bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Expression of α1, α2, α3, and α5GABAAReceptor Subunits in Seizure-Prone and Seizure-Resistant Rat Models of Temporal Lobe Epilepsy
Authors: Poulter Michael O. , Brown Leslie A. , Tynan Stephen , Willick Gordon , William Ross , McIntyre Dan C.
Published: April 2018

Temporal lobe epilepsy remains one of the most widespread seizure disorders in man, the etiology of which is controversial. Using new rat models of temporal lobe epilepsy that are either prone or resistant to develop complex partial seizures, we provide evidence that this seizure susceptibility may arise from arrested development of the GABAAreceptor system. In seizure-prone (Fast kindling) and seizure-resistant (Slow kindling) rat models, both the mRNA and protein levels of the major α subunit expressed in adult brain (α1), as well as those highly expressed during development (α2, α3, and α5), were differentially expressed in both models compared with normal controls. We found that α1subunit mRNA expression in the Fast kindling strain was approximately half the abundance of control rats, whereas in the Slow kindling strain, it was ∼70% greater than that of controls. However, Fast rats overexpressed the α2, α3, and α5(“embryonic”) subunits, having a density 50–70% greater than controls depending on brain area, whereas the converse was true of Slow rats. Using subunit-specific antibodies to α1and α5subunits, quantitative immunoblots and immunocytochemistry revealed a concordance with the mRNA levels. α1protein expression was ∼50% less than controls in the Fast strain, whereas it was 200% greater in the Slow strain. In contrast, α5subunit protein expression was greater in the Fast strain than either the control or Slow strain. These data suggest that a major predispositional factor in the development of temporal lobe epilepsy could be a failure to complete the normal switch from the GABAAreceptor α subunits highly expressed during development (α2, α3, and α5) to those highly expressed in adulthood (α1).
Temperature Modulation Reveals Three Distinct Stages of Wallerian Degeneration
Authors: Tsao Jack W. , George Edwin B. , Griffin John W.
Published: April 2018

After peripheral nerve transection, axons distal to the cut site rapidly degenerate, a process termed Wallerian degeneration. In wild-type mice the compound action potential (CAP) disappears by 3 d. Previous studies have demonstrated that cold temperatures and lower extracellular calcium ion (Ca2+) concentrations can slow the rate of Wallerian degeneration. We have incubated isolated sciatic nerve segments from wild-type and C57BL/Wld mice (which carry a gene slowing Wallerian degeneration)in vitroat 25 and 37°C. At 25°C we found that the degeneration rate of wild-type axons was slowed dramatically, with the CAP preserved up to 7 d post-transection. In contrast, at 37°C the CAPs were minimal at 2 d. When the temperature of wild-type nerves was raised to 37°C after 24–72 hr at 25°C, degeneration occurred within the subsequent 24 hr. Wld nerves, too, were preserved longer at 25°C but, on return to 37°C, degenerated promptly. Cooling the nerve within 12 hr after axotomy enhanced axonal preservation. Neither wild-type nor Wld nerves showed different degeneration rates when they were incubated with 250 μmor 5 or 10 mmextracellular Ca2+for 1–2 d, suggesting that an abrupt increase in intracellular Ca2+occurs at the time of axonal destruction. Wallerian degeneration, thus, appears to progress through three distinct stages. Initiation occurs at the time of injury with subsequent temperature-dependent and -independent phases. Nerves appear to remain intact and are able to exclude Ca2+from entering until an as yet unknown process finally increases axolemmal permeability.
Changes in Expression of the DNA Repair Protein Complex DNA-Dependent Protein Kinase after Ischemia and Reperfusion
Authors: Shackelford Deborah A. , Tobaru Takaaki , Zhang Shengjia , Zivin Justin A.
Published: April 2018

Reperfusion of ischemic tissue causes an immediate increase in DNA damage, including base lesions and strand breaks. Damage is reversible in surviving regions indicating that repair mechanisms are operable. DNA strand breaks are repaired by nonhomologous end joining in mammalian cells. This process requires DNA-dependent protein kinase (DNA-PK), composed of heterodimeric Ku antigen and a 460,000 Da catalytic subunit (DNA-PKcs). In this study, a rabbit spinal cord model of reversible ischemia was used to demonstrate the effect of acute CNS injury on the activity and expression of DNA-dependent protein kinase. The DNA-binding activity of Ku antigen, analyzed by an electrophoretic mobility shift assay, increased during reperfusion after a short ischemic insult (15 min of occlusion), from which the animals recover neurological function. After severe ischemic injury (60 min of occlusion) and reperfusion that results in permanent paraplegia, Ku DNA binding was reduced. Protein levels of the DNA-PK components—Ku70, Ku80, and DNA-PKcs—were monitored by immunoblotting. After 60 min of occlusion, the amount of DNA-PKcs and the enzyme poly(ADP-ribose) polymerase (PARP) decreased with the same time course during reperfusion. Concurrently 150 and 120 kDa fragments were immunostained by an anti-DNA-PKcs monoclonal antibody. This antibody was shown to cross-react with α-fodrin breakdown products. The 120 kDa fodrin peptide is associated with caspase-3 activation during apoptosis. Both DNA-PKcs and PARP are also substrates for caspase-3-like activities. The results are consistent with a model in which after a short ischemic insult, DNA repair proteins such as DNA-PK are activated. After severe ischemic injury, DNA damage overwhelms repair capabilities, and cell death programs are initiated.
Sequestration of G-Protein βγ Subunits by Different G-Protein α Subunits Blocks Voltage-Dependent Modulation of Ca2+Channels in Rat Sympathetic Neurons
Authors: Jeong Seong-Woo , Ikeda Stephen R.
Published: April 2018

The membrane-delimited and voltage-dependent inhibition of N-type Ca2+channels is mediated by Gβγ subunits. Previously, exogenous excess GDP-bound GαoAhas been shown to dramatically attenuate the norepinephrine (NE)-mediated Ca2+current inhibition by sequestration of Gβγ subunits in rat superior cervical ganglion (SCG) neurons. In the present study, we determined whether the attenuation of NE-mediated modulation is specific to GαoAor shared by a number of closely related (Gαtr, GαoB, Gαi1, Gαi2, Gαi3, Gαz) or unrelated (Gαs, Gαq, Gα11, Gα16, Gα12, Gα13) Gα subunits. Individual Gα subunits from different subfamilies were transiently overexpressed in SCG neurons by intranuclear injection of mammalian expression vectors encoding the desired protein. Strikingly, all Gα subunits except Gαznearly blocked basal facilitation and NE-mediated modulation. Likewise, VIP-mediated Ca2+current inhibition, which is mediated by cholera toxin-sensitive G-protein, was also completely suppressed by a number of Gα subunits overexpressed in neurons. Gαsexpression produced either enhancement or attenuation of the VIP-mediated modulation—an effect that seemed to depend on the expression level. The onset of the nonhydrolyzable GTP analog, guanylylimidodiphosphate-mediated facilitation was significantly delayed by overexpression of different GDP-bound Gα subunits. Taken together, these data suggest that a wide variety of Gα subunits are capable of forming heterotrimers with endogenous Gβγ subunits mediating voltage-dependent Ca2+channel inhibition. In conclusion, coupling specificity in signal transduction is unlikely to arise as a result of restricted Gα/Gβγ interaction.
Upregulation of Surface α4β2 Nicotinic Receptors Is Initiated by Receptor Desensitization after Chronic Exposure to Nicotine
Authors: Fenster Catherine P. , Whitworth Terri L. , Sheffield Elise B. , Quick Michael W. , Lester Robin A. J.
Published: April 2018

It is hypothesized that desensitization of neuronal nicotinic acetylcholine receptors (nAChRs) induced by chronic exposure to nicotine initiates upregulation of nAChR number. To test this hypothesis directly, oocytes expressing α4β2 receptors were chronically incubated (24–48 hr) in nicotine, and the resulting changes in specific [3H]nicotine binding to surface receptors on intact oocytes were compared with functional receptor desensitization. Four lines of evidence strongly support the hypothesis. (1) The half-maximal nicotine concentration necessary to produce desensitization (9.7 nm) was the same as that needed to induce upregulation (9.9 nm). (2) The concentration of [3H]nicotine for half-maximal binding to surface nAChRs on intact oocytes was also similar (11.1 nm), as predicted from cyclical desensitization models. (3) Functional desensitization of α3β4 receptors required 10-fold higher nicotine concentrations, and this was mirrored by a 10-fold shift in concentrations necessary for upregulation. (4) Mutant α4β2 receptors that do not recover fully from desensitization, but not wild-type channels, were upregulated after acute (1 hr) applications of nicotine. Interestingly, the nicotine concentration required for half-maximal binding of α4β2 receptors in total cell membrane homogenates was 20-fold lower than that measured for surface nAChRs in intact oocytes. These data suggest that cell homogenate binding assays may not accurately reflect thein vivodesensitization affinity of surface nAChRs and may account for some of the previously reported differences in the efficacy of nicotine for inducing nAChR desensitization and upregulation.
Olfactory Adaptation Depends on the Trp Ca2+Channel inDrosophila
Authors: Störtkuhl Klemens F. , Hovemann Bernhard T. , Carlson John R.
Published: April 2018

Olfactory adaptation is shown to occur inDrosophila, at both behavioral and physiological levels. In a behavioral paradigm, the extent of adaptation is shown to depend on the dose and duration of the adapting stimulus. Half-maximal adaptation occurred after 15 sec of exposure to an odor, and recovery occurred with a half-time of 1.5 min, under a set of test conditions. Cross-adaptation was observed among all odor combinations tested, although to a lesser extent than when the same odor was used as both the adapting and the test stimulus. Mutants of the transient receptor potential (Trp) Ca2+channel were normal in olfactory response, but defective in olfactory adaptation, when measured either behaviorally or in tests of antennal physiology. These results indicate that olfactory response and adaptation can be distinguished. Trp expression was detected in the developing antenna but, surprisingly, not in the mature antenna. These results, together with temperature-shift analysis of a temperature-sensitivetrpmutant, provide evidence of a role of Trp in olfactory system development.
Mapping the Agonist Binding Site of the GABAAReceptor: Evidence for a β-Strand
Authors: Boileau Andrew J. , Evers Amy R. , Davis Anson F. , Czajkowski Cynthia
Published: April 2018

GABAAreceptors, along with the receptors for acetylcholine, glycine, and serotonin, are members of a ligand-gated ion channel superfamily (Ortells and Lunt, 1995). Because of the paucity of crystallographic information for these ligand-gated channels, little is known about the structure of their binding sites or how agonist binding is transduced into channel gating. We used the substituted cysteine accessibility method to obtain secondary structural information about the GABA binding site and to systematically identify residues that line its surface. Each residue from α1Y59 to K70 was mutated to cysteine and expressed with wild-type β2subunits inXenopusoocytes or HEK 293 cells. The sulfhydryl-specific reagentN-biotinylaminoethyl methanethiosulfonate (MTSEA-Biotin) was used to covalently modify the cysteine-substituted residues. Receptors with cysteines substituted at positions α1T60, D62, F64, R66, and S68 reacted with MTSEA-Biotin, and α1F64C, R66C, and S68C were protected from reaction by agonist. We conclude that α1F64, R66, and S68 line part of the GABA binding site. The alternating pattern of accessibility of consecutive engineered cysteines to reaction with MTSEA-Biotin indicates that the region from α1Y59 to S68 is a β-strand.
Specification of Somatosensory Area Identity in Cortical Explants
Authors: Gitton Yorick , Cohen-Tannoudji Michel , Wassef Marion
Published: April 2018

The H-2Z1 transgene is restricted to a subset of layer IV neurons in the postnatal mouse cortex and delineates exactly the somatosensory area. Expression of the H-2Z1 transgene was used as an areal marker to determine when the parietal cortex becomes committed to a somatosensory identity. We have shown previously that grafts dissected from embryonic day 13.5 (E13.5) H-2Z1 cortex and transplanted into the cortex of nontransgenic newborns express H-2Z1 according to their site of origin. Expression was not modified on heterotopic transplantation (Cohen-Tannoudji et al., 1994). In the present study, whole cortical explants were isolated at E12.5 from noncortical tissues. The explants developed a regionalized expression of H-2Z1, indicating that regionalization takes place and is maintainedin vitro. We used this property and confronted embryonic H-2Z1 cortex with presumptive embryonic sources of regionalizing signals in anin vitrografting procedure. A great majority of E11.5–E13.5 grafts maintained their presumptive expression of H-2Z1 when grafted heterotopically on nontransgenic E13.5–E15.5 explants. However, a significantly lower proportion of E11.5 parietal grafts expressed H-2Z1 in occipital compared with parietal cortex, indicating that somatosensory identity may be partially plastic at E11.5. Earlier stages could not be tested because the E10.5 grafts failed to developin vitro. The data suggest that commitment to the expression of a somatosensory area-specific marker coincides with the onset of neurogenesis and occurs well before the birth of the non-GABAergic neurons that express H-2Z1in vivo.
Cell-Surface Glycoprotein of Oligodendrocyte Progenitors Involved in Migration
Authors: Niehaus Antje , Stegmüller Judith , Diers-Fenger Marianne , Trotter Jacqueline
Published: April 2018

Myelination by oligodendrocytes in the CNS involves the migration to and recognition and ensheathment of axons. These distinct developmental phases of myelination are assumed to involve the interplay of a precisely regulated set of cell adhesion molecules expressed by both neurons and glial cells. These molecules remain largely unelucidated. In this paper we have identified a large (330 kDa) glycoprotein expressed by murine oligodendrocyte progenitor cellsin vitroandin vivothat is downregulated as oligodendrocytes mature. Antigen-positive oligodendrocyte progenitor cells purified by panning develop into myelin-associated glycoprotein-positive oligodendrocytes and also adhere to cultured neurons. Polyclonal antibodies directed against the protein reduce the migration of oligodendrocyte progenitor cells. The observations suggest that the AN2 antigen may play a role in early stages of myelination.
OCD-Like Behaviors Caused by a Neuropotentiating Transgene Targeted to Cortical and Limbic D1+ Neurons
Authors: Campbell Keith M. , de Lecea Luis , Severynse Diana M. , Caron Marc G. , McGrath Michael J. , Sparber Sheldon B. , Sun Li-Yan , Burton Frank H.
Published: April 2018

To study the behavioral role of neurons containing the D1 dopamine receptor (D1+), we have used a genetic neurostimulatory approach. We generated transgenic mice that express an intracellular form of cholera toxin (CT), a neuropotentiating enzyme that chronically activates stimulatory G-protein (Gs) signal transduction and cAMP synthesis, under the control of the D1 promoter. Because the D1 promoter, like other CNS-expressed promoters, confers transgene expression that is regionally restricted to different D1+ CNS subsets in different transgenic lines, we observed distinct but related psychomotor disorders in different D1CT-expressing founders. In a D1CT line in which transgene expression was restricted to the following D1+ CNS regions—the piriform cortex layer II, layers II–III of somatosensory cortical areas, and the intercalated nucleus of the amygdala—D1CT mice showed normal CNS and D1+ neural architecture but increased cAMP content in whole extracts of the piriform and somatosensory cortex. These mice also exhibited a constellation of compulsive behavioral abnormalities that strongly resembled human cortical-limbic–induced compulsive disorders such as obsessive–compulsive disorder (OCD). These compulsive behaviors included episodes of perseverance or repetition of any and all normal behaviors, repetitive nonaggressive biting of siblings during grooming, and repetitive leaping. These results suggest that chronic potentiation of cortical and limbic D1+ neurons thought to induce glutamatergic output to the striatum causes behaviors reminiscent of those in human cortical-limbic–induced compulsive disorders.
Interleukin-1β Immunoreactivity and Microglia Are Enhanced in the Rat Hippocampus by Focal Kainate Application: Functional Evidence for Enhancement of Electrographic Seizures
Authors: Vezzani Annamaria , Conti Mirko , De Luigi Ada , Ravizza Teresa , Moneta Daniela , Marchesi Francesco , De Simoni Maria Grazia
Published: April 2018

Using immunocytochemistry and ELISA, we investigated the production of interleukin (IL)-1β in the rat hippocampus after focal application of kainic acid inducing electroencephalographic (EEG) seizures and CA3 neuronal cell loss. Next, we studied whether EEG seizures per se induced IL-1β and microglia changes in the hippocampus using bicuculline as a nonexcitotoxic convulsant agent. Finally, to address the functional role of this cytokine, we measured the effect of human recombinant (hr)IL-1β on seizure activity as one marker of the response to kainate.

Three and 24 hr after unilateral intrahippocampal application of 0.19 nmol of kainate, IL-1β immunoreactivity was enhanced in glia in the injected and the contralateral hippocampi. At 24 hr, IL-1β concentration increased by 16-fold (p< 0.01) in the injected hippocampus. Reactive microglia was enhanced with a pattern similar to IL-1β immunoreactivity. Intrahippocampal application of 0.77 nmol of bicuculline methiodide, which induces EEG seizures but not cell loss, enhanced IL-1β immunoreactivity and microglia, although to a less extent and for a shorter time compared with kainate. One nanogram of (hr)IL-1β intrahippocampally injected 10 min before kainate enhanced by 226% the time spent in seizures (p< 0.01). This effect was blocked by coinjection of 1 μg (hr)IL-1β receptor antagonist or 0.1 ng of 3-((+)-2-carboxypiperazin-4-yl)-propyl-1-phosphonate, selective antagonists of IL-1β and NMDA receptors, respectively.

Thus, convulsant and/or excitotoxic stimuli increase the production of IL-1β in microglia-like cells in the hippocampus. In addition, exogenous application of IL-1β prolongs kainate-induced hippocampal EEG seizures by enhancing glutamatergic neurotransmission.
Spatial Summation in the Receptive Fields of MT Neurons
Authors: Britten Kenneth H. , Heuer Hilary W.
Published: April 2018

Receptive fields (RFs) of cells in the middle temporal area (MT or V5) of monkeys will often encompass multiple objects under normal image viewing. We therefore have studied how multiple moving stimuli interact when presented within and near the RF of single MT cells. We used moving Gabor function stimuli, <1° in spatial extent and ∼100 msec in duration, presented on a grid of possible locations over the RF of the cell. Responses to these stimuli were typically robust, and their small spatial and temporal extent allowed detailed mapping of RFs and of interactions between stimuli. The responses to pairs of such stimuli were compared against the responses to the same stimuli presented singly. The responses were substantially less than the sum of the responses to the component stimuli and were well described by a power-law summation model with divisive inhibition. Such divisive inhibition is a key component of recently proposed “normalization” models of cortical physiology and is presumed to arise from lateral interconnections within a region. One open question is whether the normalization occurs only once in primary visual cortex or multiple times in different cortical areas. We addressed this question by exploring the spatial extent over which one stimulus would divide the response to another and found effective normalization from stimuli quite far removed from the RF center. This supports models under which normalization occurs both in MT and in earlier stages.
Basolateral Amygdala Noradrenergic Influences on Memory Storage Are Mediated by an Interaction between β- and α1-Adrenoceptors
Authors: Ferry Barbara , Roozendaal Benno , McGaugh James L.
Published: April 2018

Extensive evidence indicates that norepinephrine modulates memory storage through an activation of β-adrenoceptors in the basolateral nucleus of the amygdala (BLA). Recent findings suggest that the effects of β-adrenergic activation on memory storage are influenced by α1-adrenoceptor stimulation. Pharmacological findings indicate that activation of postsynaptic α1-adrenoceptors potentiates β-adrenoceptor-mediated activation of cAMP formation. The present study examined whether inactivation of α1-adrenoceptors in the BLA would alter the dose–response effects on memory storage of intra-BLA infusions of a β-adrenoceptor agonist, as well as that of a synthetic cAMP analog. Male Sprague Dawley rats received bilateral microinfusions into the BLA of either the β-adrenoceptor agonist clenbuterol (3–3000 pmol in 0.2 μl) or 8-bromoadenosine 3′:5′-cyclic monophosphate (8-bromo-cAMP) (0.2–7 nmol in 0.2 μl) alone or together with the α1-adrenoceptor antagonist prazosin (0.2 nmol) immediately after training in an inhibitory avoidance task. Retention was tested 48 hr later. Clenbuterol induced a dose-dependent enhancement of retention, and prazosin attenuated the dose–response effects of clenbuterol. Posttraining intra-BLA infusions of 8-bromo-cAMP also induced a dose-dependent enhancement of retention latencies. However, concurrent infusion of prazosin did not alter the dose–response effects of 8-bromo-cAMP. These findings are consistent with the view that α1-adrenoceptors affect memory storage by modulating β-adrenoceptor activation in the BLA. Moreover, these findings are consistent with those of pharmacological studies indicating that β-adrenoceptors modulate memory storage by a direct coupling to adenylate cyclase, whereas α1-receptors act indirectly by influencing the β-adrenoceptor-mediated influence on cAMP formation.
Ca2+Permeation of AMPA Receptors in Cerebellar Neurons Expressing Glu Receptor 2
Authors: Brorson James R. , Zhang Zehui , Vandenberghe Wim
Published: April 2018

AMPA receptors in cultured cerebellar neurons were characterized by whole-cell electrophysiological studies and single cell PCR-based quantitation of subunit mRNA expression. Purkinje neurons consistently expressed high levels of Glu receptor 2 (GluR2) mRNA and AMPA receptors with low but nonzero Ca2+permeability. Other cerebellar neurons expressed AMPA receptors with a wide range of Ca2+permeability and of fractional GluR2. These properties correlated on a cell-by-cell basis. Their relationship was well fit by a model that assumed stochastic assembly of subunits and GluR2 dominance in controlling divalent cation permeation, suggesting that AMPA receptor properties in individual neurons may be determined primarily by relative levels of subunit transcription. A fraction of receptors, lacking GluR2, can contribute a highly Ca2+-permeable component to AMPA receptor responses, even in cells expressing GluR2.
Receptors of the Glial Cell Line-Derived Neurotrophic Factor Family of Neurotrophic Factors Signal Cell Survival through the Phosphatidylinositol 3-Kinase Pathway in Spinal Cord Motoneurons
Authors: Soler Rosa M. , Dolcet Xavier , Encinas Mario , Egea Joaquim , Bayascas Jose R. , Comella Joan X.
Published: April 2018

The members of the glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors (GDNF, neurturin, persephin, and artemin) are able to promotein vivoandin vitrosurvival of different neuronal populations, including spinal cord motoneurons. These factors signal via multicomponent receptors that consist of the Ret receptor tyrosine kinase plus a member of the GDNF family receptor α (GRFα) family of glycosylphosphatidylinositol-linked coreceptors. Activation of the receptor induces Ret phosphorylation that leads the survival-promoting effects. Ret phosphorylation causes the activation of several intracellular pathways, but the biological effects caused by the activation of each of these pathways are still unknown. In the present work, we describe the ability of the GDNF family members to promote chicken motoneuron survival in culture. We show the presence of Ret and GFRα-1, GFRα-2, and GFRα-4 in chicken motoneurons usingin situhybridization and reverse transcription-PCR techniques. By Western blot analysis and kinase assays, we demonstrate the ability of these factors to induce the phosphatidylinositol 3 kinase (PI 3-kinase) and the extracellular regulated kinase (ERK)–mitogen-activated protein (MAP) kinase pathways activation. To characterize the involvement of these pathways in the survival effect, we used the PI 3-kinase inhibitor LY 294002 and the MAP kinase and ERK kinase (MEK) inhibitor PD 98059. We demonstrate that LY 294002, but not PD 98059, prevents GDNF-, neurturin-, and persephin-induced motoneuron survival, suggesting that PI 3-kinase intracellular pathway is responsible in mediating the neurotrophic effect.
A2AAdenosine Receptor Deficiency Attenuates Brain Injury Induced by Transient Focal Ischemia in Mice
Authors: Chen Jiang-Fan , Huang Zhihong , Ma Jianya , Zhu JinMin , Moratalla Rosario , Standaert David , Moskowitz Michael A. , Fink J. Stephen , Schwarzschild Michael A.
Published: April 2018

Extracellular adenosine critically modulates ischemic brain injury, at least in part through activation of the A1adenosine receptor. However, the role played by the A2Areceptor has been obscured by intrinsic limitations of A2Aadenosinergic agents. To overcome these pharmacological limitations, we explored the consequences of deleting the A2Aadenosine receptor on brain damage after transient focal ischemia. Cerebral morphology, as well as vascular and physiological measures (before, during, and after ischemia) did not differ between A2Areceptor knock-out and wild-type littermates. The volume of cerebral infarction, as well as the associated neurological deficit induced by transient filament occlusion of the middle cerebral artery, were significantly attenuated in A2Areceptor knock-out mice. This neuroprotective phenotype of A2Areceptor-deficient mice was observed in different genetic backgrounds, confirming A2Areceptor disruption as its cause. Together with complimentary pharmacological studies, these data suggest that A2Areceptors play a prominent role in the development of ischemic injury within brain and demonstrate the potential for anatomical and functional neuroprotection against stroke by A2Areceptor antagonists.
An R-Type Ca2+Current in Neurohypophysial Terminals Preferentially Regulates Oxytocin Secretion
Authors: Wang Gang , Dayanithi Govindan , Newcomb Robert , Lemos José R.
Published: April 2018

Multiple types of voltage-dependent Ca2+channels are involved in the regulation of neurotransmitter release (Tsien et al., 1991; Dunlap et al., 1995). In the nerve terminals of the neurohypophysis, the roles of L-, N-, and P/Q-type Ca2+channels in neuropeptide release have been identified previously (Wang et al., 1997a). Although the L- and N-type Ca2+currents play equivalent roles in both vasopressin and oxytocin release, the P/Q-type Ca2+current only regulates vasopressin release. An oxytocin-release and Ca2+current component is resistant to the L-, N-, and P/Q-type Ca2+channel blockers but is inhibited by Ni2+. A new polypeptide toxin, SNX-482, which is a specific α1E-type Ca2+channel blocker (Newcomb et al., 1998), was used to characterize the biophysical properties of this resistant Ca2+current component and its role in neuropeptide release. This resistant component was dose dependently inhibited by SNX-482, with an IC50of 4.1 nm. Furthermore, SNX-482 did not affect the other Ca2+current types in these CNS terminals. Like the N- and P/Q-type Ca2+currents, this SNX-482-sensitive transient Ca2+current is high-threshold activated and shows moderate steady-state inactivation. At the same concentrations, SNX-482 blocked the component of oxytocin, but not of vasopressin, release that was resistant to the other channel blockers, indicating a preferential role for this type of Ca2+current in oxytocin release from neurohypophysial terminals. Our results suggest that an α1Eor “R”-type Ca2+channel exists in oxytocinergic nerve terminals and, thus, functions in controlling only oxytocin release from the rat neurohypophysis.
Pharmacological Isolation of the Synaptic and Nonsynaptic Components of the GABA-Mediated Biphasic Response in Rat CA1 Hippocampal Pyramidal Cells
Authors: Smirnov Sergei , Paalasmaa Pekka , Uusisaari Marylka , Voipio Juha , Kaila Kai
Published: April 2018

High-frequency stimulation (HFS) applied to stratum radiatum of a rat hippocampal slice in the presence of ionotropic glutamate receptor antagonists evokes a biphasic GABAAreceptor-dependent response in CA1 pyramidal neurons, with a brief hyperpolarizing IPSP (hIPSP) followed by a long-lasting depolarization. We show now that it is possible to pharmacologically separate the hIPSP and late depolarization from one another. In neurons intracellularly perfused for 1–2 hr with F−as the major anion and no ATP, the hIPSP (and the corresponding current, hIPSC) evoked by HFS was blocked, whereas neither the late depolarization nor its underlying current was attenuated. In contrast, internal perfusion with a high concentration (5 mm) of the impermeant lidocaine derivative QX-314 selectively abolished the depolarizing component of the biphasic response and also strongly reduced depolarizations evoked by extracellular microinjection of K+. Bath application of quinine (0.2–0.5 mm) or quinidine (0.1 mm) resulted in a pronounced inhibition of the HFS-induced extracellular K+concentration ([K+]o) transient but not of the bicarbonate-dependent alkaline shift in extracellular pH. The attenuation of the [K+]otransient was closely paralleled by a suppression of the HFS-evoked depolarization but not of the hIPSP. Quini(di)ne did not affect depolarizations induced by exogenous K+either.

These data provide direct pharmacological evidence for the view that the HFS-induced biphasic response of the pyramidal neuron is composed of mechanistically distinct components: a direct GABAAreceptor-mediated phase, which is followed by a slow, nonsynaptic [K+]o-mediated depolarization. The bicarbonate-dependent, activity-induced [K+]otransient can be blocked by quini(di)ne, whereas its depolarizing action in the pyramidal neuron is inhibited by internal QX-314. The presence of fundamentally distinct components in GABAAreceptor-mediated actions evoked by HFS calls for further investigations of their functional role(s) in standard experimental maneuvers, such as those used in studies of synaptic plasticity and induction ofγoscillations.
Receptor Subtype-Induced Targeting and Subtype-Specific Internalization of Human α2-Adrenoceptors in PC12 Cells
Authors: Olli-Lähdesmäki Tuire , Kallio Jaana , Scheinin Mika
Published: April 2018

The three α2-adrenergic receptor subtypes have distinct tissue distributions, desensitization properties, and, in some cell types, subtype-specific subcellular localization and trafficking properties. The subtypes also differ in their neuronal physiology. Therefore, we have investigated the localization and targeting of human α2-adrenoceptors (α2-AR) in PC12 cells, which were transfected to express the α2-AR subtypes A, B, and C. Inspection of the receptors by indirect immunofluorescence and confocal microscopy showed that α2A-AR were mainly targeted to the tips of the neurites, α2B-AR were evenly distributed in the plasma membrane, and α2C-AR were mostly located in an intracellular perinuclear compartment. After agonist treatment, α2A- and α2B-AR were internalized into partly overlapping populations of intracellular vesicles. Receptor subtype-specific changes in PC12 cell morphology were also discovered: expression of α2A-AR, but not of α2B- or α2C-AR, induced differentiation-like changes in cells not treated with NGF. Also α2B-AR were targeted to the tips of neurites when they were coexpressed in the same cells with α2A-AR, indicating that the targeting of receptors to the tips of neurites is a consequence of a change in PC12 cell membrane protein trafficking that the α2A-subtype induces. The marked agonist-induced internalization of α2A-AR observed in both nondifferentiated and differentiated PC12 cells contrasts with earlier results from non-neuronal cells and points out the importance of the cellular environment for receptor endocytosis and trafficking. The targeting of α2A-AR to nerve terminals in PC12 cells is in line with the putative physiological role of this receptor subtype as a presynaptic autoreceptor.
Multiorgan Autonomic Dysfunction in Mice Lacking the β2 and the β4 Subunits of Neuronal Nicotinic Acetylcholine Receptors
Authors: Xu Wei , Orr-Urtreger Avi , Nigro Filippo , Gelber Shari , Sutcliffe Cara Ballard , Armstrong Dawna , Patrick James W. , Role Lorna W. , Beaudet Arthur L. , De Biasi Mariella
Published: April 2018

Transcripts for the β2 and the β4 nicotinic acetylcholine receptor (nAChR) subunits are found throughout the CNS and the peripheral nervous system. These two β subunits can form heteromultimeric channels with any of the α2, α3, α4, or α5 subunits in heterologous expression systems. Nonetheless, the subunit composition of native nAChRs and the role of different nAChR subtypesin vivoremain unclear. We prepared null mutations for the β2 and the β4 genes and bred β2−/−β4−/− mice by mating mice of identical β2−/−β4+/− or β2+/−β4−/− genotype. The β2−/− and the β4−/− single-mutant mice grow to adulthood with no visible phenotypic abnormalities. The β2−/−β4−/− double mutants survive to birth but have impaired growth and increased perinatal mortality. They also present enlarged bladders with dribbling urination and develop urinary infection and bladder stones. The ocular pupils are widely dilated and do not constrict in response to light. Histological studies revealed no significant abnormalities of brain or peripheral tissues except for hyperplasia in the bladder mucosa of β4−/− and β2−/−β4−/− mutants. Bladder strips from β2−/−β4−/− mice did not respond to nicotine but contracted when stimulated with a muscarinic agonist or electric field stimulation. Bladder strips from β4 mutants did not respond to nicotine despite the absence of major bladder dysfunctionin vivo. Acetylcholine-activated whole-cell currents were absent in superior cervical ganglion neurons from β2−/−β4−/− mice and reduced in neurons from β4−/− mice. Although there is apparent redundancy and a superficially normal phenotype in β2−/− and β4−/− mice, physiological studies indicate major deficits in the β4−/− mice. Our previous description of a similar phenotype in α3−/− mice and the current data suggest that the α3 and the β4 subunits are major components in autonomic nAChRs. The phenotype of the β2−/−β4−/− and α3−/− mice resembles the autosomal recessive megacystis-microcolon-hypoperistalsis syndrome in humans.
Glucocorticoid Receptor Expression in the Spinal Cord after Traumatic Injury in Adult Rats
Authors: Yan Ping , Xu Jian , Li Qun , Chen Sawei , Kim Gyeong-Moon , Hsu Chung Y. , Xu Xiao Ming
Published: April 2018

Methylprednisolone (MP), a glucocorticoid, is the only effective therapeutic agent used in the clinical treatment of acute spinal cord injury (SCI). MP given within 8 hr after SCI significantly improves neurological function. Although the glucocorticoid receptor (GR) is suggested to mediate MP actions, limited knowledge is available on its expression and possible function after SCI. Presently, the expression of GR was studied in a weight-drop SCI model in adult rats. Immunohistochemistry and Western blot analysis revealed an increase in GR protein expression as early as 15 min after injury. GR expression sharply increased at 4 hr (22-fold), peaked at 8 hr (56-fold), rapidly declined at 1 d, and returned to the baseline level at and after 3 d. During its peak expression, GR was localized in neural somata and dendrites but not in axons and their terminals. GR immunoreactivity was also found in oligodendrocytes and astrocytes. Interestingly, other cell types, such as endothelial cells, were GR-negative. An increase in the binding activity of nuclear proteins to the glucocorticoid responsive element was also observed after SCI, demonstrating a functional element of GR activation. Finally, colocalization of GR and tumor necrosis factor α (TNF-α), an inflammatory cytokine, was observed in neurons and glial cells, consistent with MP regulation of TNF-α in this model. Thus, the transient expression of high levels of GR after SCI may provide new insights into the anti-inflammatory action of MP.
Lesions of an Avian Forebrain Nucleus That Disrupt Song Development Alter Synaptic Connectivity and Transmission in the Vocal Premotor Pathway
Authors: Kittelberger J. Matthew , Mooney Richard
Published: April 2018

The avian forebrain nucleus, the lateral magnocellular nucleus of the anterior neostriatum (LMAN), is necessary for normal song development because LMAN lesions made in juvenile birds disrupt song production but do not disrupt song when made in adults. Although these age-limited behavioral effects implicate LMAN in song learning, a potential confound is that LMAN lesions could disrupt normal vocal motor function independent of any learning role by altering LMAN′s premotor target, the song nucleus, the robust nucleus of the archistriatum (RA). To date, however, no studies have examined directly the effects of LMAN lesions on the circuitry of the RA. We report here that juvenile LMAN lesions rapidly and profoundly affect RA, altering synaptic connectivity within this nucleus, including descending inputs from the song nucleusHVc. Specifically, morphological assays of the dendritic spines of RA projection neurons and axon terminal boutons arising from HVc show a numerical decline in the density of connections in RA in LMAN-lesioned juveniles compared with controls. Concurrently, LMAN lesions alter excitatory transmission within the juvenile RA: after LMAN lesions, the stimulus–response relationship between HVc fibers and RA neurons steepens, and the amplitude of spontaneous monophasic EPSCs increases. Rather than arresting RA in a juvenile state, LMAN lesions transform the structure and function of RA and its connections, such that it is distinct from that of the normal juvenile. In many ways, RA circuitry in LMAN-lesioned juveniles resembles that of normal adults, suggesting that LMAN lesions induce a premature maturation of the vocal motor pathway, which may lead to a loss of behavioral plasticity and abnormal song development.
Regulation of Neuregulin-Mediated Acetylcholine Receptor Synthesis by Protein Tyrosine Phosphatase SHP2
Authors: Tanowitz Michael , Si Jutong , Yu De-Hua , Feng Gen-Sheng , Mei Lin
Published: April 2018

Synapse-specific expression of the nicotinic acetylcholine receptor (AChR) is believed to be mediated by neuregulin, an epidermal growth factor-like trophic factor released by somatic motoneurons at the neuromuscular junction (NMJ). Neuregulin stimulates ErbB2, ErbB3, and ErbB4, members of the ErbB family of receptor tyrosine kinases. SHP2 is a cytoplasmic protein tyrosine phosphatase containing two Src homology 2 domains near its N terminus, and has been shown to be a positive mediator of mitogenic responses to various growth factors. We found that SHP2 interacted with ErbB2 and ErbB3 after neuregulin stimulation of muscle cells. Expression of SHP2 in C2C12 mouse muscle cells attenuated the neuregulin-induced expression of an AChR ε-promoter reporter gene, whereas a catalytically inactive SHP2 mutant or a mutant lacking the N-terminal Src homology 2 (SH2) domain enhanced reporter expression, suggesting that SHP2 negatively regulates the neuregulin signaling pathway. In fibroblast cells that express a mutant SHP2 with a targeted deletion of the N-terminal SH2 domain, neuregulin-mediated activation of the Ras/Raf/extracellular signal-regulated kinase cascade was enhanced. Furthermore, we found that SHP2 immunoreactivity colocalized with the staining of α-bungarotoxin, a marker of the NMJ. These results demonstrate a negative role of SHP2 in the neuregulin signal that leads to AChR gene expression at the NMJ.
Membrane Recycling in the Neuronal Growth Cone Revealed by FM1–43 Labeling
Authors: Diefenbach Thomas J. , Guthrie Peter B. , Stier Heike , Billups Brian , Kater S. B.
Published: April 2018

Membrane dynamics within the chick ciliary neuronal growth cone were investigated by using the membrane-impermeant dye FM1–43. A depolarization-evoked endocytosis was observed that shared many properties with the synaptic vesicle recycling previously described at the presynaptic terminal. In addition, in the absence of depolarization a basal level of constitutive endocytotic activity was observed at ∼30% of the rate of evoked endocytosis. This constitutive endocytosis accounted for large amounts of membrane retrieval: the equivalent of the entire growth cone surface area could be internalized within a 30 min period. Endosomes generated via constitutive and evoked processes were highly mobile and could move considerable distances both within the growth cone and out to the neurite. In addition to their different requirements for formation, evoked and constitutive endosomes displayed a significant difference in release properties. After a subsequent depolarization of labeled growth cones, evoked endosomes were released although constitutive endosomes were not released. Furthermore, treatment with latrotoxin released evoked endosomes, but not constitutive endosomes. Although the properties of evoked endosomes are highly reminiscent of synaptic vesicles, constitutive endosomes appear to be a separate pool resulting from a distinct and highly active process within the neuronal growth cone.
Highly Specific Neuron Loss Preserves Lateral Inhibitory Circuits in the Dentate Gyrus of Kainate-Induced Epileptic Rats
Authors: Buckmaster Paul S. , Jongen-Rêlo Ana L.
Published: April 2018

Patients with temporal lobe epilepsy display neuron loss in the hilus of the dentate gyrus. This has been proposed to be epileptogenic by a variety of different mechanisms. The present study examines the specificity and extent of neuron loss in the dentate gyrus of kainate-treated rats, a model of temporal lobe epilepsy. Kainate-treated rats lose an average of 52% of their GAD-negative hilar neurons (putative mossy cells) and 13% of their GAD-positive cells (GABAergic interneurons) in the dentate gyrus. Interneuron loss is remarkably specific; 83% of the missing GAD-positive neurons are somatostatin-immunoreactive. Of the total neuron loss in the hilus, 97% is attributed to two cell types—mossy cells and somatostatinergic interneurons. The retrograde tracer wheat germ agglutinin (WGA)-apoHRP-gold was used to identify neurons with appropriate axon projections for generating lateral inhibition. Previously, it was shown that lateral inhibition between regions separated by 1 mm persists in the dentate gyrus of kainate-treated rats with hilar neuron loss. Retrogradely labeled GABAergic interneurons are found consistently in sections extending 1 mm septotemporally from the tracer injection site in control and kainate-treated rats. Retrogradely labeled putative mossy cells are found up to 4 mm from the injection site, but kainate-treated rats have fewer than controls, and in several kainate-treated rats virtually all of these cells are missing. These findings support hypotheses of temporal lobe epileptogenesis that involve mossy cell and somatostatinergic neuron loss and suggest that lateral inhibition in the dentate gyrus does not require mossy cells but, instead, may be generated directly by GABAergic interneurons.
Regulation of Learning by EphA Receptors: a Protein Targeting Study
Authors: Gerlai R. , Shinsky N. , Shih A. , Williams P. , Winer J. , Armanini M. , Cairns B. , Winslow J. , Gao W.-Q. , Phillips H. S.
Published: April 2018

EphA family receptor tyrosine kinases and their ephrin-A ligands are involved in patterning axonal connections during brain development, but until now a role for these molecules in the mature brain had not been elucidated. Here, we show that both the EphA5 receptor and its ephrin-A ligands (2 and 5) are expressed in the adult mouse hippocampus, and the EphA5 protein is present in a phosphorylated form. Because there are no pharmacological agents available for EphA receptors, we designed recombinant immunoadhesins that specifically bind to the receptor binding site of the ephrin-A ligand (antagonist) or the ligand binding site of the EphA receptor (agonist) and thus target EphA function. We demonstrate that intrahippocampal infusion of an EphA antagonist immunoadhesin leads to impaired performance in two behavioral paradigms, T-maze spontaneous alternation and context-dependent fear conditioning, sensitive to hippocampal function, whereas activation of EphA by infusion of an agonist immunoadhesin results in enhanced performance on these tasks. Because the two behavioral tasks have different motivational, perceptual, and motor requirements, we infer the changes were not caused by these performance factors but rather to cognitive alterations. We also find bidirectional changes in gene expression and in electrophysiological measures of synaptic efficacy that correlate with the behavioral results. Thus, EphA receptors and their ligands are implicated as mediators of plasticity in the adult mammalian brain.
Auditory Thalamus, Dorsal Hippocampus, Basolateral Amygdala, and Perirhinal Cortex Role in the Consolidation of Conditioned Freezing to Context and to Acoustic Conditioned Stimulus in the Rat
Authors: Sacchetti Benedetto , Lorenzini Carlo Ambrogi , Baldi Elisabetta , Tassoni Giovanna , Bucherelli Corrado
Published: April 2018

On the basis of previous experimental evidence, it is known that the auditory thalamus (AT), the dorsal hippocampus (DH), the basolateral amygdala (BLA), and the perirhinal cortex (PC) are involved in the mnemonic processing of conditioned freezing. In particular, BLA and PC appear to be involved both in conditioned stimulus (CS) and context conditioned freezing. Through AT, the auditory CS is sent to other sites, whereas DH is involved in context conditioning. Nevertheless, the existing evidence does not make it possible to assess AT, DH, BLA, and PC involvement during the consolidation phase of conditioned freezing. To address this question, fully reversible tetrodotoxin (TTX) inactivation was performed on adult male Wistar rats having undergone CS and context fear training. Anesthetized animals were injected stereotaxically with TTX (either 5 or 10 ng in 0.5 or 1.0 μl of saline, according to site dimensions) at increasing post-acquisition delays. Context and CS freezing durations were measured during retention testing, always performed 48 and 72 hr after TTX administration. The results showed that AT inactivation does not disrupt consolidation of either contextual or auditory fear memories. In contrast, inactivation of the other three structures disrupted consolidation. For the DH, this disruption was specific to contextual cues and only occurred when inactivation was performed early (up to 1.5 hr) after training. The BLA and PC were shown to be involved in the consolidation of both contextual and auditory fear. Their involvement persisted for longer periods of time (2 d for BLA and 8 d for PC). These findings provide information to build a temporal profile for the post-training processing of fear memories in structures known to be important for this form of learning. The results are discussed in relation to previous studies on conditioned freezing and other aversive conditioned response neural correlates.