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Unconjugated bilirubin exposure impairs hippocampal long-term synaptic plasticity.

Chang FY, Lee CC, Huang CC, Hsu KS - PLoS ONE (2009)

Bottom Line: UCB treatment produced a significant decrease in the levels of NR1, NR2A and NR2B subunits of N-methyl-D-aspartate (NMDA) receptors through a calpain-mediated proteolytic cleavage mechanism.Pretreatment of the hippocampal slice cultures with NMDA receptor antagonist or calpain inhibitors effectively prevented the UCB-induced impairment of LTP and LTD.Our results indicate that the proteolytic cleavage of NMDA receptor subunits by calpain may play a critical role in mediating the UCB-induced impairment of long-term synaptic plasticity in the hippocampus.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.

ABSTRACT

Background: Jaundice is one of the most common problems encountered in newborn infants, due to immaturity of hepatic conjugation and transport processes for bilirubin. Although the majority of neonatal jaundice is benign, some neonates with severe hyperbilirubinemia develop bilirubin encephalopathy or kernicterus. Accumulation of unconjugated bilirubin (UCB) in selected brain regions may result in temporary or permanent impairments of auditory, motor, or cognitive function; however, the molecular mechanisms by which UCB elicits such neurotoxicity are still poorly understood. The present study is undertaken to investigate whether prolonged exposure of rat organotypic hippocampal slice cultures to UCB alters the induction of long-term synaptic plasticity.

Methodology/principal findings: Using electrophysiological recording techniques, we find that exposure of hippocampal slice cultures to clinically relevant concentrations of UCB for 24 or 48 h results in an impairment of CA1 long-term potentiation (LTP) and long-term depression (LTD) induction in a time- and concentration-dependent manner. Hippocampal slice cultures stimulated with UCB show no changes in the secretion profiles of the pro-inflammatory cytokines, interleukin-1beta and tumor necrosis factor-alpha, or the propidium ioide uptake. UCB treatment produced a significant decrease in the levels of NR1, NR2A and NR2B subunits of N-methyl-D-aspartate (NMDA) receptors through a calpain-mediated proteolytic cleavage mechanism. Pretreatment of the hippocampal slice cultures with NMDA receptor antagonist or calpain inhibitors effectively prevented the UCB-induced impairment of LTP and LTD.

Conclusion/significance: Our results indicate that the proteolytic cleavage of NMDA receptor subunits by calpain may play a critical role in mediating the UCB-induced impairment of long-term synaptic plasticity in the hippocampus. These observations provide new insights into the molecular mechanisms underlying UCB-induced impairment of hippocampal synaptic plasticity which, in turn, might provide opportunities for the development of novel therapeutic strategies that targets these pathways for treatment.

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Related in: MedlinePlus

Effect of prolonged UCB exposure on NMDA/AMPA ratio of excitatory postsynaptic currents (EPSCs) and AMPA receptor-mediated miniature EPSCs (mEPSCs).(A) NMDA/AMPA ratio of EPSC (at +50 mV) was determined by subtracting the averaged traces obtained in 50 µM D-APV from those collected in its absence and was found to be significantly lower in slices treated with 10 µM UCB for 24 or 48 h compared with control slices. The intensity of each stimulation was adjusted to evoke the same peak amplitude of EPSCs (≈250 pA) in each slice culture. Representative traces show EPSCs before (1) and after application of D-APV (50 µM) (2) in control and 10 µM UCB-treated slices. The NMDA receptor-mediated component (3) was derived by subtracting the AMPA receptor-mediated component (2) from the compound EPSC (1). (B) Representative traces and input-output curves of AMPA receptor-mediated EPSC (EPSCAMPA; at −70 mV in the presence of 20 µM bicuculline methiodide and 50 µM D-APV) versus stimulus intensity (µA) at the Schaffer collateral-CA1 synapses of hippocampal slice cultures in the absence (control) or presence of 10 µM UCB for 48 h. (C) Representative traces and input-output curves of NMDA receptor-mediated EPSC (EPSCNMDA; at −60 mV in the presence of 20 µM bicuculline methiodide and 20 µM CNQX) versus stimulus intensity (µA) at the Schaffer collateral-CA1 synapses of hippocampal slice cultures in the absence (control) or presence of 10 µM UCB for 48 h. (D) Representative voltage-clamp recordings of AMPA receptor-mediated mEPSCs (at −70 mV in the presence of 20 µM bicuculline methiodide, 50 µM D-APV, and 1 µM tetrodotoxin) from control slices (left) or slices treated with 10 µM UCB for 24 h (middle) or 48 h (right). The bar graphs show mean±SEM of the effects of UCB on the average frequency and amplitude of mEPSCs. Number of experiments is indicated in the parenthesis. *p<0.05 as compared with the control group by one-way ANOVA (Tukey-Kramer test).
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pone-0005876-g003: Effect of prolonged UCB exposure on NMDA/AMPA ratio of excitatory postsynaptic currents (EPSCs) and AMPA receptor-mediated miniature EPSCs (mEPSCs).(A) NMDA/AMPA ratio of EPSC (at +50 mV) was determined by subtracting the averaged traces obtained in 50 µM D-APV from those collected in its absence and was found to be significantly lower in slices treated with 10 µM UCB for 24 or 48 h compared with control slices. The intensity of each stimulation was adjusted to evoke the same peak amplitude of EPSCs (≈250 pA) in each slice culture. Representative traces show EPSCs before (1) and after application of D-APV (50 µM) (2) in control and 10 µM UCB-treated slices. The NMDA receptor-mediated component (3) was derived by subtracting the AMPA receptor-mediated component (2) from the compound EPSC (1). (B) Representative traces and input-output curves of AMPA receptor-mediated EPSC (EPSCAMPA; at −70 mV in the presence of 20 µM bicuculline methiodide and 50 µM D-APV) versus stimulus intensity (µA) at the Schaffer collateral-CA1 synapses of hippocampal slice cultures in the absence (control) or presence of 10 µM UCB for 48 h. (C) Representative traces and input-output curves of NMDA receptor-mediated EPSC (EPSCNMDA; at −60 mV in the presence of 20 µM bicuculline methiodide and 20 µM CNQX) versus stimulus intensity (µA) at the Schaffer collateral-CA1 synapses of hippocampal slice cultures in the absence (control) or presence of 10 µM UCB for 48 h. (D) Representative voltage-clamp recordings of AMPA receptor-mediated mEPSCs (at −70 mV in the presence of 20 µM bicuculline methiodide, 50 µM D-APV, and 1 µM tetrodotoxin) from control slices (left) or slices treated with 10 µM UCB for 24 h (middle) or 48 h (right). The bar graphs show mean±SEM of the effects of UCB on the average frequency and amplitude of mEPSCs. Number of experiments is indicated in the parenthesis. *p<0.05 as compared with the control group by one-way ANOVA (Tukey-Kramer test).

Mentions: Having established that prolonged UCB exposure impairs the induction of both LTP and LTD, we next asked whether these effects are mediated by an alteration of NMDA receptor function, which is critical determinant for the induction of LTP and LTD in the hippocampal CA1 region [16], [18]. To address this issue, we compared the ratio of NMDA to AMPA receptor components of evoked excitatory postsynaptic currents (EPSCs) in CA1 pyramidal neurons of control and UCB-treated slices. We recorded EPSCs when the cell was clamped at +50 mV. In this condition, both NMDA and AMPA receptors are activated by synaptically released glutamate, and their respective contribution to the EPSCs was determined by pharmacological application of NMDA receptor antagonist D-APV (50 µM). Exposure of slices to 10 µM UCB for 24 h (0.71±0.04, n = 6; p<0.05) or 48 h (0.49±0.08, n = 6; p<0.05) underwent a significant reduction in the NMDA/AMPA ratio when compared with control slices (0.87±0.05, n = 7) (Figure 3A).


Unconjugated bilirubin exposure impairs hippocampal long-term synaptic plasticity.

Chang FY, Lee CC, Huang CC, Hsu KS - PLoS ONE (2009)

Effect of prolonged UCB exposure on NMDA/AMPA ratio of excitatory postsynaptic currents (EPSCs) and AMPA receptor-mediated miniature EPSCs (mEPSCs).(A) NMDA/AMPA ratio of EPSC (at +50 mV) was determined by subtracting the averaged traces obtained in 50 µM D-APV from those collected in its absence and was found to be significantly lower in slices treated with 10 µM UCB for 24 or 48 h compared with control slices. The intensity of each stimulation was adjusted to evoke the same peak amplitude of EPSCs (≈250 pA) in each slice culture. Representative traces show EPSCs before (1) and after application of D-APV (50 µM) (2) in control and 10 µM UCB-treated slices. The NMDA receptor-mediated component (3) was derived by subtracting the AMPA receptor-mediated component (2) from the compound EPSC (1). (B) Representative traces and input-output curves of AMPA receptor-mediated EPSC (EPSCAMPA; at −70 mV in the presence of 20 µM bicuculline methiodide and 50 µM D-APV) versus stimulus intensity (µA) at the Schaffer collateral-CA1 synapses of hippocampal slice cultures in the absence (control) or presence of 10 µM UCB for 48 h. (C) Representative traces and input-output curves of NMDA receptor-mediated EPSC (EPSCNMDA; at −60 mV in the presence of 20 µM bicuculline methiodide and 20 µM CNQX) versus stimulus intensity (µA) at the Schaffer collateral-CA1 synapses of hippocampal slice cultures in the absence (control) or presence of 10 µM UCB for 48 h. (D) Representative voltage-clamp recordings of AMPA receptor-mediated mEPSCs (at −70 mV in the presence of 20 µM bicuculline methiodide, 50 µM D-APV, and 1 µM tetrodotoxin) from control slices (left) or slices treated with 10 µM UCB for 24 h (middle) or 48 h (right). The bar graphs show mean±SEM of the effects of UCB on the average frequency and amplitude of mEPSCs. Number of experiments is indicated in the parenthesis. *p<0.05 as compared with the control group by one-way ANOVA (Tukey-Kramer test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2690688&req=5

pone-0005876-g003: Effect of prolonged UCB exposure on NMDA/AMPA ratio of excitatory postsynaptic currents (EPSCs) and AMPA receptor-mediated miniature EPSCs (mEPSCs).(A) NMDA/AMPA ratio of EPSC (at +50 mV) was determined by subtracting the averaged traces obtained in 50 µM D-APV from those collected in its absence and was found to be significantly lower in slices treated with 10 µM UCB for 24 or 48 h compared with control slices. The intensity of each stimulation was adjusted to evoke the same peak amplitude of EPSCs (≈250 pA) in each slice culture. Representative traces show EPSCs before (1) and after application of D-APV (50 µM) (2) in control and 10 µM UCB-treated slices. The NMDA receptor-mediated component (3) was derived by subtracting the AMPA receptor-mediated component (2) from the compound EPSC (1). (B) Representative traces and input-output curves of AMPA receptor-mediated EPSC (EPSCAMPA; at −70 mV in the presence of 20 µM bicuculline methiodide and 50 µM D-APV) versus stimulus intensity (µA) at the Schaffer collateral-CA1 synapses of hippocampal slice cultures in the absence (control) or presence of 10 µM UCB for 48 h. (C) Representative traces and input-output curves of NMDA receptor-mediated EPSC (EPSCNMDA; at −60 mV in the presence of 20 µM bicuculline methiodide and 20 µM CNQX) versus stimulus intensity (µA) at the Schaffer collateral-CA1 synapses of hippocampal slice cultures in the absence (control) or presence of 10 µM UCB for 48 h. (D) Representative voltage-clamp recordings of AMPA receptor-mediated mEPSCs (at −70 mV in the presence of 20 µM bicuculline methiodide, 50 µM D-APV, and 1 µM tetrodotoxin) from control slices (left) or slices treated with 10 µM UCB for 24 h (middle) or 48 h (right). The bar graphs show mean±SEM of the effects of UCB on the average frequency and amplitude of mEPSCs. Number of experiments is indicated in the parenthesis. *p<0.05 as compared with the control group by one-way ANOVA (Tukey-Kramer test).
Mentions: Having established that prolonged UCB exposure impairs the induction of both LTP and LTD, we next asked whether these effects are mediated by an alteration of NMDA receptor function, which is critical determinant for the induction of LTP and LTD in the hippocampal CA1 region [16], [18]. To address this issue, we compared the ratio of NMDA to AMPA receptor components of evoked excitatory postsynaptic currents (EPSCs) in CA1 pyramidal neurons of control and UCB-treated slices. We recorded EPSCs when the cell was clamped at +50 mV. In this condition, both NMDA and AMPA receptors are activated by synaptically released glutamate, and their respective contribution to the EPSCs was determined by pharmacological application of NMDA receptor antagonist D-APV (50 µM). Exposure of slices to 10 µM UCB for 24 h (0.71±0.04, n = 6; p<0.05) or 48 h (0.49±0.08, n = 6; p<0.05) underwent a significant reduction in the NMDA/AMPA ratio when compared with control slices (0.87±0.05, n = 7) (Figure 3A).

Bottom Line: UCB treatment produced a significant decrease in the levels of NR1, NR2A and NR2B subunits of N-methyl-D-aspartate (NMDA) receptors through a calpain-mediated proteolytic cleavage mechanism.Pretreatment of the hippocampal slice cultures with NMDA receptor antagonist or calpain inhibitors effectively prevented the UCB-induced impairment of LTP and LTD.Our results indicate that the proteolytic cleavage of NMDA receptor subunits by calpain may play a critical role in mediating the UCB-induced impairment of long-term synaptic plasticity in the hippocampus.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.

ABSTRACT

Background: Jaundice is one of the most common problems encountered in newborn infants, due to immaturity of hepatic conjugation and transport processes for bilirubin. Although the majority of neonatal jaundice is benign, some neonates with severe hyperbilirubinemia develop bilirubin encephalopathy or kernicterus. Accumulation of unconjugated bilirubin (UCB) in selected brain regions may result in temporary or permanent impairments of auditory, motor, or cognitive function; however, the molecular mechanisms by which UCB elicits such neurotoxicity are still poorly understood. The present study is undertaken to investigate whether prolonged exposure of rat organotypic hippocampal slice cultures to UCB alters the induction of long-term synaptic plasticity.

Methodology/principal findings: Using electrophysiological recording techniques, we find that exposure of hippocampal slice cultures to clinically relevant concentrations of UCB for 24 or 48 h results in an impairment of CA1 long-term potentiation (LTP) and long-term depression (LTD) induction in a time- and concentration-dependent manner. Hippocampal slice cultures stimulated with UCB show no changes in the secretion profiles of the pro-inflammatory cytokines, interleukin-1beta and tumor necrosis factor-alpha, or the propidium ioide uptake. UCB treatment produced a significant decrease in the levels of NR1, NR2A and NR2B subunits of N-methyl-D-aspartate (NMDA) receptors through a calpain-mediated proteolytic cleavage mechanism. Pretreatment of the hippocampal slice cultures with NMDA receptor antagonist or calpain inhibitors effectively prevented the UCB-induced impairment of LTP and LTD.

Conclusion/significance: Our results indicate that the proteolytic cleavage of NMDA receptor subunits by calpain may play a critical role in mediating the UCB-induced impairment of long-term synaptic plasticity in the hippocampus. These observations provide new insights into the molecular mechanisms underlying UCB-induced impairment of hippocampal synaptic plasticity which, in turn, might provide opportunities for the development of novel therapeutic strategies that targets these pathways for treatment.

Show MeSH
Related in: MedlinePlus