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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

Persistent effect of CAR on M1 and M2 muscarinic receptors in the PPN. (A) Representative membrane potential oscillations recorded during a 1-s long ramp (black record on the left) in the presence of SB + TTX + MEC + persistent CAR and PIR. Note the higher frequency of oscillations due to M2 muscarinic receptors being activated. The power spectrum of this recording (right side) shows oscillations at gamma frequency. (B) Representative membrane potential oscillations recorded during a 1-s long ramp (black record on the left) in the presence of SB + TTX + MEC + CAR and MTO. Note slower oscillatory activity in this cell due to M1 muscarinic receptors being activated and M2 receptors being blocked. The power spectrum of this recording (right side) shows oscillations in the beta frequency range, and reduced gamma band activity. (C) Representative membrane oscillations recorded during 1 s long ramp (black record on the left) in the presence of all synaptic blockers including all the cholinergic blockers (MEC + MTO + PIR). The power spectrum on the right side shows oscillatory activity in the beta range, similar to the frequencies observed in the experiment described in (B). (D) Bar graph showing the mean frequency of membrane potential oscillations for the cells recorded under persistent exposure to CAR where one group had only M1 receptors activated (striped column, n = 7), and the other group had only M2 receptors activated (black column, n = 11). Note that both groups of cells were recorded under the same conditions (SB + TTX + MEC), except for the specific M1 vs. M2 receptor blockers (***p < 0.001; abbreviations: CAR, carbachol; SB, synaptic blockers; TTX, tetrodotoxin; MEC, mecamylamine; MTO, methoctramine; PIR, pirenzepine).
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Figure 3: Persistent effect of CAR on M1 and M2 muscarinic receptors in the PPN. (A) Representative membrane potential oscillations recorded during a 1-s long ramp (black record on the left) in the presence of SB + TTX + MEC + persistent CAR and PIR. Note the higher frequency of oscillations due to M2 muscarinic receptors being activated. The power spectrum of this recording (right side) shows oscillations at gamma frequency. (B) Representative membrane potential oscillations recorded during a 1-s long ramp (black record on the left) in the presence of SB + TTX + MEC + CAR and MTO. Note slower oscillatory activity in this cell due to M1 muscarinic receptors being activated and M2 receptors being blocked. The power spectrum of this recording (right side) shows oscillations in the beta frequency range, and reduced gamma band activity. (C) Representative membrane oscillations recorded during 1 s long ramp (black record on the left) in the presence of all synaptic blockers including all the cholinergic blockers (MEC + MTO + PIR). The power spectrum on the right side shows oscillatory activity in the beta range, similar to the frequencies observed in the experiment described in (B). (D) Bar graph showing the mean frequency of membrane potential oscillations for the cells recorded under persistent exposure to CAR where one group had only M1 receptors activated (striped column, n = 7), and the other group had only M2 receptors activated (black column, n = 11). Note that both groups of cells were recorded under the same conditions (SB + TTX + MEC), except for the specific M1 vs. M2 receptor blockers (***p < 0.001; abbreviations: CAR, carbachol; SB, synaptic blockers; TTX, tetrodotoxin; MEC, mecamylamine; MTO, methoctramine; PIR, pirenzepine).

Mentions: We tested the role of M2 receptors using pirenzepine (PIR, 10 μM), after 20 min of CAR application (i.e., CAR-mediated persistent effect). Figure 3A shows an example of membrane oscillations recorded during 1 s long ramps in the presence of CAR + PIR (n = 11). The power spectrum on the right shows the frequency of oscillations to be 33 Hz, in the gamma range. In contrast, cells recorded in a similar extracellular condition, except that the M1 receptor blocker PIR was replaced with the M2 receptor blocker MTO, recordings showed reduced gamma band oscillations induced by 1 s long ramps during exposure to CAR + MTO (Figure 3B; n = 7). The power spectrum of this record showed a PPN neuron with large amplitude oscillations in the beta range (21 Hz), and almost no gamma band activity (Figure 3B, right). Furthermore, when we added all of the cholinergic blockers mentioned above (MEC, PIR, and MTO) to the extracellular solution with synaptic blockers and TTX (Figure 3C; n = 5), the cells oscillated at lower (beta) frequencies, that were similar to those previously reported (3). Statistical analysis showed that PPN cells oscillated significantly faster under persistent application of CAR (Figure 3D) when only M2 receptors were available (MEC + PIR + CAR, 40 ± 1 Hz, black bar, n = 11), compared to when only M1 receptors were available (MEC + MTO + CAR, 23 ± 1 Hz, striped bar, n = 7) (One-way ANOVA; df = 17, t = 8.7, p < 0.001). These data indicate that persistent activation of M2 muscarinic receptors will trigger the activation of intracellular second messengers that ultimately affect calcium channels responsible for mediating gamma frequency oscillatory activity. We then undertook studies to identify the intracellular mechanisms underlying this phenomenon.


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)

Persistent effect of CAR on M1 and M2 muscarinic receptors in the PPN. (A) Representative membrane potential oscillations recorded during a 1-s long ramp (black record on the left) in the presence of SB + TTX + MEC + persistent CAR and PIR. Note the higher frequency of oscillations due to M2 muscarinic receptors being activated. The power spectrum of this recording (right side) shows oscillations at gamma frequency. (B) Representative membrane potential oscillations recorded during a 1-s long ramp (black record on the left) in the presence of SB + TTX + MEC + CAR and MTO. Note slower oscillatory activity in this cell due to M1 muscarinic receptors being activated and M2 receptors being blocked. The power spectrum of this recording (right side) shows oscillations in the beta frequency range, and reduced gamma band activity. (C) Representative membrane oscillations recorded during 1 s long ramp (black record on the left) in the presence of all synaptic blockers including all the cholinergic blockers (MEC + MTO + PIR). The power spectrum on the right side shows oscillatory activity in the beta range, similar to the frequencies observed in the experiment described in (B). (D) Bar graph showing the mean frequency of membrane potential oscillations for the cells recorded under persistent exposure to CAR where one group had only M1 receptors activated (striped column, n = 7), and the other group had only M2 receptors activated (black column, n = 11). Note that both groups of cells were recorded under the same conditions (SB + TTX + MEC), except for the specific M1 vs. M2 receptor blockers (***p < 0.001; abbreviations: CAR, carbachol; SB, synaptic blockers; TTX, tetrodotoxin; MEC, mecamylamine; MTO, methoctramine; PIR, pirenzepine).
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Figure 3: Persistent effect of CAR on M1 and M2 muscarinic receptors in the PPN. (A) Representative membrane potential oscillations recorded during a 1-s long ramp (black record on the left) in the presence of SB + TTX + MEC + persistent CAR and PIR. Note the higher frequency of oscillations due to M2 muscarinic receptors being activated. The power spectrum of this recording (right side) shows oscillations at gamma frequency. (B) Representative membrane potential oscillations recorded during a 1-s long ramp (black record on the left) in the presence of SB + TTX + MEC + CAR and MTO. Note slower oscillatory activity in this cell due to M1 muscarinic receptors being activated and M2 receptors being blocked. The power spectrum of this recording (right side) shows oscillations in the beta frequency range, and reduced gamma band activity. (C) Representative membrane oscillations recorded during 1 s long ramp (black record on the left) in the presence of all synaptic blockers including all the cholinergic blockers (MEC + MTO + PIR). The power spectrum on the right side shows oscillatory activity in the beta range, similar to the frequencies observed in the experiment described in (B). (D) Bar graph showing the mean frequency of membrane potential oscillations for the cells recorded under persistent exposure to CAR where one group had only M1 receptors activated (striped column, n = 7), and the other group had only M2 receptors activated (black column, n = 11). Note that both groups of cells were recorded under the same conditions (SB + TTX + MEC), except for the specific M1 vs. M2 receptor blockers (***p < 0.001; abbreviations: CAR, carbachol; SB, synaptic blockers; TTX, tetrodotoxin; MEC, mecamylamine; MTO, methoctramine; PIR, pirenzepine).
Mentions: We tested the role of M2 receptors using pirenzepine (PIR, 10 μM), after 20 min of CAR application (i.e., CAR-mediated persistent effect). Figure 3A shows an example of membrane oscillations recorded during 1 s long ramps in the presence of CAR + PIR (n = 11). The power spectrum on the right shows the frequency of oscillations to be 33 Hz, in the gamma range. In contrast, cells recorded in a similar extracellular condition, except that the M1 receptor blocker PIR was replaced with the M2 receptor blocker MTO, recordings showed reduced gamma band oscillations induced by 1 s long ramps during exposure to CAR + MTO (Figure 3B; n = 7). The power spectrum of this record showed a PPN neuron with large amplitude oscillations in the beta range (21 Hz), and almost no gamma band activity (Figure 3B, right). Furthermore, when we added all of the cholinergic blockers mentioned above (MEC, PIR, and MTO) to the extracellular solution with synaptic blockers and TTX (Figure 3C; n = 5), the cells oscillated at lower (beta) frequencies, that were similar to those previously reported (3). Statistical analysis showed that PPN cells oscillated significantly faster under persistent application of CAR (Figure 3D) when only M2 receptors were available (MEC + PIR + CAR, 40 ± 1 Hz, black bar, n = 11), compared to when only M1 receptors were available (MEC + MTO + CAR, 23 ± 1 Hz, striped bar, n = 7) (One-way ANOVA; df = 17, t = 8.7, p < 0.001). These data indicate that persistent activation of M2 muscarinic receptors will trigger the activation of intracellular second messengers that ultimately affect calcium channels responsible for mediating gamma frequency oscillatory activity. We then undertook studies to identify the intracellular mechanisms underlying this phenomenon.

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