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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

Mechanisms of ACh-induced GABA release from A17 cells. (A) Representative traces of ACh-evoked IPSCs in RB cells (1 mM, 1 s) in control conditions and after (left) removing extracellular Ca2+, (middle) applying the unspecific VGCC blocker Cd2+ (200 μM) or (right) after specific blockade of L-type VGCCs with verapamil (20 μM). (B) Traces showing the effects of disrupting intracellular calcium signaling by (left) a blocker of ryanodine receptor channels (ruthenium red, RR 40 μM), (middle) an inhibitor of the endoplasmic reticulum Ca2+-ATPase pump (cyclopiazonic acid, CPA 30 μM) and (right) an agonist of ryanodine receptors (4-chloro-m-cresol, 4-CMC 500 μM) on the responses to ACh in RB cells. (C) Bar graph displaying normalized average ACh response amplitudes in RB cells after perfusion with solutions affecting presynaptic calcium dynamics. *p < 0.05, **p < 0.01, and ***p < 0.001, for two-tailed paired t-tests. Differences between groups were significant when verapamil was compared with CPA or 4-CMC, and when Cd2+ is compared with 4-CMC (One-Way ANOVA on ranks with post hoc Dunn's test). (D) Left, montage of images from an A17 cell filled with 100 μM OGB-1 (scale bar 10 μm). Analyzed regions of interest are denoted by colored frames and correspond to Ca2+ responses traces after (middle) puff application of nicotine (Nic 1 mM, 500 ms) or (right) after a 60 mV voltage step of 200 ms duration. Bottom, black traces show current responses after the respective stimulation paradigms.
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Figure 5: Mechanisms of ACh-induced GABA release from A17 cells. (A) Representative traces of ACh-evoked IPSCs in RB cells (1 mM, 1 s) in control conditions and after (left) removing extracellular Ca2+, (middle) applying the unspecific VGCC blocker Cd2+ (200 μM) or (right) after specific blockade of L-type VGCCs with verapamil (20 μM). (B) Traces showing the effects of disrupting intracellular calcium signaling by (left) a blocker of ryanodine receptor channels (ruthenium red, RR 40 μM), (middle) an inhibitor of the endoplasmic reticulum Ca2+-ATPase pump (cyclopiazonic acid, CPA 30 μM) and (right) an agonist of ryanodine receptors (4-chloro-m-cresol, 4-CMC 500 μM) on the responses to ACh in RB cells. (C) Bar graph displaying normalized average ACh response amplitudes in RB cells after perfusion with solutions affecting presynaptic calcium dynamics. *p < 0.05, **p < 0.01, and ***p < 0.001, for two-tailed paired t-tests. Differences between groups were significant when verapamil was compared with CPA or 4-CMC, and when Cd2+ is compared with 4-CMC (One-Way ANOVA on ranks with post hoc Dunn's test). (D) Left, montage of images from an A17 cell filled with 100 μM OGB-1 (scale bar 10 μm). Analyzed regions of interest are denoted by colored frames and correspond to Ca2+ responses traces after (middle) puff application of nicotine (Nic 1 mM, 500 ms) or (right) after a 60 mV voltage step of 200 ms duration. Bottom, black traces show current responses after the respective stimulation paradigms.

Mentions: Next, we investigated the mechanisms through which nAChR activation induces Ca2+ entry into A17 cells, by studying ACh-evoked IPSCs in RB cells. Perfusion with Ca2+-free solutions importantly reduced the amplitude of ACh-evoked IPSCs in RB cells (0 Ca2+, 29.5 ± 6% of control, n = 4, p = 0.015, Figures 5A left, C; 0 Ca2+ + 1 mM EGTA, 25.3 ± 5.2% of control, n = 4, p = 0.003, Figures S1C, 5C). Because heteromeric nAChRs form channels with relatively low Ca2+ permeability (Fucile, 2004), Ca2+ influx should be provided by a different membrane conductance. Indeed, general blockers of VGCCs effectively reduced the response of RB cells to ACh (Cd2+ 200 μM, 14.5 ± 2.8% of control, n = 9, p = 0.001, Figures 5A middle, C; Co2+ 1 mM, 19.5 ± 1.2% of control, n = 4, p = 0.01, Figures S1D, 5C). More specifically, inhibition of L-type VGCCs, which are known to be present in synaptic varicosities of A17 cells (Grimes et al., 2009), almost completely abolished ACh responses (verapamil 20 μM, 7.3 ± 1.4% of control, n = 5, p = 0.02, Figures 5A right, C; nifedipine 30 μM, 17 ± 3.7% of control, n = 5, p = 0.0007, Figures S1E, 5C). This indicates a prevalent role of L-type VGCCs in cholinergic release of GABA from A17 cells, contrary to their limited participation when glutamate is the excitatory neurotransmitter (verapamil 20 μM, 91.8 ± 4.1% of control responses to glutamate [200 μM, 200 ms], n = 3, Figure S1F; see also Chávez et al., 2006; Grimes et al., 2009). On the other hand, Ca2+-induced Ca2+ release (CICR) from intracellular stores could also contribute by enhancing the cytoplasmatic Ca2+ concentration after an initial influx of the ion, as reported for glutamate-evoked GABA release from A17 cells (Chávez et al., 2006; Chávez and Diamond, 2008). Indeed, perfusion with an antagonist of ryanodine receptors produced a significant decrease in the amplitude of IPSCs triggered by ACh (ruthenium red, RR 40 μM, 26.7 ± 3% of control, n = 5, p = 0.002; Figures 5B left, C). Consistently, depletion of Ca2+ from the endoplasmic reticulum using the Ca2+-ATPase antagonist cyclopiazonic acid (CPA 30 μM) or the ryanodine receptors agonist 4-chloro-methyl-cresol (4-CMC 500 μM) significantly diminished IPSCs evoked by ACh in RB cells (35.5 ± 5.6% and 50.4 ± 6.3% of control response, p = 0.043 and 0.049, respectively, n = 4 for both conditions; Figures 5B,C).


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)

Mechanisms of ACh-induced GABA release from A17 cells. (A) Representative traces of ACh-evoked IPSCs in RB cells (1 mM, 1 s) in control conditions and after (left) removing extracellular Ca2+, (middle) applying the unspecific VGCC blocker Cd2+ (200 μM) or (right) after specific blockade of L-type VGCCs with verapamil (20 μM). (B) Traces showing the effects of disrupting intracellular calcium signaling by (left) a blocker of ryanodine receptor channels (ruthenium red, RR 40 μM), (middle) an inhibitor of the endoplasmic reticulum Ca2+-ATPase pump (cyclopiazonic acid, CPA 30 μM) and (right) an agonist of ryanodine receptors (4-chloro-m-cresol, 4-CMC 500 μM) on the responses to ACh in RB cells. (C) Bar graph displaying normalized average ACh response amplitudes in RB cells after perfusion with solutions affecting presynaptic calcium dynamics. *p < 0.05, **p < 0.01, and ***p < 0.001, for two-tailed paired t-tests. Differences between groups were significant when verapamil was compared with CPA or 4-CMC, and when Cd2+ is compared with 4-CMC (One-Way ANOVA on ranks with post hoc Dunn's test). (D) Left, montage of images from an A17 cell filled with 100 μM OGB-1 (scale bar 10 μm). Analyzed regions of interest are denoted by colored frames and correspond to Ca2+ responses traces after (middle) puff application of nicotine (Nic 1 mM, 500 ms) or (right) after a 60 mV voltage step of 200 ms duration. Bottom, black traces show current responses after the respective stimulation paradigms.
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Figure 5: Mechanisms of ACh-induced GABA release from A17 cells. (A) Representative traces of ACh-evoked IPSCs in RB cells (1 mM, 1 s) in control conditions and after (left) removing extracellular Ca2+, (middle) applying the unspecific VGCC blocker Cd2+ (200 μM) or (right) after specific blockade of L-type VGCCs with verapamil (20 μM). (B) Traces showing the effects of disrupting intracellular calcium signaling by (left) a blocker of ryanodine receptor channels (ruthenium red, RR 40 μM), (middle) an inhibitor of the endoplasmic reticulum Ca2+-ATPase pump (cyclopiazonic acid, CPA 30 μM) and (right) an agonist of ryanodine receptors (4-chloro-m-cresol, 4-CMC 500 μM) on the responses to ACh in RB cells. (C) Bar graph displaying normalized average ACh response amplitudes in RB cells after perfusion with solutions affecting presynaptic calcium dynamics. *p < 0.05, **p < 0.01, and ***p < 0.001, for two-tailed paired t-tests. Differences between groups were significant when verapamil was compared with CPA or 4-CMC, and when Cd2+ is compared with 4-CMC (One-Way ANOVA on ranks with post hoc Dunn's test). (D) Left, montage of images from an A17 cell filled with 100 μM OGB-1 (scale bar 10 μm). Analyzed regions of interest are denoted by colored frames and correspond to Ca2+ responses traces after (middle) puff application of nicotine (Nic 1 mM, 500 ms) or (right) after a 60 mV voltage step of 200 ms duration. Bottom, black traces show current responses after the respective stimulation paradigms.
Mentions: Next, we investigated the mechanisms through which nAChR activation induces Ca2+ entry into A17 cells, by studying ACh-evoked IPSCs in RB cells. Perfusion with Ca2+-free solutions importantly reduced the amplitude of ACh-evoked IPSCs in RB cells (0 Ca2+, 29.5 ± 6% of control, n = 4, p = 0.015, Figures 5A left, C; 0 Ca2+ + 1 mM EGTA, 25.3 ± 5.2% of control, n = 4, p = 0.003, Figures S1C, 5C). Because heteromeric nAChRs form channels with relatively low Ca2+ permeability (Fucile, 2004), Ca2+ influx should be provided by a different membrane conductance. Indeed, general blockers of VGCCs effectively reduced the response of RB cells to ACh (Cd2+ 200 μM, 14.5 ± 2.8% of control, n = 9, p = 0.001, Figures 5A middle, C; Co2+ 1 mM, 19.5 ± 1.2% of control, n = 4, p = 0.01, Figures S1D, 5C). More specifically, inhibition of L-type VGCCs, which are known to be present in synaptic varicosities of A17 cells (Grimes et al., 2009), almost completely abolished ACh responses (verapamil 20 μM, 7.3 ± 1.4% of control, n = 5, p = 0.02, Figures 5A right, C; nifedipine 30 μM, 17 ± 3.7% of control, n = 5, p = 0.0007, Figures S1E, 5C). This indicates a prevalent role of L-type VGCCs in cholinergic release of GABA from A17 cells, contrary to their limited participation when glutamate is the excitatory neurotransmitter (verapamil 20 μM, 91.8 ± 4.1% of control responses to glutamate [200 μM, 200 ms], n = 3, Figure S1F; see also Chávez et al., 2006; Grimes et al., 2009). On the other hand, Ca2+-induced Ca2+ release (CICR) from intracellular stores could also contribute by enhancing the cytoplasmatic Ca2+ concentration after an initial influx of the ion, as reported for glutamate-evoked GABA release from A17 cells (Chávez et al., 2006; Chávez and Diamond, 2008). Indeed, perfusion with an antagonist of ryanodine receptors produced a significant decrease in the amplitude of IPSCs triggered by ACh (ruthenium red, RR 40 μM, 26.7 ± 3% of control, n = 5, p = 0.002; Figures 5B left, C). Consistently, depletion of Ca2+ from the endoplasmic reticulum using the Ca2+-ATPase antagonist cyclopiazonic acid (CPA 30 μM) or the ryanodine receptors agonist 4-chloro-methyl-cresol (4-CMC 500 μM) significantly diminished IPSCs evoked by ACh in RB cells (35.5 ± 5.6% and 50.4 ± 6.3% of control response, p = 0.043 and 0.049, respectively, n = 4 for both conditions; Figures 5B,C).

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