Neuronale Plastizität im limbischen System adulter Ratten und deren Modifikation in Epilepsiemodellen

In den letzten Jahrhunderten wurde postuliert, dass der komplexen Verarbeitungsleistung des adulten Säugetiergehirns ein stabiles Netzwerkgefüge zugrunde liegen würde. Diese Hypothese wurde kontinuierlich durch die Erkenntnis ersetzt, dass adulte Säugetiergehirne permanent einer massiven strukturellen und synaptischen Plastizität unterliegen. Teil dieser Plastizität ist eine lebenslang in hohem Maße stattfindende Neubildung von Nervenzellen in zwei regional begrenzten Gehirnarealen, den primären neurogenen Zonen. Daneben existieren multipotente neuronale Vorläuferzellen in diversen Bereichen des adulten Säugetiergehirns deren Potential neue Neurone im naiven Gehirn zu bilden, vielfach diskutiert wird. Im ersten Teil der vorliegenden Arbeit (Publikation 1) wurde das neurogene Potential des piriformen Cortex (PC) adulter Ratten untersucht. Der PC ist der größte Anteil des olfaktorischen Cortex und entscheidend an der Verarbeitung verschiedener Riecheindrücke beteiligt. Es konnte eine Zellneubildung mit neuronalen Charakteristika im PC nachgewiesen werden, die im Vergleich zu den beiden primären neurogenen Zonen jedoch um ein Vielfaches geringer war. Weiterhin konnte die Existenz von neuronalen Vorläuferzellen abgesichert werden, aus denen sich die neugebildeten Neurone direkt bilden könnten. Weiterführende Untersuchungen ergaben, dass die neugebildeten Zellen jedoch nicht über einen langen Zeitraum erhalten bleiben. Im Vergleich zu freilebenden Tieren werden Laborratten unter deprivierten Bedingungen gehalten. Da in der Vergangenheit gezeigt wurde, dass eine Umwelt mit gesteigerten Sinneseindrücken die Überlebensrate von neugebildeten Neuronen in den primären neurogenen Zonen steigert, kann weiterhin vermutet werden, dass die Überlebensdauer der neugebildeten Zellen im PC unter natürlichen Umweltbedingungen ebenfalls gesteigert ist. Im zweiten Teil der Arbeit (Publikation 2) wurden spezifische pathophysiologische Mechanismen neurogeneseabhängiger plastischer Veränderungen im Hippocampus zweier Rattenmodelle mit Epileptogenese charakterisiert. Während ihrer Entwicklung bilden neuronale Vorläuferzellen im adulten Säugetierhippocampus transient kurze basale Dendriten aus. In epileptischen Tieren und in Epilepsiepatienten persistieren diese Dendriten und weisen weitere morphofunktionelle Eigenschaften auf, die als prokonvulsive plastische Netzwerkveränderungen interpretiert werden. Daher wurde die Generierung dieser sog. hilaren oder persistierenden basalen Dendriten direkt mit dem Auftreten von epileptischen Anfällen in Verbindung gebracht. Genauere Untersuchungen, die diese Vermutung stützen, fehlen jedoch. Die Daten der vorliegenden Arbeit belegen, dass persistierende basale Dendriten charakteristisch für ein chronisches epileptogenes, neuronales Netzwerk und nicht unmittelbar eine Folge von epileptischen Anfällen sind. Spontane wiederkehrende Anfälle resultieren jedoch in einer weiteren Steigerung der Anzahl dieser Dendriten. Basierend auf der Hypothese, dass die Persisitenz der Dendriten die Epilepsieprogression und den Erkrankungsgrad steigert, kann weiterhin gefolgert werden, dass auch in Phasen der Anfallsfreiheit diese Form aberranter neurogeneseabhängige Plastizität zu einer Progression der Epilepsie beiträgt. Die Ergebnisse dieser Untersuchungen demonstrieren einheitlich die weit reichende Bedeutung neurogeneseabhängiger plastischer Veränderungen limbischer Strukturen unter physiologischen und pathophysiologischen Bedingungen. Eine weiterführende Aufklärung des regenerativen Potentials der transienten Nervenzellneubildung im PC ist aus Sicht einer möglichen Therapie diverser neurologischer Erkrankungen von Relevanz. Das endogene Reservoir multipotenter Vorläuferzellen in dieser und weiterer Gehirnregionen könnte für den funktionellen Ersatz erkrankungsbedingt untergegangener Neurone verwendet werden. Neben diesem regenerativen Potential existieren Hinweise auf eine pathophysiologische Bedeutung der neurogeneseassoziierten Plastizität bei Epilepsien. Die weiterführende Charakterisierung und die Aufklärung der funktionellen Relevanz dieser permissiven Alterationen sind für die Entwicklung einer kausalen Therapie sowie einer Epilepsieprophylaxe von Bedeutung

Differences in trait impulsivity do not bias the response to pharmacological drug challenge in the rat five-choice serial reaction time task.

RATIONALE: Maladaptive impulsivity is symptomatic of several neuropsychiatric disorders including schizophrenia, attention-deficit hyperactivity disorder (ADHD), and substance abuse disorders; paradigms designed to assess the underlying neurobiology of this behavior are essential for the discovery of novel therapeutic agents. Various models may be used to assess impulsivity as measured by the five-choice serial reaction time task (5-CSRTT), including variable inter-trial interval (ITI) sessions, the selection of extreme high and low impulsivity phenotypes from a large outbred population of rats, as well as pharmacological challenges. OBJECTIVES: The aim of this study is to evaluate if pharmacological challenge models for impulsivity are biased by underlying differences in impulsivity phenotype. METHODS: Extreme high and low impulsivity phenotypes were selected in the 5-CSRTT, and dose-dependent effects of various pharmacological challenges, namely MK-801, yohimbine, and cocaine, were evaluated on task performance, specifically accuracy and premature responses. RESULTS: All three compounds increased premature responding, while a decrease in attentional performance occurred following MK-801 and yohimbine administration. No differences in drug-induced impulsivity between rats selected for high or low impulsivity or in parameters indicative of attentional performance could be determined. CONCLUSIONS: Our findings indicate that different pharmacological challenges increase impulsivity on the 5-CSRTT, with modest effects on attention. These effects were not influenced by underlying differences in impulsivity phenotype, which is an important prerequisite to reliably use these challenge models to screen and profile compounds with putative anti-impulsive characteristics

Dissociable effects of mGluR5 allosteric modulation on distinct forms of impulsivity in rats: interaction with NMDA receptor antagonism

Rationale: Impaired N-methyl-D-aspartate (NMDA) receptor signalling underlies several psychiatric disorders that express high levels of impulsivity. Although synergistic interactions exist between NMDA receptors and metabotropic glutamate receptor 5 (mGluR5), the significance of this interaction for impulsivity is unknown. Objective This study aims to investigate the effects of negative and positive allosteric mGluR5 modulation (NAM/PAM) on trait impulsivity and impulsivity evoked by NMDA receptor antagonism in rats. Methods: Motor and choice impulsivity were assessed using the five-choice serial reaction time task (5-CSRTT) and delayeddiscounting task (DDT), respectively. The effects of RO4917523 and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) (NAMs) and ADX47273 (PAM) were investigated in non-impulsive rats and in trait high- and low-impulsive rats. The effects of these compounds on impulsivity induced by NMDA receptor antagonism (MK801) in the 5-CSRTT were also investigated. Results: RO4917523 (0.1–1 mg/kg) decreased premature responding and increased omissions but had no effect on locomotor activity up to 0.1 mg/kg. MTEP significantly increased omissions, decreased accuracy and slowed responding but had no effect on premature responding. ADX47273 decreased premature responding at doses that had no effect on locomotor activity. MK801 increased premature responding and impaired attentional accuracy; these deficits were dose dependently rescued by ADX47273 pre-treatment. Allosteric modulation of mGluR5 had no significant effect on choice impulsivity, nor did it modulate general task performance. Conclusions: These findings demonstrate that mGluR5 allosteric modulation selectively dissociates motor and choice impulsivity. We further show that mGluR5 PAMs may have therapeutic utility in selectively targeting specific aspects of impulsivity and executive dysfunction.This research was supported by a Medical Research Council (MRC) grant to JWD (G0701500) and a grant from Boehringer Ingelheim Pharma GmbH & Co. KG. This work was carried out in the Behavioural and Clinical Neuroscience Institute (BCNI) at Cambridge University with joint support from the MRC (G1000183) and Wellcome Trust (093875/Z/10/Z) and at Boehringer Ingelheim Pharma GmbH & Co. KG, Germany. We thank David Theobald, Johannes Freudenreich, Peter Schorn, Alfie Wearn and Benjamin Jaehnke for technical support and Gert Kramer, Dr. Holger Rosenbrock and Dr. Cornelia Dorner-Ciossek for helpful scientific discussions. The authors declare that the experiments performed in this manuscript followed the principles of laboratory animal care and are in compliance with the current laws of the UK and Germany.This is the author accepted manuscript. The final version is available from Springer via http://dx.doi.org/10.1007/s00213-015-3984-

Dissociable effects of mGluR5 allosteric modulation on distinct forms of impulsivity in rats: interaction with NMDA receptor antagonism.

RATIONALE: Impaired N-methyl-D-aspartate (NMDA) receptor signalling underlies several psychiatric disorders that express high levels of impulsivity. Although synergistic interactions exist between NMDA receptors and metabotropic glutamate receptor 5 (mGluR5), the significance of this interaction for impulsivity is unknown. OBJECTIVE: This study aims to investigate the effects of negative and positive allosteric mGluR5 modulation (NAM/PAM) on trait impulsivity and impulsivity evoked by NMDA receptor antagonism in rats. METHODS: Motor and choice impulsivity were assessed using the five-choice serial reaction time task (5-CSRTT) and delayed-discounting task (DDT), respectively. The effects of RO4917523 and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) (NAMs) and ADX47273 (PAM) were investigated in non-impulsive rats and in trait high- and low-impulsive rats. The effects of these compounds on impulsivity induced by NMDA receptor antagonism (MK801) in the 5-CSRTT were also investigated. RESULTS: RO4917523 (0.1-1 mg/kg) decreased premature responding and increased omissions but had no effect on locomotor activity up to 0.1 mg/kg. MTEP significantly increased omissions, decreased accuracy and slowed responding but had no effect on premature responding. ADX47273 decreased premature responding at doses that had no effect on locomotor activity. MK801 increased premature responding and impaired attentional accuracy; these deficits were dose dependently rescued by ADX47273 pre-treatment. Allosteric modulation of mGluR5 had no significant effect on choice impulsivity, nor did it modulate general task performance. CONCLUSIONS: These findings demonstrate that mGluR5 allosteric modulation selectively dissociates motor and choice impulsivity. We further show that mGluR5 PAMs may have therapeutic utility in selectively targeting specific aspects of impulsivity and executive dysfunction.This research was supported by a Medical Research Council (MRC) grant to JWD (G0701500) and a grant from Boehringer Ingelheim Pharma GmbH & Co. KG. This work was carried out in the Behavioural and Clinical Neuroscience Institute (BCNI) at Cambridge University with joint support from the MRC (G1000183) and Wellcome Trust (093875/Z/10/Z) and at Boehringer Ingelheim Pharma GmbH & Co. KG, Germany. We thank David Theobald, Johannes Freudenreich, Peter Schorn, Alfie Wearn and Benjamin Jaehnke for technical support and Gert Kramer, Dr. Holger Rosenbrock and Dr. Cornelia Dorner-Ciossek for helpful scientific discussions. The authors declare that the experiments performed in this manuscript followed the principles of laboratory animal care and are in compliance with the current laws of the UK and Germany.This is the author accepted manuscript. The final version is available from Springer via http://dx.doi.org/10.1007/s00213-015-3984-

Selective and interactive effects of D2 receptor antagonism and positive allosteric mGluR4 modulation on waiting impulsivity

BACKGROUND: Metabotropic glutamate receptor 4 (mGluR4) and dopamine D2 receptors are specifically expressed within the indirect pathway neurons of the striato-pallidal-subthalamic pathway. This unique expression profile suggests that mGluR4 and D2 receptors may play a cooperative role in the regulation and inhibitory control of behaviour. We investigated this possibility by testing the effects of a functionally-characterised positive allosteric mGluR4 modulator, 4-((E)-styryl)-pyrimidin-2-ylamine (Cpd11), both alone and in combination with the D2 receptor antagonist eticlopride, on two distinct forms of impulsivity. METHODS: Rats were trained on the five-choice serial reaction time task (5-CSRTT) of sustained visual attention and segregated according to low, mid, and high levels of motor impulsivity (LI, MI and HI, respectively), with unscreened rats used as an additional control group. A separate group of rats was trained on a delay discounting task (DDT) to assess choice impulsivity. RESULTS: Systemic administration of Cpd11 dose-dependently increased motor impulsivity and impaired attentional accuracy on the 5-CSRTT in all groups tested. Eticlopride selectively attenuated the increase in impulsivity induced by Cpd11, but not the accompanying attentional impairment, at doses that had no significant effect on behavioural performance when administered alone. Cpd11 also decreased choice impulsivity on the DDT (i.e. increased preference for the large, delayed reward) and decreased locomotor activity. CONCLUSIONS: These findings demonstrate that mGluR4s, in conjunction with D2 receptors, affect motor- and choice-based measures of impulsivity, and therefore may be novel targets to modulate impulsive behaviour associated with a number of neuropsychiatric syndromes.This research was supported by a Medical Research Council (MRC) grant to JWD (G0701500) and a grant from Boehringer Ingelheim Pharma GmbH & Co. KG. This work was carried out at Boehringer Ingelheim Pharma GmbH & Co. KG in Germany and the Behavioural and Clinical Neuroscience Institute (BCNI) at Cambridge University. The BCNI is jointly supported by the MRC (G1000183) and Wellcome Trust (093875/Z/10/Z)

Activation of the Innate Immune Checkpoint CLEC5A on Myeloid Cells in the Absence of Danger Signals Modulates Macrophages' Function but Does Not Trigger the Adaptive T Cell Immune Response

C-Type lectin receptor 5A (CLEC5A) is a spleen tyrosine kinase- (Syk-) coupled pattern recognition receptor expressed on myeloid cells and involved in the innate immune response to viral and bacterial infections. Activation of the CLEC5A receptor with pathogen-derived antigens leads to a secretion of proinflammatory mediators such as TNF-α and IL-6 that may provoke a systemic cytokine storm, and CLEC5A gene polymorphisms are associated with the severity of DV infection. In addition, the CLEC5A receptor was mentioned in the context of noninfectious disorders like chronic obstructive pulmonary disease (COPD) or arthritis. Altogether, CLEC5A may be considered as an innate immune checkpoint capable to amplify proinflammatory signals, and this way contributes to infection or to aseptic inflammation. In this study, we determined CLEC5A receptor expression on different macrophage subsets (in vitro and ex vivo) and the functional consequences of its activation in aseptic conditions. The CLEC5A surface expression appeared the highest on proinflammatory M1 macrophages while intermediate on tumor-associated phenotypes (M2c or TAM). In contrast, the CLEC5A expression on ex vivo-derived alveolar macrophages from healthy donors or macrophages from ovarian cancer patients was hardly detectable. Targeting CLEC5A on noninflammatory macrophages with an agonistic α-CLEC5A antibody triggered a release of proinflammatory cytokines, resembling a response to dengue virus, and led to phenotypic changes in myeloid cells that may suggest their reprogramming towards a proinflammatory phenotype, e.g., upregulation of CD80 and downregulation of CD163. Interestingly, the CLEC5A agonist upregulated immune-regulatory molecules like CD206, PD-L1, and cytokines like IL-10, macrophage-derived chemokine (MDC/CCL22), and thymus and activation chemokine (TARC/CCL17) which are associated with an anti-inflammatory or a protumorigenic macrophage phenotype. In the absence of concomitant pathogenic or endogenous danger signals, the CLEC5A receptor activation did not amplify an autologous T cell response, which may represent a protective innate mechanism to avoid an undesirable autoimmune adaptive response

Cellular localization of Y-box binding protein 1 in brain tissue of rats, macaques, and humans

Background: The Y-box binding protein 1 (YB-1) is considered to be one of the key regulators of transcription and translation. However, so far only limited knowledge exists regarding its cellular distribution in the adult brain. Results: Analysis of YB-1 immunolabelling as well as double-labelling with the neuronal marker NeuN in rat brain tissue revealed a predominant neuronal expression in the dentate gyrus, the cornu ammonis pyramidal cell layer, layer III of the piriform cortex as well as throughout all layers of the parahippocampal cortex. In the hilus of the hippocampus single neurons expressed YB-1. The neuronal expression pattern was comparable in the hippocampus and parahippocampal cortex of adult macaques and humans. Double-labelling of YB-1 with the endothelial cell marker Glut-1, the multidrug transporter P-glycoprotein, and the astrocytic marker GFAP did not indicate a co-localization. Following status epilepticus in rats, no induction of YB-1 occurred in brain capillary endothelial cells and neurons. Conclusion: In conclusion, our study demonstrates that YB-1 is predominantly expressed in neurons in the adult brain of rats, macaques and humans. Lack of a co-localization with Glut-1 and P-glycoprotein argues against a direct role of YB-1 in the regulation of blood-brain barrier P-glycoprotein

Seizure-induced up-regulation of P-glycoprotein at the blood-brain barrier through glutamate and cyclooxygenase-2 signaling.

ABSTRACT Increased expression of drug efflux transporters at the bloodbrain barrier accompanies epileptic seizures and complicates therapy with antiepileptic drugs. This study is concerned with identifying mechanistic links that connect seizure activity to increased P-glycoprotein expression at the blood-brain barrier. In this regard, we tested the hypothesis that seizures increase brain extracellular glutamate, which signals through an N-methyl-D-aspartate (NMDA) receptor and cyclooxygenase-2 (COX-2) in brain capillaries to increase blood-brain barrier P-glycoprotein expression. Consistent with this hypothesis, exposing isolated rat or mouse brain capillaries to glutamate for 15 to 30 min increased P-glycoprotein expression and transport activity hours later. These increases were blocked by 5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate) (MK-801), an NMDA receptor antagonist, and by celecoxib, a selective COX-2 inhibitor; no such glutamate-induced increases were seen in brain capillaries from COX-2-null mice. In rats, intracerebral microinjection of glutamate caused locally increased P-glycoprotein expression in brain capillaries. Moreover, using a pilocarpine status epilepticus rat model, we observed seizure-induced increases in capillary P-glycoprotein expression that were attenuated by administration of indomethacin, a COX inhibitor. Our findings suggest that brain uptake of some antiepileptic drugs can be enhanced through COX-2 inhibition. Moreover, they provide insight into one mechanism that underlies drug resistance in epilepsy and possibly other central nervous system disorders. Up to 40% of epileptic patients respond poorly if at all to conventional pharmacotherapy, and impaired drug uptake into the brain is considered to be one important contributor to therapeutic failure The present study is concerned with mechanistic links that connect seizure activity to increased P-glycoprotein expression. Our goals are to identify therapeutic targets that can be manipulated to prevent seizure-induced transporter overexpression and to improve pharmacotherapy with antiepileptic drugs. The combined in vitro/in vivo experiments are focuse

Seizure-induced up-regulation of P-glycoprotein at the blood-brain barrier through glutamate and cyclooxygenase-2 signaling.

ABSTRACT Increased expression of drug efflux transporters at the bloodbrain barrier accompanies epileptic seizures and complicates therapy with antiepileptic drugs. This study is concerned with identifying mechanistic links that connect seizure activity to increased P-glycoprotein expression at the blood-brain barrier. In this regard, we tested the hypothesis that seizures increase brain extracellular glutamate, which signals through an N-methyl-D-aspartate (NMDA) receptor and cyclooxygenase-2 (COX-2) in brain capillaries to increase blood-brain barrier P-glycoprotein expression. Consistent with this hypothesis, exposing isolated rat or mouse brain capillaries to glutamate for 15 to 30 min increased P-glycoprotein expression and transport activity hours later. These increases were blocked by 5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate) (MK-801), an NMDA receptor antagonist, and by celecoxib, a selective COX-2 inhibitor; no such glutamate-induced increases were seen in brain capillaries from COX-2-null mice. In rats, intracerebral microinjection of glutamate caused locally increased P-glycoprotein expression in brain capillaries. Moreover, using a pilocarpine status epilepticus rat model, we observed seizure-induced increases in capillary P-glycoprotein expression that were attenuated by administration of indomethacin, a COX inhibitor. Our findings suggest that brain uptake of some antiepileptic drugs can be enhanced through COX-2 inhibition. Moreover, they provide insight into one mechanism that underlies drug resistance in epilepsy and possibly other central nervous system disorders. Up to 40% of epileptic patients respond poorly if at all to conventional pharmacotherapy, and impaired drug uptake into the brain is considered to be one important contributor to therapeutic failure The present study is concerned with mechanistic links that connect seizure activity to increased P-glycoprotein expression. Our goals are to identify therapeutic targets that can be manipulated to prevent seizure-induced transporter overexpression and to improve pharmacotherapy with antiepileptic drugs. The combined in vitro/in vivo experiments are focuse