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Endocannabinoids Mediate Muscarinic Acetylcholine Receptor-Dependent Long-Term Depression in the Adult Medial Prefrontal Cortex.

Martin HG, Bernabeu A, Lassalle O, Bouille C, Beurrier C, Pelissier-Alicot AL, Manzoni OJ - Front Cell Neurosci (2015)

Bottom Line: Cholinergic inputs into the prefrontal cortex (PFC) are associated with attention and cognition; however there is evidence that acetylcholine also has a role in PFC dependent learning and memory.Yet, when endogenous acetylcholine was released from local cholinergic afferents in the PFC using optogenetics, it failed to trigger eCB-LTD.Together these results shed new light on the mechanisms of synaptic plasticity in the adult PFC and expand on the actions of endogenous cholinergic signaling.

View Article: PubMed Central - PubMed

Affiliation: Aix-Marseille Université Marseille, France ; Institut de Neurobiologie de la Méditerranée UMR_S 901 Marseille, France ; INMED UMR_S 901 Marseille, France.

ABSTRACT
Cholinergic inputs into the prefrontal cortex (PFC) are associated with attention and cognition; however there is evidence that acetylcholine also has a role in PFC dependent learning and memory. Muscarinic acetylcholine receptors (mAChR) in the PFC can induce synaptic plasticity, but the underlying mechanisms remain either opaque or unresolved. We have characterized a form of mAChR mediated long-term depression (LTD) at glutamatergic synapses of layer 5 principal neurons in the adult medial PFC. This mAChR LTD is induced with the mAChR agonist carbachol and inhibited by selective M1 mAChR antagonists. In contrast to other cortical regions, we find that this M1 mAChR mediated LTD is coupled to endogenous cannabinoid (eCB) signaling. Inhibition of the principal eCB CB1 receptor blocked carbachol induced LTD in both rats and mice. Furthermore, when challenged with a sub-threshold carbachol application, LTD was induced in slices pretreated with the monoacylglycerol lipase (MAGL) inhibitor JZL184, suggesting that the eCB 2-arachidonylglyerol (2-AG) mediates M1 mAChR LTD. Yet, when endogenous acetylcholine was released from local cholinergic afferents in the PFC using optogenetics, it failed to trigger eCB-LTD. However coupling patterned optical and electrical stimulation to generate local synaptic signaling allowed the reliable induction of LTD. The light-electrical pairing induced LTD was M1 mAChR and CB1 receptor mediated. This shows for the first time that connecting excitatory synaptic activity with coincident endogenously released acetylcholine controls synaptic gain via eCB signaling. Together these results shed new light on the mechanisms of synaptic plasticity in the adult PFC and expand on the actions of endogenous cholinergic signaling.

No MeSH data available.


Related in: MedlinePlus

Endogenously released acetylcholine coupled to excitatory synaptic activity induces CB1R mediated LTD in mouse mPFC. (A) Time course of normalized field EPSPs in ChR2:ACh mouse challenged with a coupled light—EPSP 1 Hz stimulation train (1200 pulses (blue bar), protocol above). Coupled light and EPSP time course (clear circles, n = 11), light alone without EPSP time course (gray, n = 5). (B) Normalized field EPSPs from individual experiments at baseline (Pre) and 40 min after (Post) light—EPSP stimulation protocol, in black group average (**P < 0.01). Above: example traces pre (black) and post (orange) light—EPSP protocol (scale bar: 5 ms, 0.1 mV). (C) Summary plot of percent LTD calculated 40 min after end of stimulation protocol (*P < 0.05). (D) ChR2:ACh mouse field EPSP time course challenged with light—EPSP stimulation protocol (blue bar) in presence of M1 mAChR antagonist pirenzepine (0.5 μM, n = 5). Example traces above, pre (black) and post (orange) stimulation protocol. (E) Time course showing field EPSPs in response to light/EPSP stimulation protocol in ChR2:ACh mouse in presence of CB1R antagonist AM251 (4 μM, n = 6). (F) Summary bar chart of percent LTD 40 min after light—EPSP stimulation protocol.
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Figure 5: Endogenously released acetylcholine coupled to excitatory synaptic activity induces CB1R mediated LTD in mouse mPFC. (A) Time course of normalized field EPSPs in ChR2:ACh mouse challenged with a coupled light—EPSP 1 Hz stimulation train (1200 pulses (blue bar), protocol above). Coupled light and EPSP time course (clear circles, n = 11), light alone without EPSP time course (gray, n = 5). (B) Normalized field EPSPs from individual experiments at baseline (Pre) and 40 min after (Post) light—EPSP stimulation protocol, in black group average (**P < 0.01). Above: example traces pre (black) and post (orange) light—EPSP protocol (scale bar: 5 ms, 0.1 mV). (C) Summary plot of percent LTD calculated 40 min after end of stimulation protocol (*P < 0.05). (D) ChR2:ACh mouse field EPSP time course challenged with light—EPSP stimulation protocol (blue bar) in presence of M1 mAChR antagonist pirenzepine (0.5 μM, n = 5). Example traces above, pre (black) and post (orange) stimulation protocol. (E) Time course showing field EPSPs in response to light/EPSP stimulation protocol in ChR2:ACh mouse in presence of CB1R antagonist AM251 (4 μM, n = 6). (F) Summary bar chart of percent LTD 40 min after light—EPSP stimulation protocol.

Mentions: Cholinergic afferents from the basal forebrain richly innervate the mPFC (Figure 1A). Therefore we asked if endogenous release of acetylcholine might also induce M1 mAChR mediated LTD. We adopted an optogenetic approach, using mice that selectively express channelrhodopsin in cholinergic neurons (ChR2:ACh mouse) and stimulated local acetylcholine release in the mPFC with a fiber optic. Previously, a 20 min electrical paired-pulse 1 Hz protocol has proven successful in inducing M1 mAChR LTD in juvenile animals (Volk et al., 2007; Huang and Hsu, 2010). We adapted the same protocol to our light induced acetylcholine release experiments, stimulating paired-pulse release (50 ms interval) at 1 Hz for 20 min. However we found that light induced acetylcholine release alone did not cause a depression in responses from layer 5 neurons (Figure 5A). Field EPSPs 40 min after the stimulation protocol were not different from baseline values (100.5 ± 9.9%, n = 5). A possible confounding issue in our light protocol is that compared to our carbachol protocol, there is no concurrent EPSP induction during the acetylcholine challenge. Hence, we wondered if coupling our light protocol with a single evoked EPSP timed to the second light pulse might permit LTD induction (Figure 5A, insert). We incubated our slices in the NMDAR antagonist D-AP5 (50 μM) to avoid any low frequency stimulation effects and repeated the 1 Hz paired-pulse protocol with a coincident EPSP. In this scenario we saw a strong depression of field EPSPs that persisted for the length of the recording (Figure 5A). On average responses as a percent of baseline were depressed to 75.3 ± 7.2% (n = 11) 40 min after the coupled light EPSP protocol. We analyzed individual experiments before and after the protocol and found a significant depression in responses (Figure 5B; t(10) = 4.12, P = 0.002). Importantly when we repeated the same protocol without light induced acetylcholine release we failed to observe a depression of responses (fEPSP percent of baseline: 108.0 ± 3.0%, n = 3). We compared the percent LTD in the ChR2:ACh mouse with light and/or evoked EPSP with a control ChR2 mouse challenged with the same light—EPSP protocol and identified a significant effect (Figure 5C; one-way ANOVA F(3,20) = 4.73, P = 0.012). Post hoc analysis indicated this was due to a significant depression in ChR2:ACh mice with the coupled light—EPSP protocol that was not found in ChR2 mice or in mice without coupled light and synaptic activity (P < 0.05, Holm-Sidak’s multiple comparisons test). Thus the induction of endogenous acetylcholine mediated LTD in the mPFC requires the coupling of acetylcholine release with a coincident post-synaptic potential.


Endocannabinoids Mediate Muscarinic Acetylcholine Receptor-Dependent Long-Term Depression in the Adult Medial Prefrontal Cortex.

Martin HG, Bernabeu A, Lassalle O, Bouille C, Beurrier C, Pelissier-Alicot AL, Manzoni OJ - Front Cell Neurosci (2015)

Endogenously released acetylcholine coupled to excitatory synaptic activity induces CB1R mediated LTD in mouse mPFC. (A) Time course of normalized field EPSPs in ChR2:ACh mouse challenged with a coupled light—EPSP 1 Hz stimulation train (1200 pulses (blue bar), protocol above). Coupled light and EPSP time course (clear circles, n = 11), light alone without EPSP time course (gray, n = 5). (B) Normalized field EPSPs from individual experiments at baseline (Pre) and 40 min after (Post) light—EPSP stimulation protocol, in black group average (**P < 0.01). Above: example traces pre (black) and post (orange) light—EPSP protocol (scale bar: 5 ms, 0.1 mV). (C) Summary plot of percent LTD calculated 40 min after end of stimulation protocol (*P < 0.05). (D) ChR2:ACh mouse field EPSP time course challenged with light—EPSP stimulation protocol (blue bar) in presence of M1 mAChR antagonist pirenzepine (0.5 μM, n = 5). Example traces above, pre (black) and post (orange) stimulation protocol. (E) Time course showing field EPSPs in response to light/EPSP stimulation protocol in ChR2:ACh mouse in presence of CB1R antagonist AM251 (4 μM, n = 6). (F) Summary bar chart of percent LTD 40 min after light—EPSP stimulation protocol.
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Figure 5: Endogenously released acetylcholine coupled to excitatory synaptic activity induces CB1R mediated LTD in mouse mPFC. (A) Time course of normalized field EPSPs in ChR2:ACh mouse challenged with a coupled light—EPSP 1 Hz stimulation train (1200 pulses (blue bar), protocol above). Coupled light and EPSP time course (clear circles, n = 11), light alone without EPSP time course (gray, n = 5). (B) Normalized field EPSPs from individual experiments at baseline (Pre) and 40 min after (Post) light—EPSP stimulation protocol, in black group average (**P < 0.01). Above: example traces pre (black) and post (orange) light—EPSP protocol (scale bar: 5 ms, 0.1 mV). (C) Summary plot of percent LTD calculated 40 min after end of stimulation protocol (*P < 0.05). (D) ChR2:ACh mouse field EPSP time course challenged with light—EPSP stimulation protocol (blue bar) in presence of M1 mAChR antagonist pirenzepine (0.5 μM, n = 5). Example traces above, pre (black) and post (orange) stimulation protocol. (E) Time course showing field EPSPs in response to light/EPSP stimulation protocol in ChR2:ACh mouse in presence of CB1R antagonist AM251 (4 μM, n = 6). (F) Summary bar chart of percent LTD 40 min after light—EPSP stimulation protocol.
Mentions: Cholinergic afferents from the basal forebrain richly innervate the mPFC (Figure 1A). Therefore we asked if endogenous release of acetylcholine might also induce M1 mAChR mediated LTD. We adopted an optogenetic approach, using mice that selectively express channelrhodopsin in cholinergic neurons (ChR2:ACh mouse) and stimulated local acetylcholine release in the mPFC with a fiber optic. Previously, a 20 min electrical paired-pulse 1 Hz protocol has proven successful in inducing M1 mAChR LTD in juvenile animals (Volk et al., 2007; Huang and Hsu, 2010). We adapted the same protocol to our light induced acetylcholine release experiments, stimulating paired-pulse release (50 ms interval) at 1 Hz for 20 min. However we found that light induced acetylcholine release alone did not cause a depression in responses from layer 5 neurons (Figure 5A). Field EPSPs 40 min after the stimulation protocol were not different from baseline values (100.5 ± 9.9%, n = 5). A possible confounding issue in our light protocol is that compared to our carbachol protocol, there is no concurrent EPSP induction during the acetylcholine challenge. Hence, we wondered if coupling our light protocol with a single evoked EPSP timed to the second light pulse might permit LTD induction (Figure 5A, insert). We incubated our slices in the NMDAR antagonist D-AP5 (50 μM) to avoid any low frequency stimulation effects and repeated the 1 Hz paired-pulse protocol with a coincident EPSP. In this scenario we saw a strong depression of field EPSPs that persisted for the length of the recording (Figure 5A). On average responses as a percent of baseline were depressed to 75.3 ± 7.2% (n = 11) 40 min after the coupled light EPSP protocol. We analyzed individual experiments before and after the protocol and found a significant depression in responses (Figure 5B; t(10) = 4.12, P = 0.002). Importantly when we repeated the same protocol without light induced acetylcholine release we failed to observe a depression of responses (fEPSP percent of baseline: 108.0 ± 3.0%, n = 3). We compared the percent LTD in the ChR2:ACh mouse with light and/or evoked EPSP with a control ChR2 mouse challenged with the same light—EPSP protocol and identified a significant effect (Figure 5C; one-way ANOVA F(3,20) = 4.73, P = 0.012). Post hoc analysis indicated this was due to a significant depression in ChR2:ACh mice with the coupled light—EPSP protocol that was not found in ChR2 mice or in mice without coupled light and synaptic activity (P < 0.05, Holm-Sidak’s multiple comparisons test). Thus the induction of endogenous acetylcholine mediated LTD in the mPFC requires the coupling of acetylcholine release with a coincident post-synaptic potential.

Bottom Line: Cholinergic inputs into the prefrontal cortex (PFC) are associated with attention and cognition; however there is evidence that acetylcholine also has a role in PFC dependent learning and memory.Yet, when endogenous acetylcholine was released from local cholinergic afferents in the PFC using optogenetics, it failed to trigger eCB-LTD.Together these results shed new light on the mechanisms of synaptic plasticity in the adult PFC and expand on the actions of endogenous cholinergic signaling.

View Article: PubMed Central - PubMed

Affiliation: Aix-Marseille Université Marseille, France ; Institut de Neurobiologie de la Méditerranée UMR_S 901 Marseille, France ; INMED UMR_S 901 Marseille, France.

ABSTRACT
Cholinergic inputs into the prefrontal cortex (PFC) are associated with attention and cognition; however there is evidence that acetylcholine also has a role in PFC dependent learning and memory. Muscarinic acetylcholine receptors (mAChR) in the PFC can induce synaptic plasticity, but the underlying mechanisms remain either opaque or unresolved. We have characterized a form of mAChR mediated long-term depression (LTD) at glutamatergic synapses of layer 5 principal neurons in the adult medial PFC. This mAChR LTD is induced with the mAChR agonist carbachol and inhibited by selective M1 mAChR antagonists. In contrast to other cortical regions, we find that this M1 mAChR mediated LTD is coupled to endogenous cannabinoid (eCB) signaling. Inhibition of the principal eCB CB1 receptor blocked carbachol induced LTD in both rats and mice. Furthermore, when challenged with a sub-threshold carbachol application, LTD was induced in slices pretreated with the monoacylglycerol lipase (MAGL) inhibitor JZL184, suggesting that the eCB 2-arachidonylglyerol (2-AG) mediates M1 mAChR LTD. Yet, when endogenous acetylcholine was released from local cholinergic afferents in the PFC using optogenetics, it failed to trigger eCB-LTD. However coupling patterned optical and electrical stimulation to generate local synaptic signaling allowed the reliable induction of LTD. The light-electrical pairing induced LTD was M1 mAChR and CB1 receptor mediated. This shows for the first time that connecting excitatory synaptic activity with coincident endogenously released acetylcholine controls synaptic gain via eCB signaling. Together these results shed new light on the mechanisms of synaptic plasticity in the adult PFC and expand on the actions of endogenous cholinergic signaling.

No MeSH data available.


Related in: MedlinePlus