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Muscarinic modulation of high frequency oscillations in pedunculopontine neurons.

Kezunovic N, Hyde J, Goitia B, Bisagno V, Urbano FJ, Garcia-Rill E - Front Neurol (2013)

Bottom Line: We then tested the effects of the G-protein antagonist guanosine 5'-[β-thio] diphosphate trilithium salt (GDP-β-S), and the G-protein agonist 5'-[γ-thio] triphosphate trilithium salt (GTP-γ-S).We found, using a three-step protocol in voltage-clamp mode, that the increase in the frequency of oscillations induced by M2 cholinergic receptors was linked to a voltage-dependent G-protein mechanism.In summary, these results suggest that persistent cholinergic input creates a permissive activation state in the PPN that allows high frequency P/Q-type calcium channel-mediated gamma oscillations to occur.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology and Developmental Sciences, Center for Translational Neuroscience, University of Arkansas for Medical Sciences , Little Rock, AR , USA.

ABSTRACT
We previously reported that persistent application of the non-specific cholinergic agonist carbachol (CAR) increased the frequency of calcium channel-mediated oscillatory activity in pedunculopontine nucleus (PPN) neurons, which we identified as dependent on voltage-gated, high-threshold P/Q-type channels. Here, we tested the hypothesis that M2 muscarinic receptors and G-proteins associated with M2 receptors mediate the increase in oscillatory frequency in PPN neurons. We found, using depolarizing ramps, that patch clamped 9-12 day old rat PPN neurons (n = 189) reached their peak oscillatory activity around -20 mV membrane potential. Acute (short duration) application of CAR blocked the oscillatory activity through M2 muscarinic receptors, an effect blocked by atropine. However, persistent (long duration) application of CAR significantly increased the frequency of oscillatory activity in PPN neurons through M2 receptors [40 ± 1 Hz (with CAR) vs. 23 ± 1 Hz (without CAR); p < 0.001]. We then tested the effects of the G-protein antagonist guanosine 5'-[β-thio] diphosphate trilithium salt (GDP-β-S), and the G-protein agonist 5'-[γ-thio] triphosphate trilithium salt (GTP-γ-S). We found, using a three-step protocol in voltage-clamp mode, that the increase in the frequency of oscillations induced by M2 cholinergic receptors was linked to a voltage-dependent G-protein mechanism. In summary, these results suggest that persistent cholinergic input creates a permissive activation state in the PPN that allows high frequency P/Q-type calcium channel-mediated gamma oscillations to occur.

No MeSH data available.


Related in: MedlinePlus

Carbachol-mediated voltage-dependent G-protein modulation of P/Q-type voltage-gated calcium currents in PPN. (A) Three-pulse protocol used to study the voltage dependence of G-protein modulation of calcium currents (ICa) in PPN neurons after blocking N-type channels with ω-conotoxin-GVIA 2.5 μM. In the presence of synaptic receptor blockers (SB: GBZ + STR + AP5 + CNQX), TTX, MEC, and ω-CgTX-GVIA, calcium current density (ICa; pA/pF, black record) was reduced in amplitude by CAR (gray record) when the three-pulse protocol was applied as described in Figure 4. (B) CAR (30 μM) reduced the total amount of current density after the first pulse (before CAR black column, after CAR gray column; Mann–Whitney Rank Sum Test, p < 0.001). (C) Time course of activation (τON) and deactivation (τOFF) of calcium currents, obtained after fitting individual currents to the function y = y0 + a × exp (−[−Time (ms)/τ(ON or OFF)). The effects of CAR on time course were not significantly different (Kruskal–Wallis ANOVA, H = 4.33, p = 0.23). (Abbreviations: CAR, carbachol; SB, synaptic blockers; TTX, tetrodotoxin; MEC, mecamylamine).
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Figure 5: Carbachol-mediated voltage-dependent G-protein modulation of P/Q-type voltage-gated calcium currents in PPN. (A) Three-pulse protocol used to study the voltage dependence of G-protein modulation of calcium currents (ICa) in PPN neurons after blocking N-type channels with ω-conotoxin-GVIA 2.5 μM. In the presence of synaptic receptor blockers (SB: GBZ + STR + AP5 + CNQX), TTX, MEC, and ω-CgTX-GVIA, calcium current density (ICa; pA/pF, black record) was reduced in amplitude by CAR (gray record) when the three-pulse protocol was applied as described in Figure 4. (B) CAR (30 μM) reduced the total amount of current density after the first pulse (before CAR black column, after CAR gray column; Mann–Whitney Rank Sum Test, p < 0.001). (C) Time course of activation (τON) and deactivation (τOFF) of calcium currents, obtained after fitting individual currents to the function y = y0 + a × exp (−[−Time (ms)/τ(ON or OFF)). The effects of CAR on time course were not significantly different (Kruskal–Wallis ANOVA, H = 4.33, p = 0.23). (Abbreviations: CAR, carbachol; SB, synaptic blockers; TTX, tetrodotoxin; MEC, mecamylamine).

Mentions: We then studied the effects of CAR on P/Q-type calcium channels, which are the main high-threshold calcium channels involved in PPN oscillatory activity [i.e., after blocking N-type channels with ω-conotoxin-GVIA 2.5 μM; Ref. (3)]. After bath-applying CAR for >20 min, P/Q-type mediated ICa showed apparently slower activation and deactivation time courses for both the pre-pulse and post-pulse compared to the ω-CgTX-GVIA condition (Figure 5A, τON and τOFF arrows, respectively). In addition, CAR reduced ICa density (pA/pF) from −71 ± 5 to −41 ± 3 pA/pF (Figure 5B, Mann–Whitney Rank Sum Test, p < 0.001). However, mean current density values observed after CAR in the absence or presence of ω-CgTX-GVIA were not significantly different (CAR: 17 ± 5 pA/pF, n = 9; CAR + ω-CgTX-GVIA: 29 ± 3 pA/pF; Student’s t-test, Kruskal–Wallis ANOVA, H = 3.6, p = 0.06). No significant differences were observed when comparing τON/τOFF values (Figure 5C, Kruskal–Wallis ANOVA, H = 4.33, p = 0.23). These results suggest that the effects of CAR in reducing oscillations was entirely through P/Q-type calcium channels.


Muscarinic modulation of high frequency oscillations in pedunculopontine neurons.

Kezunovic N, Hyde J, Goitia B, Bisagno V, Urbano FJ, Garcia-Rill E - Front Neurol (2013)

Carbachol-mediated voltage-dependent G-protein modulation of P/Q-type voltage-gated calcium currents in PPN. (A) Three-pulse protocol used to study the voltage dependence of G-protein modulation of calcium currents (ICa) in PPN neurons after blocking N-type channels with ω-conotoxin-GVIA 2.5 μM. In the presence of synaptic receptor blockers (SB: GBZ + STR + AP5 + CNQX), TTX, MEC, and ω-CgTX-GVIA, calcium current density (ICa; pA/pF, black record) was reduced in amplitude by CAR (gray record) when the three-pulse protocol was applied as described in Figure 4. (B) CAR (30 μM) reduced the total amount of current density after the first pulse (before CAR black column, after CAR gray column; Mann–Whitney Rank Sum Test, p < 0.001). (C) Time course of activation (τON) and deactivation (τOFF) of calcium currents, obtained after fitting individual currents to the function y = y0 + a × exp (−[−Time (ms)/τ(ON or OFF)). The effects of CAR on time course were not significantly different (Kruskal–Wallis ANOVA, H = 4.33, p = 0.23). (Abbreviations: CAR, carbachol; SB, synaptic blockers; TTX, tetrodotoxin; MEC, mecamylamine).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: Carbachol-mediated voltage-dependent G-protein modulation of P/Q-type voltage-gated calcium currents in PPN. (A) Three-pulse protocol used to study the voltage dependence of G-protein modulation of calcium currents (ICa) in PPN neurons after blocking N-type channels with ω-conotoxin-GVIA 2.5 μM. In the presence of synaptic receptor blockers (SB: GBZ + STR + AP5 + CNQX), TTX, MEC, and ω-CgTX-GVIA, calcium current density (ICa; pA/pF, black record) was reduced in amplitude by CAR (gray record) when the three-pulse protocol was applied as described in Figure 4. (B) CAR (30 μM) reduced the total amount of current density after the first pulse (before CAR black column, after CAR gray column; Mann–Whitney Rank Sum Test, p < 0.001). (C) Time course of activation (τON) and deactivation (τOFF) of calcium currents, obtained after fitting individual currents to the function y = y0 + a × exp (−[−Time (ms)/τ(ON or OFF)). The effects of CAR on time course were not significantly different (Kruskal–Wallis ANOVA, H = 4.33, p = 0.23). (Abbreviations: CAR, carbachol; SB, synaptic blockers; TTX, tetrodotoxin; MEC, mecamylamine).
Mentions: We then studied the effects of CAR on P/Q-type calcium channels, which are the main high-threshold calcium channels involved in PPN oscillatory activity [i.e., after blocking N-type channels with ω-conotoxin-GVIA 2.5 μM; Ref. (3)]. After bath-applying CAR for >20 min, P/Q-type mediated ICa showed apparently slower activation and deactivation time courses for both the pre-pulse and post-pulse compared to the ω-CgTX-GVIA condition (Figure 5A, τON and τOFF arrows, respectively). In addition, CAR reduced ICa density (pA/pF) from −71 ± 5 to −41 ± 3 pA/pF (Figure 5B, Mann–Whitney Rank Sum Test, p < 0.001). However, mean current density values observed after CAR in the absence or presence of ω-CgTX-GVIA were not significantly different (CAR: 17 ± 5 pA/pF, n = 9; CAR + ω-CgTX-GVIA: 29 ± 3 pA/pF; Student’s t-test, Kruskal–Wallis ANOVA, H = 3.6, p = 0.06). No significant differences were observed when comparing τON/τOFF values (Figure 5C, Kruskal–Wallis ANOVA, H = 4.33, p = 0.23). These results suggest that the effects of CAR in reducing oscillations was entirely through P/Q-type calcium channels.

Bottom Line: We then tested the effects of the G-protein antagonist guanosine 5'-[β-thio] diphosphate trilithium salt (GDP-β-S), and the G-protein agonist 5'-[γ-thio] triphosphate trilithium salt (GTP-γ-S).We found, using a three-step protocol in voltage-clamp mode, that the increase in the frequency of oscillations induced by M2 cholinergic receptors was linked to a voltage-dependent G-protein mechanism.In summary, these results suggest that persistent cholinergic input creates a permissive activation state in the PPN that allows high frequency P/Q-type calcium channel-mediated gamma oscillations to occur.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology and Developmental Sciences, Center for Translational Neuroscience, University of Arkansas for Medical Sciences , Little Rock, AR , USA.

ABSTRACT
We previously reported that persistent application of the non-specific cholinergic agonist carbachol (CAR) increased the frequency of calcium channel-mediated oscillatory activity in pedunculopontine nucleus (PPN) neurons, which we identified as dependent on voltage-gated, high-threshold P/Q-type channels. Here, we tested the hypothesis that M2 muscarinic receptors and G-proteins associated with M2 receptors mediate the increase in oscillatory frequency in PPN neurons. We found, using depolarizing ramps, that patch clamped 9-12 day old rat PPN neurons (n = 189) reached their peak oscillatory activity around -20 mV membrane potential. Acute (short duration) application of CAR blocked the oscillatory activity through M2 muscarinic receptors, an effect blocked by atropine. However, persistent (long duration) application of CAR significantly increased the frequency of oscillatory activity in PPN neurons through M2 receptors [40 ± 1 Hz (with CAR) vs. 23 ± 1 Hz (without CAR); p < 0.001]. We then tested the effects of the G-protein antagonist guanosine 5'-[β-thio] diphosphate trilithium salt (GDP-β-S), and the G-protein agonist 5'-[γ-thio] triphosphate trilithium salt (GTP-γ-S). We found, using a three-step protocol in voltage-clamp mode, that the increase in the frequency of oscillations induced by M2 cholinergic receptors was linked to a voltage-dependent G-protein mechanism. In summary, these results suggest that persistent cholinergic input creates a permissive activation state in the PPN that allows high frequency P/Q-type calcium channel-mediated gamma oscillations to occur.

No MeSH data available.


Related in: MedlinePlus