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The regulation of M1 muscarinic acetylcholine receptor desensitization by synaptic activity in cultured hippocampal neurons.

Willets JM, Nelson CP, Nahorski SR, Challiss RA - J. Neurochem. (2007)

Bottom Line: Using a protocol where neurons are exposed to an EC(50) concentration of the muscarinic agonist methacholine (MCh) prior to (R1), and following (R2) a desensitizing pulse of a high concentration of this agonist, we have found that the reduction in M(1) mACh receptor responsiveness is decreased in quiescent (+tetrodotoxin) neurons and increased when synaptic activity is enhanced by blocking GABA(A) receptors with picrotoxin.The picrotoxin-mediated effect on M1 mACh receptor responsiveness was completely prevented by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blockade.In contrast, picrotoxin-driven translocation of myristoylated, alanine-rich C kinase substrate was accompanied by translocation of PKCbetaII, but not PKCepsilon, and was dependent on PKC and Ca2+/calmodulin.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK.

ABSTRACT
To better understand metabotropic/ionotropic integration in neurons we have examined the regulation of M1 muscarinic acetylcholine (mACh) receptor signalling in mature (> 14 days in vitro), synaptically-active hippocampal neurons in culture. Using a protocol where neurons are exposed to an EC(50) concentration of the muscarinic agonist methacholine (MCh) prior to (R1), and following (R2) a desensitizing pulse of a high concentration of this agonist, we have found that the reduction in M(1) mACh receptor responsiveness is decreased in quiescent (+tetrodotoxin) neurons and increased when synaptic activity is enhanced by blocking GABA(A) receptors with picrotoxin. The picrotoxin-mediated effect on M1 mACh receptor responsiveness was completely prevented by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blockade. Inhibition of endogenous G protein-coupled receptor kinase 2 by transfection with the non-G(q/11)alpha-binding, catalytically-inactive (D110A,K220R)G protein-coupled receptor kinase 2 mutant, decreased the extent of M1 mACh receptor desensitization under all conditions. Pharmacological inhibition of protein kinase C (PKC) activity, or chronic phorbol ester-induced PKC down-regulation had no effect on agonist-mediated receptor desensitization in quiescent or spontaneously synaptically active neurons, but significantly decreased the extent of receptor desensitization in picrotoxin-treated neurons. MCh stimulated the translocation of diacylglycerol- sensitive eGFP-PKCepsilon, but not Ca2+/diacylglycerol-sensitive eGFP-PKCbetaII in both the absence, and presence of tetrodotoxin. Under these conditions, MCh-stimulated eGFP-myristoylated, alanine-rich C kinase substrate translocation was dependent on PKC activity, but not Ca2+/calmodulin. In contrast, picrotoxin-driven translocation of myristoylated, alanine-rich C kinase substrate was accompanied by translocation of PKCbetaII, but not PKCepsilon, and was dependent on PKC and Ca2+/calmodulin. Taken together these data suggest that the level of synaptic activity may determine the different kinases recruited to regulate M1 mACh receptor desensitization in neurons.

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Effects of synaptic activity on M1 muscarinic acetylcholine (mACh) receptor responsiveness and re-sensitization assessed through inositol 1,4,5-trisphosphate (IP3) imaging of hippocampal neurons. The desensitization protocol (R1/Rmax/R2) was performed as described in the Experimental Procedures section. Representative traces showing M1 mACh receptor desensitization in the presence of (a) tetrodotoxin (TTx) (500 nmol/L); (b) no pre-addition (spontaneous synaptic activity; Spont in panel d); and (c) synaptic activity induced by picrotoxin (PiTx, 100 μmol/L). Picrotoxin was present 3 min prior to and throughout the experiment. Methacholine (R1, 10 μmol/L, 30 s; Rmax, 100 μmol/L, 60 s; R2 10 μmol/L, 30 s) was added as indicated by the bars. (d) Cumulative data for time-courses of M1 mACh receptor re-sensitization in the absence (Spont) or presence of TTx, or following PiTx addition. Data are expressed as means ± SEM for the percentage change in R2 relative to the R1 response, for 5–15 neurons taken from at least three separate hippocampal preparations. Significant differences in the R2/R1 ratio from the +TTx condition at a given time-point are indicated as *p< 0.05; **p< 0.01.
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fig01: Effects of synaptic activity on M1 muscarinic acetylcholine (mACh) receptor responsiveness and re-sensitization assessed through inositol 1,4,5-trisphosphate (IP3) imaging of hippocampal neurons. The desensitization protocol (R1/Rmax/R2) was performed as described in the Experimental Procedures section. Representative traces showing M1 mACh receptor desensitization in the presence of (a) tetrodotoxin (TTx) (500 nmol/L); (b) no pre-addition (spontaneous synaptic activity; Spont in panel d); and (c) synaptic activity induced by picrotoxin (PiTx, 100 μmol/L). Picrotoxin was present 3 min prior to and throughout the experiment. Methacholine (R1, 10 μmol/L, 30 s; Rmax, 100 μmol/L, 60 s; R2 10 μmol/L, 30 s) was added as indicated by the bars. (d) Cumulative data for time-courses of M1 mACh receptor re-sensitization in the absence (Spont) or presence of TTx, or following PiTx addition. Data are expressed as means ± SEM for the percentage change in R2 relative to the R1 response, for 5–15 neurons taken from at least three separate hippocampal preparations. Significant differences in the R2/R1 ratio from the +TTx condition at a given time-point are indicated as *p< 0.05; **p< 0.01.

Mentions: Our previous work has extensively characterized the desensitization of M1 mACh receptors in immature (< 10 days in vitro, hereafter DIV), non-synaptically active hippocampal neurons (Willets et al. 2004, 2005). Based on these findings we applied a similar protocol (see Materials and methods) to study receptor desensitization in mature (≥ 14 DIV) neurons. Comparison of the responses before (R1) and following (R2), the addition of a desensitizing pulse of MCh (100 μmol/L), showed a clear reduction in the R2 response 5 min after the desensitizing pulse under all conditions. In the presence of TTx (500 nmol/L; to block spontaneous synaptic activity), desensitization, indicated as the decrease in R2 response relative to the R1 response, was similar to our previous findings in immature neurons (Fig. 1a). However, in the absence of TTx the hippocampal cultures display spontaneous, glutamate-driven and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated synaptic activity (Bacci et al. 1999; Nash et al. 2004; Young et al. 2005), which results in AMPA receptor-mediated depolarization and Ca2+ entry via NMDA receptors and voltage-operated Ca2+ channels. In agreement with our previous data (Nash et al. 2004) such activity promotes enhanced IP3 formation, seen as spikes above the level of IP3 produced by the presence of agonist (Fig. 1b). However, in the presence of spontaneous synaptic activity the difference between R2 and R1 responses was increased (Fig. 1b), suggesting that synaptic activity may increase the rate/extent of M1 mACh receptor desensitization. To test this hypothesis, we enhanced spontaneous synaptic activity by adding picrotoxin (100 μmol/L) to block GABAA receptors and enhance the effect of glutamate-driven AMPA receptor-mediated Ca2+ excitability within the neuronal culture (Nash et al. 2004; Young et al. 2005). Picrotoxin (100 μmol/L) was added 3 min prior to the start of, and throughout the experiment. The presence of picrotoxin significantly suppressed the R2 response compared to R1, indicating that the level of synaptic activity within the neuronal culture directly effects receptor responsiveness (Fig. 1c).


The regulation of M1 muscarinic acetylcholine receptor desensitization by synaptic activity in cultured hippocampal neurons.

Willets JM, Nelson CP, Nahorski SR, Challiss RA - J. Neurochem. (2007)

Effects of synaptic activity on M1 muscarinic acetylcholine (mACh) receptor responsiveness and re-sensitization assessed through inositol 1,4,5-trisphosphate (IP3) imaging of hippocampal neurons. The desensitization protocol (R1/Rmax/R2) was performed as described in the Experimental Procedures section. Representative traces showing M1 mACh receptor desensitization in the presence of (a) tetrodotoxin (TTx) (500 nmol/L); (b) no pre-addition (spontaneous synaptic activity; Spont in panel d); and (c) synaptic activity induced by picrotoxin (PiTx, 100 μmol/L). Picrotoxin was present 3 min prior to and throughout the experiment. Methacholine (R1, 10 μmol/L, 30 s; Rmax, 100 μmol/L, 60 s; R2 10 μmol/L, 30 s) was added as indicated by the bars. (d) Cumulative data for time-courses of M1 mACh receptor re-sensitization in the absence (Spont) or presence of TTx, or following PiTx addition. Data are expressed as means ± SEM for the percentage change in R2 relative to the R1 response, for 5–15 neurons taken from at least three separate hippocampal preparations. Significant differences in the R2/R1 ratio from the +TTx condition at a given time-point are indicated as *p< 0.05; **p< 0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2658029&req=5

fig01: Effects of synaptic activity on M1 muscarinic acetylcholine (mACh) receptor responsiveness and re-sensitization assessed through inositol 1,4,5-trisphosphate (IP3) imaging of hippocampal neurons. The desensitization protocol (R1/Rmax/R2) was performed as described in the Experimental Procedures section. Representative traces showing M1 mACh receptor desensitization in the presence of (a) tetrodotoxin (TTx) (500 nmol/L); (b) no pre-addition (spontaneous synaptic activity; Spont in panel d); and (c) synaptic activity induced by picrotoxin (PiTx, 100 μmol/L). Picrotoxin was present 3 min prior to and throughout the experiment. Methacholine (R1, 10 μmol/L, 30 s; Rmax, 100 μmol/L, 60 s; R2 10 μmol/L, 30 s) was added as indicated by the bars. (d) Cumulative data for time-courses of M1 mACh receptor re-sensitization in the absence (Spont) or presence of TTx, or following PiTx addition. Data are expressed as means ± SEM for the percentage change in R2 relative to the R1 response, for 5–15 neurons taken from at least three separate hippocampal preparations. Significant differences in the R2/R1 ratio from the +TTx condition at a given time-point are indicated as *p< 0.05; **p< 0.01.
Mentions: Our previous work has extensively characterized the desensitization of M1 mACh receptors in immature (< 10 days in vitro, hereafter DIV), non-synaptically active hippocampal neurons (Willets et al. 2004, 2005). Based on these findings we applied a similar protocol (see Materials and methods) to study receptor desensitization in mature (≥ 14 DIV) neurons. Comparison of the responses before (R1) and following (R2), the addition of a desensitizing pulse of MCh (100 μmol/L), showed a clear reduction in the R2 response 5 min after the desensitizing pulse under all conditions. In the presence of TTx (500 nmol/L; to block spontaneous synaptic activity), desensitization, indicated as the decrease in R2 response relative to the R1 response, was similar to our previous findings in immature neurons (Fig. 1a). However, in the absence of TTx the hippocampal cultures display spontaneous, glutamate-driven and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated synaptic activity (Bacci et al. 1999; Nash et al. 2004; Young et al. 2005), which results in AMPA receptor-mediated depolarization and Ca2+ entry via NMDA receptors and voltage-operated Ca2+ channels. In agreement with our previous data (Nash et al. 2004) such activity promotes enhanced IP3 formation, seen as spikes above the level of IP3 produced by the presence of agonist (Fig. 1b). However, in the presence of spontaneous synaptic activity the difference between R2 and R1 responses was increased (Fig. 1b), suggesting that synaptic activity may increase the rate/extent of M1 mACh receptor desensitization. To test this hypothesis, we enhanced spontaneous synaptic activity by adding picrotoxin (100 μmol/L) to block GABAA receptors and enhance the effect of glutamate-driven AMPA receptor-mediated Ca2+ excitability within the neuronal culture (Nash et al. 2004; Young et al. 2005). Picrotoxin (100 μmol/L) was added 3 min prior to the start of, and throughout the experiment. The presence of picrotoxin significantly suppressed the R2 response compared to R1, indicating that the level of synaptic activity within the neuronal culture directly effects receptor responsiveness (Fig. 1c).

Bottom Line: Using a protocol where neurons are exposed to an EC(50) concentration of the muscarinic agonist methacholine (MCh) prior to (R1), and following (R2) a desensitizing pulse of a high concentration of this agonist, we have found that the reduction in M(1) mACh receptor responsiveness is decreased in quiescent (+tetrodotoxin) neurons and increased when synaptic activity is enhanced by blocking GABA(A) receptors with picrotoxin.The picrotoxin-mediated effect on M1 mACh receptor responsiveness was completely prevented by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blockade.In contrast, picrotoxin-driven translocation of myristoylated, alanine-rich C kinase substrate was accompanied by translocation of PKCbetaII, but not PKCepsilon, and was dependent on PKC and Ca2+/calmodulin.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK.

ABSTRACT
To better understand metabotropic/ionotropic integration in neurons we have examined the regulation of M1 muscarinic acetylcholine (mACh) receptor signalling in mature (> 14 days in vitro), synaptically-active hippocampal neurons in culture. Using a protocol where neurons are exposed to an EC(50) concentration of the muscarinic agonist methacholine (MCh) prior to (R1), and following (R2) a desensitizing pulse of a high concentration of this agonist, we have found that the reduction in M(1) mACh receptor responsiveness is decreased in quiescent (+tetrodotoxin) neurons and increased when synaptic activity is enhanced by blocking GABA(A) receptors with picrotoxin. The picrotoxin-mediated effect on M1 mACh receptor responsiveness was completely prevented by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blockade. Inhibition of endogenous G protein-coupled receptor kinase 2 by transfection with the non-G(q/11)alpha-binding, catalytically-inactive (D110A,K220R)G protein-coupled receptor kinase 2 mutant, decreased the extent of M1 mACh receptor desensitization under all conditions. Pharmacological inhibition of protein kinase C (PKC) activity, or chronic phorbol ester-induced PKC down-regulation had no effect on agonist-mediated receptor desensitization in quiescent or spontaneously synaptically active neurons, but significantly decreased the extent of receptor desensitization in picrotoxin-treated neurons. MCh stimulated the translocation of diacylglycerol- sensitive eGFP-PKCepsilon, but not Ca2+/diacylglycerol-sensitive eGFP-PKCbetaII in both the absence, and presence of tetrodotoxin. Under these conditions, MCh-stimulated eGFP-myristoylated, alanine-rich C kinase substrate translocation was dependent on PKC activity, but not Ca2+/calmodulin. In contrast, picrotoxin-driven translocation of myristoylated, alanine-rich C kinase substrate was accompanied by translocation of PKCbetaII, but not PKCepsilon, and was dependent on PKC and Ca2+/calmodulin. Taken together these data suggest that the level of synaptic activity may determine the different kinases recruited to regulate M1 mACh receptor desensitization in neurons.

Show MeSH
Related in: MedlinePlus