bluuebunny/crossref_metadata_2025 huggingface dataset:
Conditional Ablation of Cerebellar Astrocytes in Postnatal Transgenic Mice
Authors: Delaney Catherine L. , Brenner Michael , Messing Albee
Published: April 2018

Astrocytes have been proposed to have multiple roles in the development and maintenance of the vertebrate CNS. To facilitate documentation of these roles, we designed a transgene to enable their ablation at selectable times. The transgene consists of the coding region for the herpes simplex virus-thymidine kinase (HSV-TK) under the control of the human glial fibrillary acidic protein gene promoter. The HSV-TK is innocuous but converts the antiherpetic agent ganciclovir (GCV) to a toxic product that interferes with DNA replication in proliferating cells. In a developmental study, transgenic mice were treated with GCV during the first postnatal week, with evaluation at P19. Treated mice displayed severe ataxia. Histological examination revealed disrupted astrocyte development, particularly in the cerebellum, with marked secondary effects on other cell types. Cerebellar defects included a loss in the numbers of astrocytes and an overall reduction in cerebellar size and disruption of the normally well defined cellular layers. Radial glia were disordered, Purkinje cells were ectopically distributed and displayed abnormal dendritic trees, and granule cells were markedly depleted. These effects were more severe in animals treated on postnatal day 1 versus treatment at day 5. A major factor causing granule cell death was excitotoxicity attributable to activation of NMDA receptors. These results suggest a critical role for astrocytes in cerebellar development.
A Cerebral Central Pattern Generator inAplysiaand Its Connections with Buccal Feeding Circuitry
Authors: Perrins Ray , Weiss Klaudiusz R.
Published: April 2018

Different feeding-related behaviors inAplysiarequire substantial variations in the coordination of movements of two separate body parts, the lips and buccal mass. The central pattern generators (CPGs) and motoneurons that control buccal mass movements reside largely in the buccal ganglion. It was previously thought that control of the cerebral neuronal circuitry and motoneurons that generate lip movements was coordinated directly by feedback from buccal interneurons. Here, we describe cerebral lip motoneuron C15, which drives rhythmic activity in the isolated cerebral ganglion. Other lip motoneurons are active during this program, so we define it as a cerebral motor program (CMP). The C15 in each cerebral hemiganglion drives the CMP in ipsilateral neurons only, suggesting there are independent CPGs in each hemiganglion. The cerebral and buccal CPGs interact at several points. For example, cerebral-to-buccal interneurons (CBIs), which can drive the buccal CPG, receive excitatory input when the cerebral CPG is active. Likewise, C15, which can drive the cerebral CPG, is excited when the buccal CPG is active. This excitation is simultaneous in both C15s, coupling the activity in the two hemiganglionic cerebral CPGs. Therefore, there are independent cerebral and buccal CPGs, which can produce distinct rhythms, but which interact at several points. Furthermore, the connections between the cerebral and buccal CPGs alter during different forms of motor program. We suggest that such alterations in the interactions between these CPGs might contribute to the generation of the various forms of coordination of lip and buccal mass movements that are necessary during different feeding-related behaviors.
Molecular Determinants of β1Subunit-Induced Gating Modulation in Voltage-Dependent Na+Channels
Authors: Makita Naomasa , Bennett Paul B. , George Jr. Alfred L.
Published: April 2018

Recombinant brain, skeletal muscle, and heart voltage-gated Na+channel α subunits differ in their functional responses to an accessory β1subunit when coexpressed inXenopusoocytes. We exploited the distinct β1subunit responses observed for the human heart (hH1) and human skeletal muscle (hSkM1) isoforms to identify determinants of this response. Chimeric α subunits were constructed by exchanging the S5–S6 interhelical loops of each domain between hH1 and hSkM1 and then examined for effects on inactivation induced by coexpressed β1subunit in oocytes. Substitution of single S5–S6 loops in either domain 1 (D1/S5–S6) or domain 4 (D4/S5–S6) of hSkM1 by the corresponding segments of hH1 produced channels that exhibited an attenuated response to coexpressed β1subunit. Substitutions of both D1/S5–S6 and D4/S5–S6 in hSkM1 by the corresponding loops from hH1 completely abolished the effects of the β1subunit on inactivation. The reciprocal chimera in which both D1/S5–S6 and D4/S5–S6 from hSkM1 were transplanted into hH1 exhibited significant β1responsiveness (accelerated inactivation). The region within D4/S5–S6 that conferred β1responsiveness was determined to reside primarily within an extracellular loop between the putative pore-forming segment SS2 and D4/S6. Gating modulation was also demonstrated using a chimeric β subunit consisting of the extracellular domains of β1and the transmembrane and C-terminal domains of the rat brain β2subunit. These results suggest that the D1/S5–S6 and D4/S5–S6 loops in the α subunit and the extracellular domain of the β1subunit are important determinants of the β1subunit-induced gating modulation in Na+channels.
NMDA Receptor Dependence of Kindling and Mossy Fiber Sprouting: Evidence that the NMDA Receptor Regulates Patterning of Hippocampal Circuits in the Adult Brain
Authors: Sutula T. , Koch J. , Golarai G. , Watanabe Y. , McNamara J. O.
Published: April 2018

The NMDA receptor plays an important role in patterning neural connectivity in the developing brain. In the adult brain, repeated kindling stimulation of limbic pathways increases the NMDA-dependent component of synaptic transmission in granule cells of the dentate gyrus (DG) and also induces sprouting of the mossy fiber axons of granule cells that reorganizes synaptic connections in the DG. Because the NMDA antagonist MK801 impedes the progression of kindling, it was of interest to determine whether MK801 also modified mossy fiber sprouting. Low doses of MK801, which had no antiseizure effect, impaired the progression of kindling and development of mossy fiber sprouting during the initial and also more advanced stages of kindling. These observations demonstrate that the NMDA receptor is a component of a molecular pathway that influences the progression of kindling and mossy fiber sprouting and suggest that NMDA-dependent gene expression may play a role in the development of long-term structural and functional alterations induced by seizures in hippocampal circuitry. The NMDA receptor appears to play a continuing role in modifying the organization and patterns of connectivity in hippocampal circuits of the adult brain.
Apoptosis and Its Relation to the Cell Cycle in the Developing Cerebral Cortex
Authors: Thomaidou Dimitra , Mione Marina C. , Cavanagh John F. R. , Parnavelas John G.
Published: April 2018

Large numbers of dying cells are found in proliferating tissues, suggesting a link between cell death and cell division. We detected and quantified dying cells during pre- and early postnatal development of the rat cerebral cortex usingin situend labeling of DNA fragmentation [terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)] and electron microscopy. The proliferative zones that give rise to the neuronal and glial cell types of the cortex, the ventricular and, to a larger extent, the subventricular zones showed higher incidence of cell death than other regions of the developing cortex during the period of neurogenesis. Gel electrophoresis of DNA isolated from the subventricular zone of newborn animals showed a ladder pattern that is characteristic of apoptosis. The number of apoptotic cells remained high in this zone for at least 2 weeks, during which period cells continued to divide. The correlation between cell division and cell death was studied in the subventricular zone of newborn rats; cumulative labeling with bromodeoxyuridine showed that 71% of TUNEL-labeled cells had taken up this S-phase marker before undergoing cell death. Using bromodeoxyuridine and [3H]-thymidine in succession to identify a cohort of proliferating cells, we found that the clearance time of TUNEL-positive nuclei was 2 hr and 20 min. A comparison between the number of mitotic figures and that of TUNEL-positive nuclei showed that cell death affects one in every 14 cells produced by dividing ventricular zone cells at embryonic day 16 and one in every 1.5 cells produced in the subventricular zone of newborn rats. In addition, we found that most of TUNEL-positive cells were in the G1 phase of their cell cycle. We conclude that apoptosis is prominent in the proliferating neuroepithelium of the developing rat cerebral cortex and that it is related to the progression of the cell cycle.
Characterization of a CNS Cell Line, CAD, in which Morphological Differentiation Is Initiated by Serum Deprivation
Authors: Qi Yanping , Wang James K. T. , McMillian Michael , Chikaraishi Dona M.
Published: April 2018

A CNS catecholaminergic cell line, Cath.a, was established by targeted oncogenesis in transgenic mice. Cath.a cells express neuronal properties but lack neuronal morphology. Here, we describe a variant of Cath.a, called CAD (Cath.a-differentiated), in which reversible morphological differentiation can be initiated by removal of serum or exogenously added protein from the medium. In serum- or protein-free media, CAD cells stop proliferating and extend long processes. Differentiated CAD cells can be maintained without serum or protein for at least 6 weeks. CAD cells are distinct from Cath.a cells; most significant, the original immortalizing oncogene, SV40 T antigen, was spontaneously lost. By immunostaining or immunoblotting, we show that CAD cells express neuron-specific proteins, such as class III β-tubulin, GAP-43, SNAP-25, and synaptotagmin, but not GFAP. Ultrastructurally, processes from differentiated CAD cells have abundant parallel microtubules and intermediate filaments, and bear varicosities that contain both large dense-core vesicles/granules (120–160 nm) and smaller clear vesicles (60–80 nm). Additionally, CAD cells express enzymatically active tyrosine hydroxylase and accumulatel-DOPA. CAD cells exhibit biochemical and morphological characteristics of primary neurons and provide an unique tool for studying neuronal differentiation.
Regional Expression and Cellular Localization of the α1and β Subunit of High Voltage-Activated Calcium Channels in Rat Brain
Authors: Ludwig Andreas , Flockerzi Veit , Hofmann Franz
Published: April 2018

The neuronal high voltage-activated calcium channels are a family of ion channels composed from up to five different α1and four different β subunits. The neuronal distribution and subunit composition of calcium channels were investigated using subunit-specific antibodies and riboprobes. The β subunit-specific antibodies identified the presence of β1ain skeletal muscle; β2in heart; and β2, β3, and β4in brain. The β3protein was widely distributed in rat brain, with prominent labeling of olfactory bulb, cortex, hippocampus, and habenula. The β4protein was also widely expressed, most prominently in the cerebellum. β2protein was expressed at only low levels.In situhybridization with β subunit-specific riboprobes confirmed the differential expression pattern of the individual subunits. Hybridization with riboprobes specific for the α1A,α1B,α1C, and α1Dsubunits showed a broad distribution of α1Aand α1Btranscripts, whereas the expression level of α1Cand α1DmRNA was lower and more spatially restricted. The overall expression pattern and cellular localization suggested that β4may associate predominantly, but probably not exclusively, with the α1Asubunit, and β3with the α1Bsubunit. In certain brain areas such as the habenula, the β3subunit may associate with other α1subunits too. Furthermore, the β2subunit may form complexes with different α1subunits in brain and cardiac muscle. These results demonstrate that a given β subunit may associate with different α1subunits in a cell type-dependent manner, contributing to the diversity of the neuronal calcium channels.
Ligand-Gated Ion Channel Subunit Partnerships: GABAAReceptor α6Subunit Gene Inactivation Inhibits δ Subunit Expression
Authors: Jones A. , Korpi E. R. , McKernan R. M. , Pelz R. , Nusser Z. , Mäkelä R. , Mellor J. R. , Pollard S. , Bahn S. , Stephenson F. A. , Randall A. D. , Sieghart W. , Somogyi P. , Smith A. J. H. , Wisden W.
Published: April 2018

Cerebellar granule cells express six GABAAreceptor subunits abundantly (α1, α6, β2, β3, γ2, and δ) and assemble various pentameric receptor subtypes with unknown subunit compositions; however, the rules guiding receptor subunit assembly are unclear. Here, removal of intact α6protein from cerebellar granule cells allowed perturbations in other subunit levels to be studied. Exon 8 of the mouse α6subunit gene was disrupted by homologous recombination. In α6−/− granule cells, the δ subunit was selectively degraded as seen by immunoprecipitation, immunocytochemistry, and immunoblot analysis with δ subunit-specific antibodies. The δ subunit mRNA was present at wild-type levels in the mutant granule cells, indicating a post-translational loss of the δ subunit. These results provide genetic evidence for a specific association between the α6and δ subunits. Because in α6−/− neurons the remaining α1, β2/3, and γ2subunits cannot rescue the δ subunit, certain potential subunit combinations may not be found in wild-type cells.
Neuropeptide Amidation inDrosophila: Separate Genes Encode the Two Enzymes Catalyzing Amidation
Authors: Kolhekar Aparna S. , Roberts Marie S. , Jiang Ning , Johnson Richard C. , Mains Richard E. , Eipper Betty A. , Taghert Paul H.
Published: April 2018

In vertebrates, the two-step peptide α-amidation reaction is catalyzed sequentially by two enzymatic activities contained within one bifunctional enzyme called PAM (peptidylglycine α-amidating mono-oxygenase).Drosophilahead extracts contained both of these PAM-related enzyme activities: a mono-oxygenase (PHM) and a lyase (PAL). However, no bifunctional PAM protein was detected. We identified cDNAs encoding an active mono-oxygenase that is highly homologous to mammalian PHM. PHM-like immunoreactivity was found within diverse larval tissues, including the CNS, endocrine glands, and gut epithelium. Northern and Western blot analyses demonstrate RNA and protein species corresponding to the cloned PHM, but not to a bifunctional PAM, leading us to predict the existence of separatePHMandPALgenes inDrosophila. TheDrosophila PHMgene displays an organization of exons that is highly similar to the PHM-encoding portion of the ratPAMgene. Genetic analysis was consistent with the prediction of separatePHMandPALgene functions inDrosophila: a P element insertion line containing a transposon within thePHMtranscription unit displayed strikingly lower PHM enzyme levels, whereas PAL levels were increased slightly. The lethal phenotype displayed by thedPHMP element insertion indicates a widespread essential function. Reversion analysis indicated that the lethality associated with the insertion chromosome likely is attributable to the P element insertion. These combined data indicate a fundamental evolutionary divergence in the genes coding for critical neurotransmitter biosynthetic enzymes: inDrosophila, the two enzyme activities of PAM are encoded by separate genes.
A Post-Transcriptional Regulatory Mechanism Restricts Expression of the Paraneoplastic Cerebellar Degeneration Antigen cdr2 to Immune Privileged Tissues
Authors: Corradi John P. , Yang Chingwen , Darnell Jennifer C. , Dalmau Josep , Darnell Robert B.
Published: April 2018

Paraneoplastic cerebellar degeneration (PCD) is believed to be an autoimmune disorder initiated by the ectopic expression of a neuron-specific protein in breast and ovarian tumors. PCD antisera was used previously to identify several cerebellar degeneration-related (cdr) genes encoding putative PCD antigens. We have found that the cdr2 gene, which encodes a cytoplasmic leucine zipper protein of unknown function, is expressed in PCD-associated tumors, whereas other cdr genes are not; thus, cdr2 encodes the PCD tumor antigen. To determine whether the expression pattern of cdr2 is consistent with its proposed role in PCD, we have isolated the mouse homolog and examined both the mRNA and protein distribution in adult tissues. We have found that cdr2 mRNA is expressed in almost all tissues, whereas the protein is expressed only in the brain and testis. Within the brain, both the cdr2 mRNA and immunoreactivity are confined primarily to neurons in the cerebellum and brainstem, the regions most affected in PCD. These results suggest first that the tissue-specific expression of cdr2 is regulated at a post-transcriptional level. Moreover, because the brain and testis are considered to be immune-privileged sites, the expression pattern of cdr2 is compatible with the autoimmune model of PCD pathogenesis.
A Pacemaker Current in Dye-Coupled Hilar Interneurons Contributes to the Generation of Giant GABAergic Potentials in Developing Hippocampus
Authors: Strata Fabrizio , Atzori Marco , Molnar Margherita , Ugolini Gabriele , Tempia Filippo , Cherubini Enrico
Published: April 2018

The establishment of synaptic connections and their refinement during development require neural activity. Increasing evidence suggests that spontaneous bursts of neural activity within an immature network are mediated by γ-aminobutyric acid via a paradoxical excitatory action. Our data show that in the developing hippocampus such synchronous burst activity is generated in the hilar region by transiently coupled cells. These cells have been identified as neuronal elements because they fire action potentials and they are not positive for the glial fibrillary acidic protein staining. Oscillations in hilar cells are “paced” by a hyperpolarization-activated current, with properties ofIh. Coactivated interneurons synchronously release GABA, which via its excitatory action may serve a neurotrophic function during the refinement of hippocampal circuitry.
Glutamate, But Not Dopamine, Stimulates Stress-Activated Protein Kinase and AP-1-Mediated Transcription in Striatal Neurons
Authors: Schwarzschild Michael A. , Cole Rebecca L. , Hyman Steven E.
Published: April 2018

Drugs that stimulate dopamine and glutamate receptors have been shown to induce the expression of AP-1 proteins (such as c-Fos and c-Jun) in the striatum and to induce binding of these proteins to AP-1 sites on DNA, leading to the hypothesis that AP-1-mediated transcription contributes to the long-term effects of these drugs. To examine this hypothesis, we compared the regulation of AP-1-mediated transcription to the inductions of AP-1-binding activity and genes encoding AP-1 proteins in primary cultures of striatal neurons. Although glutamate, dopamine, and forskolin (an activator of adenylate cyclase) all induce c-fosmRNA and AP-1 binding, we found, surprisingly, that only glutamate induces transcription of a transfected AP-1-driven fusion gene. To explore the basis for this discrepancy, we investigated the possibility that the phosphorylation of c-Jun may also be required for AP-1-mediated transcription in striatal neurons. Glutamate, but neither dopamine nor forskolin, raises the levels of phosphorylated c-Jun as well as the activity of a Jun kinase (SAPK/JNK) in striatal cultures. Both the glutamatergic induction of AP-1-mediated transcription and activation of SAPK/JNK appear to be mediated, at least in part, via NMDA receptors. In striatal neurons, the phosphorylation of AP-1 proteins produced by glutamate may be required to convert AP-1 protein expression and binding to transcriptional activation.
Mechanisms and Effects of Intracellular Calcium Buffering on Neuronal Survival in Organotypic Hippocampal Cultures Exposed to Anoxia/Aglycemia or to Excitotoxins
Authors: Abdel-Hamid Khaled M. , Tymianski Michael
Published: April 2018

Neuronal calcium loading attributable to hypoxic/ischemic injury is believed to trigger neurotoxicity. We examined in organotypic hippocampal slice cultures whether artificially and reversibly enhancing the Ca2+buffering capacity of neurons reduces the neurotoxic sequelae of oxygen–glucose deprivation (OGD), whether such manipulation has neurotoxic potential, and whether the mechanism underlying these effects is pre- or postsynaptic. Neurodegeneration caused over 24 hr by 60 min of OGD was triggered largely by NMDA receptor activation and was attenuated temporarily by pretreating the slices with cell-permeant Ca2+buffers such as 1,2 bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid acetoxymethyl ester (BAPTA-AM). This pretreatment produced a transient, reversible increase in intracellular buffer content as demonstrated autoradiographically using slices loaded with14C-BAPTA-AM and by confocal imaging of slices loaded with the BAPTA-AM analog calcium green-acetoxymethyl ester (AM). The time courses of14C-BAPTA retention and of neuronal survival after OGD were identical, indicating that increased buffer content is necessary for the observed protective effect. Protection by Ca2+buffering originated presynaptically because BAPTA-AM was ineffective when endogenous transmitter release was bypassed by directly applying NMDA to the cultures, and because pretreatment with the low Ca2+affinity buffer 2-aminophenol-N,N,O-triacetic acid acetoxymethyl ester, which attenuates excitatory transmitter release, attenuated neurodegeneration. Thus, in cultured hippocampal slices, enhancing neuronal Ca2+buffering unequivocally attenuates or delays the onset of anoxic neurodegeneration, likely by attenuating the synaptic release of endogenous excitatory neurotransmitters (excitotoxicity).
Regulation of Purkinje Cell Alignment by Reelin as Revealed with CR-50 Antibody
Authors: Miyata Takaki , Nakajima Kazunori , Mikoshiba Katsuhiko , Ogawa Masaharu
Published: April 2018

Cerebellar Purkinje cells are generated in the ventricular zone, migrate outward, and finally form a monolayer in the cortex. Inreelermice, however, most Purkinje cells cluster abnormally in subcortical areas. Reelin, the candidatereelergene product recognized by the CR-50 monoclonal antibody, is concentrated in a cortical zone along which Purkinje cells are aligned linearly, implying that it may regulate their alignment. We used anin vitrosystem and a transplantation approach to analyze the function of Reelin.

Explant culture for 7 d of cerebella isolated from wild-type andreelermice at embryonic day 13 (E13) reproduced in a phenotype-dependent manner the two distinct arrangement patterns (linear vs clustered) of Purkinje cells. Extensive CR-50 binding to wild-type explants converted the linear pattern into areeler-like, clustered pattern. On the other hand, whenreelerexplants lacking Reelin were crowned with an artificial layer of Reelin+granule cells, some Reelin molecules were distributed into a superficial zone of thereelerexplants, and Purkinje cells formed a linear pattern along the Reelin-rich overlay. This “rescue” effect was also inhibited by CR-50. Hence, Reelin is involved in the Purkinje cell alignment, and the lack of this activity may explain the malformation inreelercerebella.

We further injected Reelin+granule cells into the fourth ventricle of E12–13 mice. Extensive incorporation of the injected Reelin+cells into the ventricular zone, but not of Reelin−cells, forced Purkinje cells of the host cerebella to form an aberrant layer, suggesting that premigratory Purkinje cells may already be responsive to Reelin or Reelin-related signals.
Conduction in Segmentally Demyelinated Mammalian Central Axons
Authors: Felts Paul A. , Baker Theresa A. , Smith Kenneth J.
Published: April 2018

The prominent symptoms associated with central demyelinating diseases such as multiple sclerosis (MS) are primarily caused by conduction deficits in affected axons. The symptoms may go into remission, but the mechanisms underlying remissions are uncertain. One factor that could be important is the restoration of conduction to affected axons, but it is not known whether demyelinated central axons resemble their peripheral counterparts in being able to conduct in the absence of repair by remyelination. In the present study we have made intra-axonal recordings from central axons affected by a demyelinating lesion, and then the axons have been labeled ionophoretically to permit their subsequent identification. Ultrastructural examination of 23 labeled preparations has established that some segmentally demyelinated central axons can conduct, and that they can do so over continuous lengths of demyelination exceeding several internodes (2500 μm). Such segmentally demyelinated central axons were found to conduct with the anticipated reduction in velocity and a refractory period of transmission (RPT) as much as 34 times the value obtained from the nondemyelinated portion of the same axon; the RPT was typically prolonged to 2–5 times the normal value. We conclude that some segmentally demyelinated central axons can conduct, and we propose that the restoration of conduction to such axons is likely to contribute to the remissions commonly observed in diseases such as MS.
Retinoic Acid Synthesis in the Developing Chick Retina
Authors: Mey Jörg , McCaffery Peter , Dräger Ursula C.
Published: April 2018

The transcriptional activator retinoic acid (RA) has been shown to influence the early patterning of the vertebrate eye. Models for the establishment of the retinofugal projection postulate gradients of cell-surface markers across the retinal surface that are expressed by ganglion cells and mediate the correct connection of fibers within central target fields. Spatial asymmetries of RA and RA-producing enzymes, as have been found in the eyes of mice and zebrafish, could induce the required asymmetry in gene expression. Here we exploited the large size of the retina of the embryonic chick to analyze the spatial and temporal characteristics of the RA system by HPLC in combination with a reporter cell assay. As in other embryonic vertebrates, the chick retina was found to contain different RA-generating enzymes segregated along the dorsoventral axis. The major RA isomer in both dorsal and ventral retina was all-transRA, and no 9-cisRA could be detected. This excludes a difference in production of these two isomers as an explanation for the expression of different RA-generating enzymes. At developmental stages embryonic days (E) 4 and 5, the ventral retina contained higher all-transRA levels than the dorsal retina. After E8, however, the difference disappeared, and in embryos at E9 and older the RA concentration was slightly higher in dorsal than ventral retina.
Rapid Seizure-Induced Reduction of Benzodiazepine and Zn2+Sensitivity of Hippocampal Dentate Granule Cell GABAAReceptors
Authors: Kapur Jaideep , Macdonald Robert L.
Published: April 2018

Fast synaptic inhibition in the forebrain is mediated primarily by GABA acting on GABAAreceptors (GABARs). GABARs are regulated by numerous positive (barbiturates, benzodiazepines, and neurosteroids) and negative (picrotoxin, bicuculline, and Zn2+) allosteric modulators. The sensitivity of GABARs to GABA and to allosteric modulators changes gradually during normal development, during development of chronic epilepsy, and after prolonged exposure to GABAR agonists. Here we report the development of rapid functional plasticity of GABARs occurring over 45 min of continuous seizures (status epilepticus) in rats. Seizures induced in rats by administration of lithium followed by pilocarpine were readily terminated by the benzodiazepine diazepam when administered early during the seizures (after 10 min of seizures). However, during status epilepticus, there was a substantial reduction of diazepam potency for termination of the seizures. To determine whether the loss of sensitivity of the animals to diazepam was caused by an alteration of GABAR functional properties, we obtained whole-cell GABAR currents from hippocampal dentate granule cells isolated acutely from control rats and from rats undergoing status epilepticus. GABAR properties were characterized by determining GABA sensitivity and the sensitivity of GABARs to regulation by benzodiazepines, barbiturates, and Zn2+. When compared with those from naive controls, GABAR currents from rats undergoing status epilepticus were less sensitive to diazepam and Zn2+but retained their sensitivity to GABA and pentobarbital. We conclude that the prolonged seizures of status epilepticus rapidly altered the functional properties of hippocampal dentate granule cell GABARs.
Presynaptic Depression at a Calyx Synapse: The Small Contribution of Metabotropic Glutamate Receptors
Authors: von Gersdorff Henrique , Schneggenburger Ralf , Weis Sibylle , Neher Erwin
Published: April 2018

Synaptic depression of evoked EPSCs was quantified with stimulation frequencies ranging from 0.2 to 100 Hz at the single CNS synapse formed by the calyx of Held in the rat brainstem. Half-maximal depression occurred at ≈1 Hz, with 10 and 100 Hz stimulation frequencies reducing EPSC amplitudes to ≈30% and ≈10% of their initial magnitude, respectively. The time constant of recovery from depression elicited by 10 Hz afferent fiber stimulation was 4.2 sec. AMPA and NMDA receptor-mediated EPSCs depressed in parallel at 1–5 Hz stimulation frequencies, suggesting that depression was induced by presynaptic mechanism(s) that reduced glutamate release. To determine the contribution of autoreceptors to depression, we studied the inhibitory effects of the metabotropic glutamate receptor (mGluR) agonists (1S, 3S)-ACPD andl-AP4 and found them to be reversed in a dose-dependent manner by (RS)-α-cyclopropyl-4-phosphonophenylglycine (CPPG), a novel and potent competitive antagonist of mGluRs. At 300 μm, CPPG completely reversed the effects ofl-AP4 and (1S, 3S)-ACPD, but reduced 5–10 Hz elicited depression by only ≈6%. CPPG-sensitive mGluRs, presumably activated by glutamate spillover during physiological synaptic transmission, thus contribute on the order of only 10% to short-term synaptic depression. We therefore suggest that the main mechanism contributing to the robust depression elicited by 5–10 Hz afferent fiber stimulation of the calyx of Held synapse is synaptic vesicle pool depletion.
Expression of Neural RNA-Binding Proteins in the Postnatal CNS: Implications of Their Roles in Neuronal and Glial Cell Development
Authors: Sakakibara Shin-ichi , Okano Hideyuki
Published: April 2018

There is an increasing interest in the role of RNA-binding proteins during neural development. Mouse-Musashi-1 (m-Msi-1) is a mouse neural RNA-binding protein with sequence similarity toDrosophila musashi(d-msi), which is essential for neural development. m-Msi-1 is highly enriched in neural precursor cells that are capable of generating both neurons and glia during embryonic CNS development. The present study characterized m-Msi-1-expressing cells in the postnatal and adult CNS. Postnatally, m-Msi-1 was expressed in proliferative neuronal precursors in the external granule cell layer of the cerebellum and in the anterior corner of the subventricular zone of the lateral ventricles. In gliogenesis, the persistent expression of m-Msi-1 was observed in cells of the astrocyte lineage ranging from proliferative glial precursors in the subventricular zone (SVZ) to differentiated astrocytes in the parenchyma. In addition, we showed that m-Msi-1 was still expressed in proliferating cells in the adult SVZ, which may contain neural precursor or stem cells. Another neural RNA-binding protein Hu (the mammalian homolog of aDrosophilaneuronal RNA-binding protein Elav) was present in postmitotic neurons throughout the development of the CNS, and its pattern of expression was compared with that of m-Msi-1. These observations imply that these two RNA-binding proteins may be involved in the development of neurons and glia by regulating gene expression at the post-transcriptional level.
Circadian Phase Shifts to Neuropeptide YIn Vitro: Cellular Communication and Signal Transduction
Authors: Biello Stephany M. , Golombek Diego A. , Schak Kathryn M. , Harrington Mary E.
Published: April 2018

Mammalian circadian rhythms originate in the hypothalamic suprachiasmatic nuclei (SCN), from which rhythmic neural activity can be recordedin vitro. Application of neurochemicals can reset this rhythm. Here we determine cellular correlates of the phase-shifting properties of neuropeptide Y (NPY) on the hamster circadian clockin vitro. Drug or control treatments were applied to hypothalamic slices containing the SCN on the first dayin vitro. The firing rates of individual cells were sampled on the second dayin vitro. Control slices exhibited a peak in firing rate in the middle of the day. Microdrop application of NPY to the SCN phase advanced the time of peak firing rate. This phase-shifting effect of NPY was not altered by block of sodium channels with tetrodotoxin or block of calcium channels with cadmium and nickel, consistent with a direct postsynaptic site of action. Pretreatment with the glutamate receptor antagonists (dl-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione disodium) also did not alter phase shifts to NPY. Blocking GABAAreceptors with bicuculline (Bic) had effects only at very high (millimolar) doses of Bic, whereas blocking GABABreceptors did not alter effects of NPY. Phase shifts to NPY were blocked by pretreatment with inhibitors of protein kinase C (PKC), suggesting that PKC activation may be necessary for these effects. Bathing the slice in low Ca2+/high Mg2+can block phase shifts to NPY, possibly via a depolarizing action. A depolarizing high K+bath can also block NPY phase shifts. The results are consistent with direct action of NPY on pacemaker neurons, mediated through a signal transduction pathway that depends on activation of PKC.
Supranormal Stimulation of D1Dopamine Receptors in the Rodent Prefrontal Cortex Impairs Spatial Working Memory Performance
Authors: Zahrt Justin , Taylor Jane R. , Mathew Rex G. , Arnsten Amy F. T.
Published: April 2018

Although previous research has emphasized the beneficial effects of dopamine (DA) on functions of the prefrontal cortex (PFC), recent studies of animals exposed to mild stress indicate that excessive DA receptor stimulation may be detrimental to the spatial working memory functions of the PFC (Arnsten and Goldman-Rakic, 1990; Murphy et al., 1994, 1996a,b, 1997). In particular, these studies have suggested that supranormal stimulation of D1receptors may contribute to the detrimental actions of DA in the PFC (Murphy et al., 1994, 1996a). The current study directly tested this hypothesis by examining the effects of infusing a full D1receptor agonist, SKF 81297, into the PFC of rats performing a spatial working memory task, delayed alternation. SKF 81297 produced a dose-related impairment in delayed-alternation performance. The impairment was reversed by pretreatment with a D1receptor antagonist, SCH 23390, consistent with drug actions at D1receptors. SCH 23390 by itself had no effect on performance, although slightly higher doses impaired performance (Murphy et al., 1994, 1996a). There was a significant relationship between infusion location and drug efficacy; animals with cannulae anterior to the PFC were not impaired by SKF 81297 infusions. Taken together, these results demonstrate that supranormal D1receptor stimulation in the PFC is sufficient to impair PFC working memory function. These cognitive data are consistent with recent electrophysiological studies of D1receptor mechanisms affecting the PFC (Williams and Goldman-Rakic, 1995; Yang and Seamans, 1996). Increased D1receptor stimulation during stress may serve to take the PFC “off-line” to allow posterior cortical and subcortical structures to regulate behavior, but may contribute to the vulnerability of the PFC in many neuropsychiatric disorders.
Systemic Morphine-Induced Fos Protein in the Rat Striatum and Nucleus Accumbens Is Regulated by μ Opioid Receptors in the Substantia Nigra and Ventral Tegmental Area
Authors: Bontempi Bruno , Sharp Frank R.
Published: April 2018

To characterize how systemic morphine induces Fos protein in dorsomedial striatum and nucleus accumbens (NAc), we examined the role of receptors in striatum, substantia nigra (SN), and ventral tegmental area (VTA). Morphine injected into medial SN or into VTA of awake rats induced Fos in neurons in ipsilateral dorsomedial striatum and NAc. Morphine injected into lateral SN induced Fos in dorsolateral striatum and globus pallidus. The morphine infusions produced contralateral turning that was most prominent after lateral SN injections. Intranigral injections of [d-Ala2,N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO), a μ opioid receptor agonist, and of bicuculline, a GABAAreceptor antagonist, induced Fos in ipsilateral striatum. Fos induction in dorsomedial striatum produced by systemic administration of morphine was blocked by (1) SN and VTA injections of the μ1opioid antagonist naloxonazine and (2) striatal injections of either MK 801, an NMDA glutamate receptor antagonist, or SCH 23390, a D1dopamine receptor antagonist.

Fos induction in dorsomedial striatum and NAc after systemic administration of morphine seems to be mediated by dopamine neurons in medial SN and VTA that project to medial striatum and NAc, respectively. Systemic morphine is proposed to act on μ opioid receptors located on GABAergic interneurons in medial SN and VTA. Inhibition of these GABA interneurons disinhibits medial SN and VTA dopamine neurons, producing dopamine release in medial striatum and NAc. This activates D1dopamine receptors and coupled with the coactivation of NMDA receptors possibly from cortical glutamate input induces Fos in striatal and NAc neurons. The modulation of target gene expression by Fos could influence addictive behavioral responses to opiates.
Defective Learning in Mutants of theDrosophilaGene for a Regulatory Subunit of cAMP-Dependent Protein Kinase
Authors: Goodwin Stephen F. , Del Vecchio Maria , Velinzon Klara , Hogel Catherine , Russell Steven R. H. , Tully Tim , Kaiser Kim
Published: April 2018

Disruptions of aDrosophilagene encoding a regulatory subunit of cAMP-dependent protein kinase homologous to mammalian RIβ (dPKA-RI) were targeted to the first (noncoding) exon of dPKA-RI via site-selected P element mutagenesis. Flies homozygous for either of two mutant alleles showed specific defects in olfactory learning but not in subsequent memory decay. In contrast, olfactory acuity and shock reactivity, component behaviors required for normal odor avoidance learning, were normal in these mutants. Northern and Western blot analyses of mRNA and protein extracted from adult heads have revealed a complex lesion of thePKA-RIlocus, including expression of a novel product and over- or underexpression of wild-type products in mutants. Western blot analysis revealed reductions in RI protein in mutants. PKA activity in the absence of exogenous cAMP also was significantly higher than normal in homogenates from mutant adult heads. These two mutant alleles failed to complement each other for each of these phenotypic defects, eliminating second-site mutations as a possible explanation. These results establish a role for an RI regulatory subunit of PKA in Pavlovian olfactory conditioning.
Differential Dependence on GluR2 Expression of Three Characteristic Features of AMPA Receptors
Authors: Washburn Mark S. , Numberger Markus , Zhang Sunan , Dingledine Raymond
Published: April 2018

The GluR2 subunit controls three key features of ion flux through the AMPA subtype of glutamate receptors—calcium permeability, inward rectification, and channel block by external polyamines, but whether each of these features is equally sensitive to GluR2 abundance is unknown. The relations among these properties were compared in native AMPA receptors expressed by acutely isolated hippocampal interneurons and in recombinant receptors expressed byXenopusoocytes. The shape of current–voltage (I–V) relations between −100 and +50 mV for either recombinant or native AMPA receptors was well described by a Woodhull block model in which the affinity for internal polyamine varied over a 1000-fold range in different cells. In oocytes injected with mixtures of GluR2:non-GluR2 mRNA, the relative abundance of GluR2 required to reduce the log of internal blocker affinity by 50% was two- to fourfold higher than that needed to half-maximally reduce divalent permeability or channel block by external polyamines. Likewise, in interneurons the affinity of externally applied argiotoxin for its blocking site was a steep function of internal blocker affinity. These results indicate that the number of GluR2 subunits in AMPA receptors is variable in both oocytes and interneurons. More GluR2 subunits in an AMPA receptor are required to maximally reduce internal blocker affinity than to abolish calcium permeability or external polyamine channel block. Accordingly, single-cell RT-PCR showed that approximately one-half of the physiologically characterized interneurons exhibiting inwardly rectifying AMPA receptors expressed detectable levels of edited GluR2. The physiological effects of a moderate change in GluR2 relative abundance, such as occurs after ischemia or seizures or after chronic exposure to morphine, thus will be dependent on the ambient GluR2 level in a cell-specific manner.
The Circumventricular Organs Form a Potential Neural Pathway for Lactate Sensitivity: Implications for Panic Disorder
Authors: Shekhar Anantha , Keim Stanley R.
Published: April 2018

Patients with panic disorder experience panic attacks after intravenous sodium lactate infusions by an as yet unexplained mechanism. Lactate elicits a panic-like response in rats with chronic dysfunction of GABA neurotransmission in the dorsomedial hypothalamus (DMH). The circumventricular organs, organum vasculosum lamina terminalis (OVLT) and subfornical organ (SFO), are potential sites that could detect increases in plasma lactate levels and activate the DMH. To test this, we obtained baseline heart rate (HR) and blood pressure (BP) responses to lactate infusions in rats fit with femoral arterial and venous catheters. Next, unilateral chronic injection cannulae connected to an Alzet infusion pump filled with the GABA synthesis inhibitorl-allylglycine (l-AG) were implanted into the DMH. Another chronic injection cannula was implanted into the region of the OVLT, SFO, or an adjacent control site, the median preoptic area (MePOA). These rats were tested once again with lactate infusions after injection of either artificial cerebrospinal fluid (ACSF) or tetrodotoxin (TTX) into the CVO sites. Injecting TTX into the OVLT completely blocked the lactate-induced response, whereas TTX injections into the SFO or MePOA did not. Also, direct injections of lactate (100 or 500 nl) into the OVLT elicited robust anxiety-like responses in these rats. These results suggest that the OVLT may be the primary site that detects lactate infusions, activating an anxiety-like response in a compromised DMH, and provide the first neuroanatomical basis for lactate response in panic disorder.
The Cerebellum and Red Nucleus Are Not Required forIn VitroClassical Conditioning of the Turtle Abducens Nerve Response
Authors: Anderson Curtis W. , Keifer Joyce
Published: April 2018

The role of the cerebellum during motor learning is a controversial issue. Many authors have suggested that the cerebellum and its connections with the red nucleus are essential for the acquisition of the conditioned eye blink reflex. Although there is little argument that the cerebellum is an important component to the generation of the conditioned response (CR), a number of studies have suggested that the cerebellum is not essential for conditioning. Using anin vitromodel of the classically conditioned turtle abducens nerve response, we investigated the effect of cerebellar and red nucleus lesions on the acquisition, extinction, and reacquisition of CRs. Neural discharge was recorded from the abducens nerve after a single shock unconditioned stimulus (US) was applied to the ipsilateral trigeminal nerve. When the US was paired with a conditioned stimulus (CS) applied to the posterior eighth, or auditory, nerve, a positive slope of CR acquisition was recorded in the abducens nerve. After extinction stimuli in which the CS and US were alternated, the number of CRs decreased to near zero. When the CS and US were once again paired, reacquisition at a faster rate was recorded. The CRs showed unusual timing features compared with preparations in which the cerebellum was intact; they had significantly shorter latencies and showed burst-like responses. These data demonstrate that it is possible to classically condition thisin vitropreparation in the absence of the cerebellum and red nucleus. However, the latencies of CRs were found to be dramatically altered in the cerebellar-lesioned preparations, suggesting that the cerebellum does play a role in the timing of the CR.
Synaptic Transmission Deficits inCaenorhabditis elegansSynaptobrevin Mutants
Authors: Nonet Michael L. , Saifee Owais , Zhao Hongjuan , Rand James B. , Wei Liping
Published: April 2018

Synaptobrevins are vesicle-associated proteins implicated in neurotransmitter release by both biochemical studies and perturbation experiments that use botulinum toxins. To test these modelsin vivo, we have isolated and characterized the first synaptobrevin mutants in metazoans and show that neurotransmission is severely disrupted in mutant animals. Mutants lackingsnb-1die just after completing embryogenesis. The dying animals retain some capability for movement, although they are extremely uncoordinated and incapable of feeding. We also have isolated and characterized several hypomorphicsnb-1mutants. Although fully viable, these mutants exhibit a variety of behavioral abnormalities that are consistent with a general defect in the efficacy of synaptic transmission. The viable mutants are resistant to the acetylcholinesterase inhibitor aldicarb, indicating that cholinergic transmission is impaired. Extracellular recordings from pharyngeal muscle also demonstrate severe defects in synaptic transmission in the mutants. The molecular lesions in the hypomorphic alleles reside on the hydrophobic face of a proposed amphipathic–helical region implicated biochemically in interacting with the t-SNAREs syntaxin and SNAP-25. Finally, we demonstrate that double mutants lacking both the v-SNAREs synaptotagmin andsnb-1are phenotypically similar tosnb-1mutants and less severe than syntaxin mutants. Our work demonstrates that synaptobrevin is essential for viability and is required for functional synaptic transmission. However, our analysis also suggests that transmitter release is not completely eliminated by removal of either one or both v-SNAREs.