Interferon-gamma-induced changes in synaptic activity and AMPA receptor clustering in hippocampal cultures

Extended release of interferon-gamma (IFN-gamma) in the nervous system during immunological and infectious conditions may trigger demyelinating disorders and cause disturbances in brain function. The aim of this study was to examine the effects of IFN-gamma on neuronal function in rat hippocampal cell cultures by using whole cell patch clamp analysis together with quantitative immunocytochemistry. Acute application of IFN-gamma to differentiated neurons in culture caused no immediate neurophysiological responses, but recordings after 48 h of incubation displayed an increase in frequency of AMPA receptor (AMPAR)-mediated spontaneous excitatory postsynaptic currents (EPSCs). Quantitative immunocytochemistry for the AMPAR subunit GluR1 showed no alteration in receptor clustering at this time point. However, prolonged treatment with IFN-gamma for 2 weeks resulted in a significant reduction in AMPAR clustering on dendrites but no marked differences in EPSC frequency between treated neurons and controls could be observed. On the other hand, treatment of hippocampal neurons for 4 weeks, instituted at an immature stage (1 day in culture), caused a significant reduction in spontaneous EPSC frequency. These neurons developed with no overt alterations in dendritic arborization or in the appearance of dendritic spines as visualized by alpha-actinin immunocytochemistry. Nonetheless, there was a marked reduction in AMPAR clustering on dendrites. These observations show that a key immunomodulatory molecule, IFN-gamma, can cause long-term modifications of synaptic activity and perturb glutamate receptor clustering.     

Umsatz von Trichloräthylen im Organismus

Zusammenfassung Bei Versuchen mit Hunden und Ratten sind Trichloräthylen, Trichloräthylalkohol, Trichloräthylaldehyd (Chloral) und Trichloressigsäure durch subcutane Injektion zugeführt und die ausgeschiedene Menge von Trichloressigsäure im Urin untersucht worden. Von den 3 zuerst genannten Stoffen werden nur wenige Prozent mit dem Urin in Form von Trichloressigsäure ausgeschieden. Nach Zufuhr von Natriumtrichloracetat werden 1/3–1/2 als Trichloressigsäure ausgeschieden. Zufuhr von Disulfiram per os bei Ratten hemmt die Trichloressigsäureausscheidung im Urin sehr stark nach Zufuhr von Trichloräthylen und Trichloräthylalkohol. Eine deutliche, aber nicht ganz so ausgesprochene Herabsetzung wird nach der Zufuhr von Trichloräthylaldehyd (Chloral) festgestellt. Die Ausscheidung nach Zufuhr von Natriumtrichloracetat wird nicht durch Disulfiram beeinflußt. Die erhaltenen Resultate sind wahrscheinlich darauf zurückzuführen, daß Disulfiram die Oxydation des Trichloräthylaldehyds hemmt. Der Reaktionsverlauf für den Umsatz von Trichloräthylen im Organismus dürfte daher wahrscheinlich als eine Oxydation via Trichloräthylalkohol und Trichloräthylaldehyd zu Trichloressigsäure anzusehen sein.Herrn Professor Heinrich Zangger zum 80. Geburtstag gewidmet.     

Memantine increases brain production of kynurenic acid via protein kinase A-dependent mechanism

We describe a novel aspect of action of memantine ex vivo, in the brain cortical slices and in vitro, in mixed glial cultures. The drug potently increased the production of kynurenic acid, an endogenous tryptophan metabolite blocking N-methyl-D-aspartate (NMDA) and nicotinic alpha7 receptors. In cortical slices memantine, an open-channel NMDA blocker (100-150 microM), but not the competitive NMDA receptor antagonist, LY235959 increased the production of kynurenic acid. Neither SCH23390, D1 receptor antagonist (50 microM) nor raclopride, D2 receptor antagonist (10 microM) changed the memantine-induced effects. Propranolol (100 microM) has partially reduced its action. Selective cAMP-dependent protein kinase (PKA) inhibitor, KT5720 (1 microM), but not selective protein kinase C (PKC) inhibitor, NPC15437 (30 microM) totally reversed the action of memantine. In mixed glial cultures, 2-24 h incubation with memantine (2-50 microM) enhanced the production of kynurenic acid. Memantine (up to 0.5 mM) has not affected the activity of kynurenic acid biosynthetic enzymes. The obtained data suggest that memantine enhances the production of kynurenic acid in PKA-mediated way. This effect may partially contribute to the therapeutic actions of memantine and be of a potential clinical importance.     

Interferon-γ-induced changes in synaptic activity and AMPA receptor clustering in hippocampal cultures

Extended release of interferon-γ (IFN-γ) in the nervous system during immunological and infectious conditions may trigger demyelinating disorders and cause disturbances in brain function. The aim of this study was to examine the effects of IFN-γ on neuronal function in rat hippocampal cell cultures by using whole cell patch clamp analysis together with quantitative immunocytochemistry. Acute application of IFN-γ to differentiated neurons in culture caused no immediate neurophysiological responses, but recordings after 48 h of incubation displayed an increase in frequency of AMPA receptor (AMPAR)-mediated spontaneous excitatory postsynaptic currents (EPSCs). Quantitative immunocytochemistry for the AMPAR subunit GluR1 showed no alteration in receptor clustering at this time point. However, prolonged treatment with IFN-γ for 2 weeks resulted in a significant reduction in AMPAR clustering on dendrites but no marked differences in EPSC frequency between treated neurons and controls could be observed. On the other hand, treatment of hippocampal neurons for 4 weeks, instituted at an immature stage (1 day in culture), caused a significant reduction in spontaneous EPSC frequency. These neurons developed with no overt alterations in dendritic arborization or in the appearance of dendritic spines as visualized by α-actinin immunocytochemistry. Nonetheless, there was a marked reduction in AMPAR clustering on dendrites. These observations show that a key immunomodulatory molecule, IFN-γ, can cause long-term modifications of synaptic activity and perturb glutamate receptor clustering.     

Changes in calcium currents and GABAergic spontaneous activity in cultured rat hippocampal neurons after a neurotropic influenza A virus infection

In order to study mechanisms by which a neurotropic strain of influenza A virus (A/WSN/33) may affect neuronal function or cause nerve cell death, hippocampal cultures from embryonic rats were infected with this virus. Approximately 70% of the neurons in the infected cultures became immunopositive for viral antigens and showed reduced voltage-dependent Ca(2+) currents in whole-cell patch clamp recordings, but no changes in other membrane properties or in cytosolic Ca(2+) concentration were seen. These immunopositive neurons underwent apoptosis 3-4 days after infection. Ca(2+) channel inhibitors had no significant effect on neuronal survival. The immunonegative population of neurons survived, but displayed increased frequency of miniature inhibitory postsynaptic currents of gamma-amino-butyric acid origin compared with controls. The frequency of alpha-amino-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA) receptor-mediated miniature excitatory postsynaptic currents was not altered. Viral nucleoproteins, overexpressed using the Semliki Forest virus system, were localized to the dendritic spines as shown by double immunolabeling with actinin, but did not by themselves cause neuronal death or changes in synaptic transmission as measured by AMPA-mediated excitatory postsynaptic currents. Our results show that an influenza A virus infection can cause selective neurophysiological changes in hippocampal neurons and that these can persist even after the viral antigens have been cleared.     

β-adrenergic enhancement of brain kynurenic acid production mediated via cAMP-related protein kinase A signaling

The central levels of endogenous tryptophan metabolite kynurenic acid (KYNA), an antagonist of N-methyl-d-aspartate (NMDA) and α7-nicotinic receptors, affect glutamatergic and dopaminergic neurotransmission. Here, we demonstrate that selective agonists of β₁-receptors (xamoterol and denopamine), β₂-receptors (formoterol and albuterol), α- and β-receptors (epinephrine), 8pCPT-cAMP and 8-Br-cAMP (analogues of cAMP) increase the production of KYNA in rat brain cortical slices and in mixed glial cultures. Neither betaxolol, β₁-adrenergic antagonist, nor timolol, a non-selective β_1,2-adrenergic antagonist has influenced synthesis of KYNA in both paradigms. In contrast, KT5720, a selective inhibitor of protein kinase A (PKA), strongly reduced KYNA formation in cortical slices (2–10 µM) and in glial cultures (100 nM). β-adrenergic antagonists and KT5720 prevented the β-adrenoceptor agonists-induced increases of KYNA synthesis. In vivo, β-adrenergic agonist clenbuterol (0.1–1.0 mg/kg) increased the cortical endogenous level of KYNA; the effect was blocked with propranolol (10 mg/kg). β-adrenoceptors agonists, cAMP analogues and KT5720 did not affect directly the activity of KAT I or KAT II measured in partially purified cortical homogenate. In contrast, the exposure of intact cultured glial cells to pCPT-cAMP, 8-Br-cAMP and formoterol has lead to an enhanced action of KATs. These findings demonstrate that β-adrenoceptor-mediated enhancement of KYNA production is a cAMP- and PKA-dependent event. PKA activity appears to be an essential signal affecting KYNA formation. Described here novel mechanism regulating KYNA availability may be of a potential importance, considering that various stimuli, among them clinically used drugs, activate cAMP/PKA pathway, and thus could counteract the central deficits of KYNA.     

New insight into the antidepressants action: modulation of kynurenine pathway by increasing the kynurenic acid/3-hydroxykynurenine ratio

Altered function of kynurenine pathway has emerged recently as one of the factors contributing to the pathogenesis of depression. Neuroprotective kynurenic acid (KYNA) and neurotoxic 3-hydroxykynurenine (3-HK) are two immediate metabolites of l-kynurenine. Here, we aimed to assess the hypothesis that antidepressant drugs that may change brain KYNA/3-HK ratio. In primary astroglial cultures, fluoxetine, citalopram, amitriptyline and imipramine (1–10 μM) increased de novo production of KYNA and diminished 3-HK synthesis (24 and 48, but not 2 h). RT-PCR studies revealed that Kat1, Kat2 and kynurenine-3-monooxygenase (Kmo) gene expressions were not altered after 2 h. At 24 h, the expression of Kat1 and Kat2 genes was enhanced by all studied drugs, whereas Kmo expression was diminished by citalopram, fluoxetine and amitriptyline, but not imipramine. After 48 h, the expression of Kat1 and Kat2 was further up-regulated, and Kmo expression was down-regulated by all antidepressants. The ratio KYNA/3-HK was increased by fluoxetine, citalopram, amitriptyline and imipramine in a time-dependent manner—the effect was not observed after 2 h, modest after 24 h and robust after 48 h incubation time. Our findings indicate that the action of antidepressants may involve re-establishing of the beneficial ratio between KYNA and 3-HK. Shift in the kynurenine pathway, observed after prolonged exposure to antidepressant drugs, may partly explain their delayed therapeutic effectiveness.     

Activation of kynurenine pathway in ex vivo fibroblasts from patients with bipolar disorder or schizophrenia: Cytokine challenge increases production of 3-hydroxykynurenine

Accumulating data suggest a causative link between immune stimulation, disturbed metabolism of tryptophan, and pathogenesis of bipolar disorder and schizophrenia. The goal of this study was to examine the production of kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK) and the expression of kynurenine pathway enzymes involved in their synthesis and metabolism in cultured skin fibroblasts obtained from patients with bipolar disorder, schizophrenia or from healthy control individuals. The assessment was performed under basal conditions or following treatment with interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, or their combinations, in cells exposed to exogenous kynurenine. In both groups of patients, the baseline production of KYNA and 3-HK was increased, as compared to control subjects. Case-treatment analyses revealed significant interactions between bipolar case status and IL-1β, IL-6, IFN-γ + TNF-α, or IFN-γ + IL-1β, as well as between schizophrenia case status and IL-1β, IFN-γ + TNF-α, or IFN-γ + IL-1β, in terms of higher 3-HK. Noteworthy, no case-treatment interactions in terms of KYNA production were found. Observed changes did not appear to correlate with the expression of genes encoding kynurenine aminotransferases (KATs), kynureninase (KYNU) or kynurenine-3-monooxygenase (KMO). The single nucleotide polymorphisms (SNPs), rs1053230 and rs2275163, in KMO influenced KYNA levels yet did not explain the case-treatment discrepancies. In conclusion, our present findings indicate the utility of skin-derived fibroblasts for kynurenines research and support the concept of kynurenine pathway alterations in bipolar disorder and schizophrenia. The increase in ratio between neurotoxic 3-HK and neuroinhibitory/neuroprotective KYNA following exposure to cytokines may account for altered neurogenesis and structural abnormalities characteristic for both diseases.     

Prolonged therapy with antidepressants increases hippocampal level of kynurenic acid and expression of Kat1 and Kat2 genes

Background

Accumulating data suggest an important role of disturbed kynurenine pathway and altered glutamatergic transmission in the pathogenesis of depression. In here, we focused on detailed analyses of kynurenic acid (KYNA) status in vivo following single and 14-day administration of selected tricyclic antidepressant drugs (TCAs) and serotonin selective reuptake inhibitors (SSRIs) in rats.

Inhibition of hippocampal synaptic transmission by impairment of Ral function

Large clostridial cytotoxins and protein overexpression were used to probe for involvement of Ras-related GTPases (guanosine triphosphate) in synaptic transmission in cultured rat hippocampal neurons. The toxins TcdA-10463 (inactivates Rho, Rac, Cdc42, Rap) and TcsL-1522 (inactivates Ral, Rac, Ras, R-Ras, Rap) both inhibited autaptic responses. In a proportion of the neurons (25%, TcdA-10463; 54%, TcsL-1522), the inhibition was associated with a shift from activity-dependent depression to facilitation, indicating that the synaptic release probability was reduced. Overexpression of a dominant negative Ral mutant, Ral A28N, caused a strong inhibition of autaptic responses, which was associated with a shift to facilitation in a majority (80%) of the neurons. These results indicate that Ral, along with at least one other non-Rab GTPase, participates in presynaptic regulation in hippocampal neurons.