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Acetylcholine induces GABA release onto rod bipolar cells through heteromeric nicotinic receptors expressed in A17 amacrine cells.

Elgueta C, Vielma AH, Palacios AG, Schmachtenberg O - Front Cell Neurosci (2015)

Bottom Line: Using whole-cell patch clamp recordings in slices of rat retina, we found that ACh application triggers GABA release onto rod bipolar (RB) cells.Activation of nAChRs induced GABA release after Ca(2+) accumulation in A17 cell dendrites and varicosities mediated by L-type voltage-gated calcium channels (VGCCs) and intracellular Ca(2+) stores.Inhibition of acetylcholinesterase depolarized A17 cells and increased spontaneous inhibitory postsynaptic currents in RB cells, indicating that endogenous ACh enhances GABAergic inhibition of RB cells.

View Article: PubMed Central - PubMed

Affiliation: Centro Interdisciplinario de Neurociencia de Valparaíso, Facultad de Ciencias, Universidad de Valparaíso Valparaíso, Chile ; Systemic and Cellular Neurophysiology, Institute of Physiology I, Albert-Ludwigs-Universität Freiburg, Germany.

ABSTRACT
Acetylcholine (ACh) is a major retinal neurotransmitter that modulates visual processing through a large repertoire of cholinergic receptors expressed on different retinal cell types. ACh is released from starburst amacrine cells (SACs) under scotopic conditions, but its effects on cells of the rod pathway have not been investigated. Using whole-cell patch clamp recordings in slices of rat retina, we found that ACh application triggers GABA release onto rod bipolar (RB) cells. GABA was released from A17 amacrine cells and activated postsynaptic GABAA and GABAC receptors in RB cells. The sensitivity of ACh-induced currents to nicotinic ACh receptor (nAChR) antagonists (TMPH ~ mecamylamine > erysodine > DhβE > MLA) together with the differential potency of specific agonists to mimic ACh responses (cytisine > RJR2403 ~ choline), suggest that A17 cells express heteromeric nAChRs containing the β4 subunit. Activation of nAChRs induced GABA release after Ca(2+) accumulation in A17 cell dendrites and varicosities mediated by L-type voltage-gated calcium channels (VGCCs) and intracellular Ca(2+) stores. Inhibition of acetylcholinesterase depolarized A17 cells and increased spontaneous inhibitory postsynaptic currents in RB cells, indicating that endogenous ACh enhances GABAergic inhibition of RB cells. Moreover, injection of neostigmine or cytisine reduced the b-wave of the scotopic flash electroretinogram (ERG), suggesting that cholinergic modulation of GABA release controls RB cell activity in vivo. These results describe a novel regulatory mechanism of RB cell inhibition and complement our understanding of the neuromodulatory control of retinal signal processing.

No MeSH data available.


Related in: MedlinePlus

Functional β4-containing nAChRs are expressed in A17 cells and mediate ACh-evoked GABA release onto RB cells. Representative traces of whole-voltage clamp recordings during localized application of ACh (1 mM, 1 s) or nicotinic agonists specific for (A) homomeric (choline 1 mM, 1 s, top), (B) α4β2 (RJR-2403 100 μM, 1 s, middle) or (C) β4 subunit-containing nAChRs (cytisine 100 μM, 1 s, bottom) in A17 (top) or RB cells (bottom). All experiments were performed using double-barreled puffer pipettes. (D) Bar plot summarizing the normalized effects of specific nicotinic agonists on A17 (gray solid bars) and RB (with bars) cells. Two-tailed paired t-test, *p < 0.05 and **p < 0.01. The normalized average effect of cytisine was significantly different when compared to choline or RJR-2403 in A17 (One-Way ANOVA with Bonferroni-adjusted pairwise comparisons) and RB cells (One-Way ANOVA on ranks with post hoc Dunn's test).
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Figure 4: Functional β4-containing nAChRs are expressed in A17 cells and mediate ACh-evoked GABA release onto RB cells. Representative traces of whole-voltage clamp recordings during localized application of ACh (1 mM, 1 s) or nicotinic agonists specific for (A) homomeric (choline 1 mM, 1 s, top), (B) α4β2 (RJR-2403 100 μM, 1 s, middle) or (C) β4 subunit-containing nAChRs (cytisine 100 μM, 1 s, bottom) in A17 (top) or RB cells (bottom). All experiments were performed using double-barreled puffer pipettes. (D) Bar plot summarizing the normalized effects of specific nicotinic agonists on A17 (gray solid bars) and RB (with bars) cells. Two-tailed paired t-test, *p < 0.05 and **p < 0.01. The normalized average effect of cytisine was significantly different when compared to choline or RJR-2403 in A17 (One-Way ANOVA with Bonferroni-adjusted pairwise comparisons) and RB cells (One-Way ANOVA on ranks with post hoc Dunn's test).

Mentions: Nicotinic receptors are assembled by different combinations of α and β subunits. The precise subunit composition defines important receptor properties such as ligand affinity, channel kinetics and ionic selectivity (Alkondon and Albuquerque, 1993), greatly affecting the influence that nAChR activation will have in cellular function. To investigate which nAChR subtypes are expressed in A17 cells and mediate postsynaptic responses in RB cells, we tested the effects of specific nicotinic pharmacological agents. Perfusion of an antagonist specific for neuronal heteromeric nAChRs (tetramethylpiperidine-4-yl-heptanoate [TMPH] 10 μM, Damaj et al., 2005; Papke et al., 2005) abolished ACh responses in both cell types (3.6 ± 0.7% of control in A17 cells, n = 5, p = 0.02; 3.2 ± 0.4% of control in RB cells, p = 0.008, n = 5, Figures 3C,G), while homomeric nAChR-specific antagonist methyllycaconitine (MLA) did not produce a significant effect neither in A17 nor in RB cells (10 nM; 96.8 ± 4.2 and 96.7 ± 2.7% of control responses, p = 0.25 and 0.17 respectively, n = 8 for both cells types, Figures 3D,G). One of the most commonly expressed heteromeric nAChRs in the CNS is the α4β2 subtype (Dani and Bertrand, 2007). The prototypical α4β2 nAChR antagonist dihydro-β-erythroiodine (DHβE 10 μM, Harvey et al., 1996) produced a small albeit significant reduction of ACh-induced currents in A17 cells (89.7 ± 3% of control, n = 15, p = 0.009) and a marked decrease in RB cell responses (61 ± 5.3% of control, n = 6, p = 0.003; Figures 3E,G). Erysodine (10 μM), another alkaloid with high affinity for α4β2 nAChRs (Decker and Anderson, 1995; Iturriaga-Vásquez et al., 2010), had a more pronounced effect in A17 and RB cells (36.1 ± 3.3% of control in A17 cells, n = 10, p = 0.0002; 17 ± 0.8% of control in RB cells, n = 10, p = 0.00003; Figures 3F,G) but did not completely block the response to ACh. Pharmacology of nicotinic receptors in A17 cells was further investigated by comparing ACh-evoked currents with those induced by specific nicotinic agonists. Choline (Chol, 1 mM), a byproduct of ACh cleavage and an α7-nAChR-specific ligand (Alkondon and Pereira, 1999), produced only a marginal response in A17 cells and had no discernible effect in RB cells (10.1 ± 3.9 and 2.5 ± 2% of ACh-evoked responses respectively, n = 5 for both cell types, p = 0.03 and 0.017; Figures 4A,D). Likewise, an agonist with specificity for α4β2 nAChRs (RJR-2403 100 μM, Papke et al., 2000) failed to activate either A17 or RB cells (7.5 ± 5% and 1.7 ± 0.6% of ACh response, n = 4 and 3, p = 0.009 and 0.004, respectively; Figures 4B,D). On the contrary, the β4 subunit-specific nicotinic agonist cytisine (100 μM; Luetje and Patrick, 1991; Papke and Heinemann, 1994) induced currents in A17 and RB cells comparable to those evoked by ACh (108.7 ± 14.7% and 113.3 ± 22.1% of ACh responses respectively, n = 5 for both cell types, p = 0.58 and 0.97; Figures 4C,D), although with a slower decay (tau 3.9 ± 0.3 vs. 28.7 ± 4.9 s, for ACh vs. cytisine-evoked currents in A17 cells, n = 5, p = 0.047). In summary, ACh responses in both cell types have a comparable pharmacological profile, further supporting that A17 cells through activation of heteromeric nAChRs, provide the majority of the ACh-triggered GABA release onto RB cells.


Acetylcholine induces GABA release onto rod bipolar cells through heteromeric nicotinic receptors expressed in A17 amacrine cells.

Elgueta C, Vielma AH, Palacios AG, Schmachtenberg O - Front Cell Neurosci (2015)

Functional β4-containing nAChRs are expressed in A17 cells and mediate ACh-evoked GABA release onto RB cells. Representative traces of whole-voltage clamp recordings during localized application of ACh (1 mM, 1 s) or nicotinic agonists specific for (A) homomeric (choline 1 mM, 1 s, top), (B) α4β2 (RJR-2403 100 μM, 1 s, middle) or (C) β4 subunit-containing nAChRs (cytisine 100 μM, 1 s, bottom) in A17 (top) or RB cells (bottom). All experiments were performed using double-barreled puffer pipettes. (D) Bar plot summarizing the normalized effects of specific nicotinic agonists on A17 (gray solid bars) and RB (with bars) cells. Two-tailed paired t-test, *p < 0.05 and **p < 0.01. The normalized average effect of cytisine was significantly different when compared to choline or RJR-2403 in A17 (One-Way ANOVA with Bonferroni-adjusted pairwise comparisons) and RB cells (One-Way ANOVA on ranks with post hoc Dunn's test).
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Figure 4: Functional β4-containing nAChRs are expressed in A17 cells and mediate ACh-evoked GABA release onto RB cells. Representative traces of whole-voltage clamp recordings during localized application of ACh (1 mM, 1 s) or nicotinic agonists specific for (A) homomeric (choline 1 mM, 1 s, top), (B) α4β2 (RJR-2403 100 μM, 1 s, middle) or (C) β4 subunit-containing nAChRs (cytisine 100 μM, 1 s, bottom) in A17 (top) or RB cells (bottom). All experiments were performed using double-barreled puffer pipettes. (D) Bar plot summarizing the normalized effects of specific nicotinic agonists on A17 (gray solid bars) and RB (with bars) cells. Two-tailed paired t-test, *p < 0.05 and **p < 0.01. The normalized average effect of cytisine was significantly different when compared to choline or RJR-2403 in A17 (One-Way ANOVA with Bonferroni-adjusted pairwise comparisons) and RB cells (One-Way ANOVA on ranks with post hoc Dunn's test).
Mentions: Nicotinic receptors are assembled by different combinations of α and β subunits. The precise subunit composition defines important receptor properties such as ligand affinity, channel kinetics and ionic selectivity (Alkondon and Albuquerque, 1993), greatly affecting the influence that nAChR activation will have in cellular function. To investigate which nAChR subtypes are expressed in A17 cells and mediate postsynaptic responses in RB cells, we tested the effects of specific nicotinic pharmacological agents. Perfusion of an antagonist specific for neuronal heteromeric nAChRs (tetramethylpiperidine-4-yl-heptanoate [TMPH] 10 μM, Damaj et al., 2005; Papke et al., 2005) abolished ACh responses in both cell types (3.6 ± 0.7% of control in A17 cells, n = 5, p = 0.02; 3.2 ± 0.4% of control in RB cells, p = 0.008, n = 5, Figures 3C,G), while homomeric nAChR-specific antagonist methyllycaconitine (MLA) did not produce a significant effect neither in A17 nor in RB cells (10 nM; 96.8 ± 4.2 and 96.7 ± 2.7% of control responses, p = 0.25 and 0.17 respectively, n = 8 for both cells types, Figures 3D,G). One of the most commonly expressed heteromeric nAChRs in the CNS is the α4β2 subtype (Dani and Bertrand, 2007). The prototypical α4β2 nAChR antagonist dihydro-β-erythroiodine (DHβE 10 μM, Harvey et al., 1996) produced a small albeit significant reduction of ACh-induced currents in A17 cells (89.7 ± 3% of control, n = 15, p = 0.009) and a marked decrease in RB cell responses (61 ± 5.3% of control, n = 6, p = 0.003; Figures 3E,G). Erysodine (10 μM), another alkaloid with high affinity for α4β2 nAChRs (Decker and Anderson, 1995; Iturriaga-Vásquez et al., 2010), had a more pronounced effect in A17 and RB cells (36.1 ± 3.3% of control in A17 cells, n = 10, p = 0.0002; 17 ± 0.8% of control in RB cells, n = 10, p = 0.00003; Figures 3F,G) but did not completely block the response to ACh. Pharmacology of nicotinic receptors in A17 cells was further investigated by comparing ACh-evoked currents with those induced by specific nicotinic agonists. Choline (Chol, 1 mM), a byproduct of ACh cleavage and an α7-nAChR-specific ligand (Alkondon and Pereira, 1999), produced only a marginal response in A17 cells and had no discernible effect in RB cells (10.1 ± 3.9 and 2.5 ± 2% of ACh-evoked responses respectively, n = 5 for both cell types, p = 0.03 and 0.017; Figures 4A,D). Likewise, an agonist with specificity for α4β2 nAChRs (RJR-2403 100 μM, Papke et al., 2000) failed to activate either A17 or RB cells (7.5 ± 5% and 1.7 ± 0.6% of ACh response, n = 4 and 3, p = 0.009 and 0.004, respectively; Figures 4B,D). On the contrary, the β4 subunit-specific nicotinic agonist cytisine (100 μM; Luetje and Patrick, 1991; Papke and Heinemann, 1994) induced currents in A17 and RB cells comparable to those evoked by ACh (108.7 ± 14.7% and 113.3 ± 22.1% of ACh responses respectively, n = 5 for both cell types, p = 0.58 and 0.97; Figures 4C,D), although with a slower decay (tau 3.9 ± 0.3 vs. 28.7 ± 4.9 s, for ACh vs. cytisine-evoked currents in A17 cells, n = 5, p = 0.047). In summary, ACh responses in both cell types have a comparable pharmacological profile, further supporting that A17 cells through activation of heteromeric nAChRs, provide the majority of the ACh-triggered GABA release onto RB cells.

Bottom Line: Using whole-cell patch clamp recordings in slices of rat retina, we found that ACh application triggers GABA release onto rod bipolar (RB) cells.Activation of nAChRs induced GABA release after Ca(2+) accumulation in A17 cell dendrites and varicosities mediated by L-type voltage-gated calcium channels (VGCCs) and intracellular Ca(2+) stores.Inhibition of acetylcholinesterase depolarized A17 cells and increased spontaneous inhibitory postsynaptic currents in RB cells, indicating that endogenous ACh enhances GABAergic inhibition of RB cells.

View Article: PubMed Central - PubMed

Affiliation: Centro Interdisciplinario de Neurociencia de Valparaíso, Facultad de Ciencias, Universidad de Valparaíso Valparaíso, Chile ; Systemic and Cellular Neurophysiology, Institute of Physiology I, Albert-Ludwigs-Universität Freiburg, Germany.

ABSTRACT
Acetylcholine (ACh) is a major retinal neurotransmitter that modulates visual processing through a large repertoire of cholinergic receptors expressed on different retinal cell types. ACh is released from starburst amacrine cells (SACs) under scotopic conditions, but its effects on cells of the rod pathway have not been investigated. Using whole-cell patch clamp recordings in slices of rat retina, we found that ACh application triggers GABA release onto rod bipolar (RB) cells. GABA was released from A17 amacrine cells and activated postsynaptic GABAA and GABAC receptors in RB cells. The sensitivity of ACh-induced currents to nicotinic ACh receptor (nAChR) antagonists (TMPH ~ mecamylamine > erysodine > DhβE > MLA) together with the differential potency of specific agonists to mimic ACh responses (cytisine > RJR2403 ~ choline), suggest that A17 cells express heteromeric nAChRs containing the β4 subunit. Activation of nAChRs induced GABA release after Ca(2+) accumulation in A17 cell dendrites and varicosities mediated by L-type voltage-gated calcium channels (VGCCs) and intracellular Ca(2+) stores. Inhibition of acetylcholinesterase depolarized A17 cells and increased spontaneous inhibitory postsynaptic currents in RB cells, indicating that endogenous ACh enhances GABAergic inhibition of RB cells. Moreover, injection of neostigmine or cytisine reduced the b-wave of the scotopic flash electroretinogram (ERG), suggesting that cholinergic modulation of GABA release controls RB cell activity in vivo. These results describe a novel regulatory mechanism of RB cell inhibition and complement our understanding of the neuromodulatory control of retinal signal processing.

No MeSH data available.


Related in: MedlinePlus