient in a cAMP phosphodiesterase and has altered memory. Outward current modulation by cAMP was investigated by acute or chronic exposure to cAMP analogs. The analysis included a scrutiny of outward current modulation by cAMP in neurons from the mushroom bodies (mrb). InDrosophila, the mrb are the centers of olfactory acquisition and retention. Based on outward current patterns, neurons were classified into four types. Downmodulation of outward currents induced by acute application of cAMP analogs was reversible and found only in type I and type IV neurons. In the general wt neuron population, approximately half of neurons exhibited cAMP-modulated, 4-aminopyridine (4-AP)-sensitive currents. On the other hand, a significantly larger fraction of mrb neurons in wt (70%) was endowed with cAMP-modulated, 4-AP-sensitive currents. Only 30% of thedncneurons displayed outward currents modulated by cAMP. The deficit of cAMP-modulated outward currents was most severe in neurons derived from the mrb ofdncindividuals. Only 4% of the mrb neurons ofdncwere cAMP-modulated. Thedncdefect can be induced by chronic exposure of wt neurons to cAMP analogs. These results document for the first time a well defined electrophysiological neuron phenotype in correlation with thedncdefect. Moreover, this study demonstrates that indncmutants such a deficiency affects most severely neurons in brain centers of acquisition and retention.
Modulation of Hippocampal Acetylcholine Release and Spontaneous Alternation Scores by Intrahippocampal Glucose Injections
Authors: Ragozzino Michael E. , Pal Shanthi N. , Unick Katharine , Stefani Mark R. , Gold Paul E.
Published: April 2018

Recent evidence indicates that systemic glucose treatment enhances memory while producing a corresponding increase in hippocampal acetylcholine (ACh) output. The present experiments examined whether unilateral intrahippocampal infusions of glucose would enhance spontaneous alternation performance and whether there would be a corresponding increase in ACh output in the ipsilateral and contralateral hippocampus. Extracellular ACh was assessed in samples collected at 12 min intervals usingin vivomicrodialysis with HPLC with electrochemical detection. Twelve minutes after a unilateral infusion of artificial cerebrospinal fluid (CSF) or glucose (6.6 mm), rats were tested in a cross maze for spontaneous alternation behavior with concurrent microdialysis collection. In two experiments, glucose infusions significantly increased alternation scores (67.5 and 59%) compared with CSF controls (42.4 and 39.5%, respectively). In both experiments, behavioral testing resulted in increased ACh output in the hippocampus. Glucose administration at the time of alternation tests enhanced ACh output beyond that of behavioral testing alone both ipsilateral (+93.8%) and contralateral (+85%) to the infusion site, as compared with ACh output (+36.1% and +55.5%) of CSF controls. Glucose infusions did not affect hippocampal ACh output in rats kept in a holding chamber. These results suggest that glucose may enhance alternation scores by modulating ACh release. The findings also indicate that unilateral glucose infusions increase hippocampal ACh output both ipsilateral and contralateral to the site of injection. Furthermore, glucose increased ACh output only during maze testing, suggesting that specific behavioral demands, perhaps requiring activation of cholinergic neurons, determine the efficacy of glucose in modulating ACh release.
μ-Opioid Peptides Inhibit Thalamic Neurons
Authors: Brunton Jennifer , Charpak Serge
Published: April 2018

Opioidergic inhibition of neurons in the centrolateral nucleus of the thalamus was investigated using anin vitrothalamic slice preparation from young rats. The μ-opioid receptor agonistd-Ala2,N-Me-Phe4,glycinol5-enkephalin (DAMGO) evoked a hyperpolarization and decrease in input resistance that was reversible, concentration-dependent, and persisted in the presence of tetrodotoxin. Application of the specific μ-receptor antagonist Cys2,Tyr3,Orn5,Pen7-amide blocked this response. The respective δ- and κ-opioid receptor agonists, (d-Pen2,d-Pen5)-enkephalin and (±)-trans-U-50488 methanesulfonate had no effect. Voltage-clamp experiments showed that DAMGO activated an inwardly rectifying potassium conductance (GKIR) characterized by rectification at hyperpolarized potentials that increased in elevated extracellular potassium concentrations, a complete block by Ba2+(1 mm), and a voltage-dependent block by Cs+. The extent of μ-opioid inhibition in other thalamic nuclei was then investigated. Widespread inhibition similar to that seen in the centrolateral nucleus was observed in a number of sensory, motor, intralaminar, and midline nuclei. Our results suggest that the net action of opioids would depend on their source: exogenous (systemically administered) opiates inhibiting the entire thalamus and favoring the shift of cell firing from tonic to bursting mode; and endogenously released opioids acting on specific thalamic nuclei, their release depending on the origin of the presynaptic input.
Cytoskeletal Actin Gates a Cl−Channel in Neocortical Astrocytes
Authors: Lascola Christopher D. , Nelson Deborah J. , Kraig Richard P.
Published: April 2018

Increases in astroglial Cl−conductance accompany changes in cell morphology and disassembly of cytoskeletal actin, but Cl−channels underlying these conductance increases have not been described. We characterize an outwardly rectifying Cl−channel in rodent neocortical cultured astrocytes and describe how cell shape and cytoskeletal actin modulate channel gating. In inside-out patch-clamp recordings from cultured astrocytes, outwardly rectifying Cl−channels either were spontaneously active or inducible in quiescent patches by depolarizing voltage steps. Average single-channel conductance was 36 pS between −60 and −80 mV and was 75 pS between 60 and 80 mV in symmetrical (150 mmNaCl) solutions. The permeability ratio (PNa/PCl) was 0.14 at lower ionic strength but increased at higher salt concentrations. Both ATP and 4,4-diisothiocyanostilbene-2,2′-disulfonic acid produced a flicker block, whereas Zn2+produced complete inhibition of channel activity.

The frequency of observing both spontaneous and inducible Cl−channel activity was markedly higher in stellate than in flat, polygonally shaped astrocytes. In addition, cytoskeletal actin modulated channel open-state probability (PO) and conductance at negative membrane potentials, controlling the degree of outward rectification. Direct application of phalloidin, which stabilizes actin, preserved lowPOand promoted lower conductance levels at negative potentials. LowerPOalso was induced by direct application of polymerized actin. The actions of phalloidin and actin were reversed by coapplication of gelsolin and cytochalasin D, respectively. These results provide the first report of an outwardly rectifying Cl−channel in neocortical astrocytes and demonstrate how changes in cell shape and cytoskeletal actin may control Cl−conductance in these cells.
Ca2+Signals Mediated by P2X-Type Purinoceptors in Cultured Cerebellar Purkinje Cells
Authors: Mateo Jesús , Garcı́a-Lecea Marta , Miras-Portugal Ma Teresa , Castro Enrique
Published: April 2018

We have studied [Ca2+]isignals elicited by extracellular ATP in cultured cells from postnatal day 7–8 rat cerebellum using single-cell fluorescence microscopy and fura-2. Putative Purkinje cells selected under phase contrast by size and characteristic cytoplasm appearance were uniquely identified by selective labeling with anti-calbindin antibodies. Extracellularly applied ATP (50 μm) evoked fast [Ca2+]irises revealed by a rapid and transient increase in fura-2F340/F380ratio in all Purkinje cells tested, whereas granule cells failed to show a response to ATP. The mean [Ca2+]iincrease was ∼400 nm, comparable to that obtained after glutamate stimulation. The response to ATP was completely abolished by removal of extracellular Ca2+with EGTA. Conversely, an increased extracellular Mn2+entry pathway was activated by ATP stimulation. These results indicate that the effect of ATP is mediated by an ionotropic P2X receptor. The action of ATP was mimicked by the analog 2-methylthio-adenosine 5′-triphosphate with similar efficacy but almost half its potency (EC50, 10.6 ± 0.7 vs 21.7 ± 1.9 μm). Other purinergic compounds tested, such as adenosine(5′)-tetraphospho-(5′)adenosine, adenosine(5′)pentaphospho-(5′)adenosine, adenosine 5′-(α,β-methylene) triphosphate, UTP, and adenosine, were completely inactive in eliciting [Ca2+]iresponses. The purinoceptor antagonists suramin and pyridoxalphosphate-6-azophenyl-2′,4′disulphonic acid effectively blocked the responses elicited by ATP. Our results demonstrate for the first time the presence of functional ionotropic P2X purinoceptors in the cerebellar Purkinje cells and indicate that their pharmacology is similar to receptors formed by P2X2subunit oligomers.
Role of the Jun Kinase Pathway in the Regulation of c-Jun Expression and Apoptosis in Sympathetic Neurons
Authors: Eilers Andreas , Whitfield Jonathan , Babij Carol , Rubin Lee L. , Ham Jonathan
Published: April 2018

When deprived of nerve growth factor (NGF), developing sympathetic neurons die by apoptosis. This death is associated with an increase in the level of c-Jun protein and is blocked by expression of a c-Jun dominant negative mutant. Here we have investigated whether NGF withdrawal activates Jun kinases, a family of stress-activated protein kinases that can stimulate the transcriptional activity of c-Jun by phosphorylating serines 63 and 73 in the transactivation domain and which can activate c-jungene expression. We found that sympathetic neurons contained high basal levels of Jun kinase activity that increased further after NGF deprivation. In contrast, p38 kinase, another stress-activated protein kinase that can also stimulate c-jungene expression, was not activated after NGF withdrawal. Consistent with Jun kinase activation, we found using a phospho-c-Jun-specific antibody that c-Jun was phosphorylated on serine 63 after NGF withdrawal. Furthermore, expression of a constitutively active form of MEK kinase 1 (MEKK1), which strongly activates the Jun kinase pathway, increased c-Jun protein levels and c-Jun phosphorylation and induced apoptosis in the presence of NGF. This death could be prevented by co-expression of SEKAL, a dominant negative mutant of SAPK/ERK kinase 1 (SEK1), an activator of Jun kinase that is a target of MEKK1. In contrast, expression of SEKALalone did not prevent c-Jun expression, increases in c-Jun phosphorylation, or cell death after NGF withdrawal. Thus, activation of Jun kinase and increases in c-Jun phosphorylation and c-Jun protein levels occur at the same time after NGF withdrawal, but c-Jun levels and phosphorylation are regulated by an SEK1-independent pathway.
NMDAR1 Glutamate Receptor Subunit Isoforms in Neostriatal, Neocortical, and Hippocampal Nitric Oxide Synthase Neurons
Authors: Weiss Sara W. , Albers David S. , Iadarola Michael J. , Dawson Ted M. , Dawson Valina L. , Standaert David G.
Published: April 2018

Nitric oxide (NO), an unconventional and diffusible neurotransmitter, is synthesized by nitric oxide synthase (NOS). NMDA glutamate receptors are potent regulators of NO synthesis. We have used dual-label immunofluorescence and confocal microscopy to examine forebrain neurons in the rat that contain high levels of neuronal NOS (nNOS) for the presence of the NMDAR1 receptor subunit protein and regions of this protein encoded by three alternative spliced segments of the NMDAR1 mRNA: N1, C1, and C2. In the neostriatum, neocortex, and hippocampus, nNOS-labeled neurons exhibit strong NMDAR1 immunoreactivity (-ir). In all three of these regions, nNOS-positive neurons are characterized by the absence of immunoreactivity for the C1 segment of NMDAR1, whereas C1-ir is abundant in most nNOS-negative neurons. In addition, nNOS-ir neurons exhibit selective staining for the alternative C2′ terminus of NMDAR1 that is produced when the C2 segment is absent. These results demonstrate directly that neurons with abundant nNOS-ir contain NMDAR1 receptor subunit proteins and that the NMDAR1 isoforms present in these cells differ from those of most other neurons in these regions. The distinct NMDA receptor phenotype of these nNOS-positive neurons is likely to contribute to both the physiological regulation of NO release by glutamate as well as to NO-mediated excitotoxic injury.
Involvement of cAMP-Dependent Protein Kinase in the Nucleus Accumbens in Cocaine Self-Administration and Relapse of Cocaine-Seeking Behavior
Authors: Self David W. , Genova Lisa M. , Hope Bruce T. , Barnhart William J. , Spencer Jennifer J. , Nestler Eric J.
Published: April 2018

cAMP-dependent protein kinase (PKA) in the nucleus accumbens (NAc) has been implicated in cocaine addiction because (1) cocaine reinforcement is mediated by dopamine receptors that modulate cAMP formation, and (2) repeated exposure to cocaine upregulates the cAMP system in NAc neurons. This study tested PKA involvement in cocaine self-administration and relapse of cocaine-seeking behavior by infusing cAMP analogs that activate or inhibit PKA into the NAc of rats. Bilateral intra-NAc infusions of the PKA inhibitorRp-cAMPS reduced baseline cocaine self-administration, shifted the dose–response curve for cocaine self-administration to the left, and induced relapse of cocaine-seeking behavior after extinction from cocaine self-administration, consistent with an enhancement of cocaine effects in each paradigm. In contrast, pretreatment with intra-NAc infusions of a PKA activator,Sp-cAMPS or dibutyryl cAMP, increased baseline cocaine self-administration during the second hour of testing and shifted the dose–response curve to the right, consistent with an antagonist-like action. After extinction from cocaine self-administration, similar infusions of Sp-cAMPS induced generalized responding at both drug-paired and inactive levers. As an index of PKA activityin vivo, NAc infusions of Rp-cAMPS reduced basal levels of dopamine-regulated phosphoprotein-32 phosphorylation and blocked amphetamine-induced increases in cAMP response element-binding protein (CREB) phosphorylation. Conversely, NAc infusions ofSp-cAMPS increased phosphorylation of CREB. Together, these results suggest that sustained upregulation of the cAMP system in the NAc after repeated cocaine exposure could underlie tolerance to cocaine reinforcement, whereas acute inhibition of this system may contribute to drug craving and relapse in addicted subjects.
Glutamate and GABA Release Are Enhanced by Different Subtypes of Presynaptic Nicotinic Receptors in the Lateral Geniculate Nucleus
Authors: Guo Jian-Zhong , Tredway Trevor L. , Chiappinelli Vincent A.
Published: April 2018

The functional role of nicotinic acetylcholine receptors (nAChRs) in the ventral lateral geniculate nucleus (LGNv) was examined in chick brain slices. Whole-cell patch-clamp recordings of neurons in the LGNv revealed the presence of bicuculline-resistant spontaneous postsynaptic currents (PSCs), which were subsequently blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an AMPA receptor antagonist. Carbachol and other nicotinic agonists produced marked increases in the frequency of the glutamatergic spontaneous PSCs in the presence of tetrodotoxin, whereas they had little or no effect on current amplitude. The nicotinic receptor antagonist dihydro-β-erythroidine (DHβE) blocked the carbachol-induced enhancement of spontaneous glutamatergic PSCs. α-bungarotoxin (α-BgTx) selectively blocked the nAChR-mediated enhancement of spontaneous glutamatergic PSCs but did not prevent nAChR-mediated enhancement of spontaneous GABAergic PSCs in the LGNv. Methyllycaconitine and strychnine, other blockers of nAChRs containing the α7 subunit, failed to inhibit carbachol’s increase of spontaneous glutamatergic and GABAergic PSCs. These results demonstrate that the LGNv neurons receive both glutamatergic and GABAergic inputs and that the release of these transmitters can be modulated by different presynaptic nAChRs. Thus, the regulation of synaptic efficacy in the brain by presynaptic nAChRs can be complex, involving multiple neurotransmitters acting on the same neuron.
Inhibition by ATP of Hippocampal Synaptic Transmission Requires Localized Extracellular Catabolism by Ecto-Nucleotidases into Adenosine and Channeling to Adenosine A1Receptors
Authors: Cunha Rodrigo A. , Sebastião Ana M. , Ribeiro J. A.
Published: April 2018

ATP analogs substituted in the γ-phosphorus (ATPγS, β,γ-imido-ATP, and β,γ-methylene-ATP) were used to probe the involvement of P2receptors in the modulation of synaptic transmission in the hippocampus, because their extracellular catabolism was virtually not detected in CA1 slices. ATP and γ-substituted analogs were equipotent to inhibit synaptic transmission in CA1 pyramid synapses (IC50of 17–22 μm). The inhibitory effect of ATP and γ-phosphorus-substituted ATP analogs (30 μm) was not modified by the P2receptor antagonist suramin (100 μm), was inhibited by 42–49% by the ecto-5′-nucleotidase inhibitor and α,β-methylene ADP (100 μm), was inhibited by 74–85% by 2 U/ml adenosine deaminase (which converts adenosine into its inactive metabolite-inosine), and was nearly prevented by the adenosine A1receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (10 nm). Stronger support for the involvement of extracellular adenosine formation as a main requirement for the inhibitory effect of ATP and γ-substituted ATP analogs was the observation that an inhibitor of adenosine uptake, dipyridamole (20 μm), potentiated by 92–124% the inhibitory effect of ATP and γ-substituted ATP analogs (10 μm), a potentiation similar to that obtained for 10 μmadenosine (113%). Thus, the present results indicate that inhibition by extracellular ATP of hippocampal synaptic transmission requires localized extracellular catabolism by ecto-nucleotidases and channeling of the generated adenosine to adenosine A1receptors.
Metabotropic Glutamate Receptors Negatively Regulate Melatonin Synthesis in Rat Pinealocytes
Authors: Yamada Hiroshi , Yatsushiro Shouki , Ishio Shougo , Hayashi Mitsuko , Nishi Tsuyoshi , Yamamoto Akitsugu , Futai Masamitsu , Yamaguchi Akihito , Moriyama Yoshinori
Published: April 2018

Rat pinealocytes receive noradrenergic innervation that stimulates melatonin synthesis in a cAMP-mediated manner. In addition to melatonin, we showed previously that pinealocytes secretel-glutamate through an exocytic mechanism. The released glutamate inhibits norepinephrine (NE)-dependent melatonin synthesis. Consistent with this observation, specific agonists of class II metabotropic glutamate receptors (mGluRs), including 1-(1S,3R)-aminocyclopentane-1,3-dicarboxylic acid (tACPD), inhibited NE-dependent melatonin synthesis, whereas agonists for other types of glutamate receptors did not. Furthermore, reverse transcription-PCR, Northern blotting, and immunohistochemistry analyses indicated expression of class II mGluR3 in pinealocytes. Inhibitory guanine nucleotide-binding protein (Gi) was also detected in pinealocytes.l-Glutamate or agonists of class II receptors decreased NE- or forskolin-dependent increase of cAMP and serotonin-N-acetyltransferase activities to similar extents. These effects were blocked by pertussis toxin or dibutyryl cAMP. These results indicate that the inhibitory cAMP cascade is involved in the glutamate-evoked inhibition of melatonin synthesis. We propose that the glutaminergic system negatively regulates NE-dependent melatonin synthesis in rat pinealocytes.
Alterations in Dopamine Release But Not Dopamine Autoreceptor Function in Dopamine D3Receptor Mutant Mice
Authors: Koeltzow Timothy E. , Xu Ming , Cooper Donald C. , Hu Xiu-Ti , Tonegawa Susumu , Wolf Marina E. , White Francis J.
Published: April 2018

Dopamine (DA) autoreceptors expressed along the somatodendritic extent of midbrain DA neurons modulate impulse activity, whereas those expressed at DA nerve terminals regulate both DA synthesis and release. Considerable evidence has indicated that these DA autoreceptors are of the D2subtype of DA receptors. However, many pharmacological studies have suggested an autoreceptor role for the DA D3receptor. This possibility was tested with mice lacking the D3receptor as a result of gene targeting. The basal firing rates of DA neurons within both the substantia nigra and ventral tegmental area were not different in D3receptor mutant and wild-type mice. The putative D3receptor-selective agonistR(+)-trans-3,4,4a,10b-tetrahydro-4-propyl-2H,5H-(1)benzopyrano(4,3-b)−1,4-oxazin-9-ol (PD 128907) was equipotent at inhibiting the activity of both populations of midbrain DA neurons in the two groups of mice. In the γ-butyrolactone (GBL) model of DA autoreceptor function, mutant and wild-type mice were identical with respect to striatal DA synthesis and its suppression by PD 128907.In vivomicrodialysis studies of DA release in ventral striatum revealed higher basal levels of extracellular DA in mutant mice but similar inhibitory effects of PD 128907 in mutant and wild-type mice. These results suggest that the effects of PD 128907 on dopamine cell function reflect stimulation of D2as opposed to D3receptors. Although D3receptors do not seem to be significantly involved in DA autoreceptor function, they may participate in postsynaptically activated short-loop feedback modulation of DA release.