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Membrane-derived phospholipids control synaptic neurotransmission and plasticity.

García-Morales V, Montero F, González-Forero D, Rodríguez-Bey G, Gómez-Pérez L, Medialdea-Wandossell MJ, Domínguez-Vías G, García-Verdugo JM, Moreno-López B - PLoS Biol. (2015)

Bottom Line: LPA increased myosin light chain phosphorylation, which is known to trigger actomyosin contraction, and reduced the number of synaptic vesicles docked to active zones in excitatory boutons.However, LPA-induced depression of GABAergic transmission was correlated with an endocytosis-independent reduction of GABAA receptors, possibly by GABAAγ2 dephosphorylation and subsequent increased lateral diffusion.We propose that lysophospholipids serve as potential local messengers that tune synaptic strength to precedent activity of the neuron.

View Article: PubMed Central - PubMed

Affiliation: Grupo de Neurodegeneración y Neuroreparación (GRUNEDERE), Área de Fisiología, Facultad de Medicina, Universidad de Cádiz, Cádiz, Spain.

ABSTRACT
Synaptic communication is a dynamic process that is key to the regulation of neuronal excitability and information processing in the brain. To date, however, the molecular signals controlling synaptic dynamics have been poorly understood. Membrane-derived bioactive phospholipids are potential candidates to control short-term tuning of synaptic signaling, a plastic event essential for information processing at both the cellular and neuronal network levels in the brain. Here, we showed that phospholipids affect excitatory and inhibitory neurotransmission by different degrees, loci, and mechanisms of action. Signaling triggered by lysophosphatidic acid (LPA) evoked rapid and reversible depression of excitatory and inhibitory postsynaptic currents. At excitatory synapses, LPA-induced depression depended on LPA1/Gαi/o-protein/phospholipase C/myosin light chain kinase cascade at the presynaptic site. LPA increased myosin light chain phosphorylation, which is known to trigger actomyosin contraction, and reduced the number of synaptic vesicles docked to active zones in excitatory boutons. At inhibitory synapses, postsynaptic LPA signaling led to dephosphorylation, and internalization of the GABAAγ2 subunit through the LPA1/Gα12/13-protein/RhoA/Rho kinase/calcineurin pathway. However, LPA-induced depression of GABAergic transmission was correlated with an endocytosis-independent reduction of GABAA receptors, possibly by GABAAγ2 dephosphorylation and subsequent increased lateral diffusion. Furthermore, endogenous LPA signaling, mainly via LPA1, mediated activity-dependent inhibitory depression in a model of experimental synaptic plasticity. Finally, LPA signaling, most likely restraining the excitatory drive incoming to motoneurons, regulated performance of motor output commands, a basic brain processing task. We propose that lysophospholipids serve as potential local messengers that tune synaptic strength to precedent activity of the neuron.

No MeSH data available.


Related in: MedlinePlus

LPA rearranges SVs at excitatory boutons in a MLCK-dependent manner.(A) Western blot of phosphorylated and total MLC protein levels (denoted as pMLC and MLC, respectively) in the HN of neonatal brain stem slices incubated for 10 min in aCSF alone (control) or supplemented with either LPA (2.5 μM), vehicle (0.2% DMSO), LPA + vehicle, or LPA + ML-7 (10 μM). α-tubulin (α-tub) expression was the internal loading reference. (B) Histogram showing the average ratio of pMLC to total MLC densitometric intensity for the control and treated slices. Ratio values were normalized relative to the control group. Columns represent the average of at least three independent experiments. *p < 0.05, one-way ANOVA on Ranks relative to control condition. (C) eEPSCsAMPA recorded from two HMNs in normal aCSF and after 10 min bath perfusion with the indicated combination of drugs. (D) Average eEPSCAMPA amplitude for the ML-7 (n = 5 HMNs) and LPA+ML-7 (n = 7 HMNs) treated groups of HMNs compared with their respective pretreatment controls (before). (E) eEPSCsAMPA evoked in HMNs by paired-pulse stimulation of VLRF before and following treatment with LPA and finally after coaddition of ML-7. (F) Changes in PPR of eEPSCsAMPA measured in HMNs exposed sequentially to LPA and LPA+ML-7. *p < 0.05, one-way RM-ANOVA relative to the control condition in D and F. (G, H) Electron micrographs of two S-type boutons (containing spherical vesicles) with asymmetric synaptic contacts on the somatic membrane of a HMN depicting details of the procedure used to examine topographically the numerical changes in SVs. The number of SVs was counted in three zones, each 0.1 μm wide, parallel to the membrane of the synaptic cleft and at successively greater distances from the a.z. (G). The first region (red dashed line) encloses an area directly adjacent to the a.z. membrane. The intermediate region (orange dashed line) was located in the interval from 0.1 μm to 0.2 μm away from the a.z. Finally, the more distant region (white dashed line) occupied an area corresponding to the distance interval from 0.2 μm to 0.3 μm. The total number of SVs contained in each bouton section was also quantified (H). (I–K) Electron micrographs of S-type boutons in contact with the somatic membrane of HMNs from neonatal rats following incubation (10 min) of brain stem slices in aCSF alone (control) or supplemented with LPA or LPA+ML-7 at concentrations indicated in A. The boxed region (red dashed line) encloses the area directly adjacent to the a.z. membrane. (L) Quantitative changes in the number of SVs (expressed as percentage change from control) are shown in each spatial compartment. Histogram bins indicate distances from the a.z. as indicated in the legend. Increment in the number of the total pool of SVs per bouton section is also illustrated (yellow bars). (M) High-magnification electron microscopy images showing in detail the SVs (membranes in contact with the presynaptic density) docked to the a.z. (arrowheads). Scale bars: G–K, 200 nm; M, 100 nm. (N) Histogram showing the linear density of docked SVs per μm of a.z. under the indicated conditions. Control, n = 133 boutons/a.z.; vehicle, n = 54/104 boutons/a.z.; LPA, n = 102 boutons/a.z.; LPA plus ML-7, n = 102 boutons/a.z. *p < 0.05, one-way ANOVA relative to the control condition. Experiments and analysis were performed as in our previous published study [8]. Plots data can be found in S1 Data.
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pbio.1002153.g005: LPA rearranges SVs at excitatory boutons in a MLCK-dependent manner.(A) Western blot of phosphorylated and total MLC protein levels (denoted as pMLC and MLC, respectively) in the HN of neonatal brain stem slices incubated for 10 min in aCSF alone (control) or supplemented with either LPA (2.5 μM), vehicle (0.2% DMSO), LPA + vehicle, or LPA + ML-7 (10 μM). α-tubulin (α-tub) expression was the internal loading reference. (B) Histogram showing the average ratio of pMLC to total MLC densitometric intensity for the control and treated slices. Ratio values were normalized relative to the control group. Columns represent the average of at least three independent experiments. *p < 0.05, one-way ANOVA on Ranks relative to control condition. (C) eEPSCsAMPA recorded from two HMNs in normal aCSF and after 10 min bath perfusion with the indicated combination of drugs. (D) Average eEPSCAMPA amplitude for the ML-7 (n = 5 HMNs) and LPA+ML-7 (n = 7 HMNs) treated groups of HMNs compared with their respective pretreatment controls (before). (E) eEPSCsAMPA evoked in HMNs by paired-pulse stimulation of VLRF before and following treatment with LPA and finally after coaddition of ML-7. (F) Changes in PPR of eEPSCsAMPA measured in HMNs exposed sequentially to LPA and LPA+ML-7. *p < 0.05, one-way RM-ANOVA relative to the control condition in D and F. (G, H) Electron micrographs of two S-type boutons (containing spherical vesicles) with asymmetric synaptic contacts on the somatic membrane of a HMN depicting details of the procedure used to examine topographically the numerical changes in SVs. The number of SVs was counted in three zones, each 0.1 μm wide, parallel to the membrane of the synaptic cleft and at successively greater distances from the a.z. (G). The first region (red dashed line) encloses an area directly adjacent to the a.z. membrane. The intermediate region (orange dashed line) was located in the interval from 0.1 μm to 0.2 μm away from the a.z. Finally, the more distant region (white dashed line) occupied an area corresponding to the distance interval from 0.2 μm to 0.3 μm. The total number of SVs contained in each bouton section was also quantified (H). (I–K) Electron micrographs of S-type boutons in contact with the somatic membrane of HMNs from neonatal rats following incubation (10 min) of brain stem slices in aCSF alone (control) or supplemented with LPA or LPA+ML-7 at concentrations indicated in A. The boxed region (red dashed line) encloses the area directly adjacent to the a.z. membrane. (L) Quantitative changes in the number of SVs (expressed as percentage change from control) are shown in each spatial compartment. Histogram bins indicate distances from the a.z. as indicated in the legend. Increment in the number of the total pool of SVs per bouton section is also illustrated (yellow bars). (M) High-magnification electron microscopy images showing in detail the SVs (membranes in contact with the presynaptic density) docked to the a.z. (arrowheads). Scale bars: G–K, 200 nm; M, 100 nm. (N) Histogram showing the linear density of docked SVs per μm of a.z. under the indicated conditions. Control, n = 133 boutons/a.z.; vehicle, n = 54/104 boutons/a.z.; LPA, n = 102 boutons/a.z.; LPA plus ML-7, n = 102 boutons/a.z. *p < 0.05, one-way ANOVA relative to the control condition. Experiments and analysis were performed as in our previous published study [8]. Plots data can be found in S1 Data.

Mentions: LPA induces smooth muscle contraction in a PLC-dependent, ROCK-independent manner that involves myosin light chain (MLC) phosphorylation by MLC kinase (MLCK) [31]. These findings point to MLCK as a potential kinase mediating the presynaptic action of LPA on excitatory neurotransmission. Accordingly, LPA increased the p-MLC:MLC ratio in the HN relative to aCSF-incubated brain stem slices, which was fully prevented by coincubation with the specific MLCK inhibitor ML-7 (Fig 5A and 5B). In concordance, though ML-7 per se did not alter the amplitude of eEPSCsAMPA, as we also recently reported [8], it fully suppressed LPA-induced alterations on eEPSCsAMPA amplitude and PPR (Fig 5C–5F). This further supports MLCK as a main molecular substrate activated by LPA signaling within excitatory presynaptic terminals.


Membrane-derived phospholipids control synaptic neurotransmission and plasticity.

García-Morales V, Montero F, González-Forero D, Rodríguez-Bey G, Gómez-Pérez L, Medialdea-Wandossell MJ, Domínguez-Vías G, García-Verdugo JM, Moreno-López B - PLoS Biol. (2015)

LPA rearranges SVs at excitatory boutons in a MLCK-dependent manner.(A) Western blot of phosphorylated and total MLC protein levels (denoted as pMLC and MLC, respectively) in the HN of neonatal brain stem slices incubated for 10 min in aCSF alone (control) or supplemented with either LPA (2.5 μM), vehicle (0.2% DMSO), LPA + vehicle, or LPA + ML-7 (10 μM). α-tubulin (α-tub) expression was the internal loading reference. (B) Histogram showing the average ratio of pMLC to total MLC densitometric intensity for the control and treated slices. Ratio values were normalized relative to the control group. Columns represent the average of at least three independent experiments. *p < 0.05, one-way ANOVA on Ranks relative to control condition. (C) eEPSCsAMPA recorded from two HMNs in normal aCSF and after 10 min bath perfusion with the indicated combination of drugs. (D) Average eEPSCAMPA amplitude for the ML-7 (n = 5 HMNs) and LPA+ML-7 (n = 7 HMNs) treated groups of HMNs compared with their respective pretreatment controls (before). (E) eEPSCsAMPA evoked in HMNs by paired-pulse stimulation of VLRF before and following treatment with LPA and finally after coaddition of ML-7. (F) Changes in PPR of eEPSCsAMPA measured in HMNs exposed sequentially to LPA and LPA+ML-7. *p < 0.05, one-way RM-ANOVA relative to the control condition in D and F. (G, H) Electron micrographs of two S-type boutons (containing spherical vesicles) with asymmetric synaptic contacts on the somatic membrane of a HMN depicting details of the procedure used to examine topographically the numerical changes in SVs. The number of SVs was counted in three zones, each 0.1 μm wide, parallel to the membrane of the synaptic cleft and at successively greater distances from the a.z. (G). The first region (red dashed line) encloses an area directly adjacent to the a.z. membrane. The intermediate region (orange dashed line) was located in the interval from 0.1 μm to 0.2 μm away from the a.z. Finally, the more distant region (white dashed line) occupied an area corresponding to the distance interval from 0.2 μm to 0.3 μm. The total number of SVs contained in each bouton section was also quantified (H). (I–K) Electron micrographs of S-type boutons in contact with the somatic membrane of HMNs from neonatal rats following incubation (10 min) of brain stem slices in aCSF alone (control) or supplemented with LPA or LPA+ML-7 at concentrations indicated in A. The boxed region (red dashed line) encloses the area directly adjacent to the a.z. membrane. (L) Quantitative changes in the number of SVs (expressed as percentage change from control) are shown in each spatial compartment. Histogram bins indicate distances from the a.z. as indicated in the legend. Increment in the number of the total pool of SVs per bouton section is also illustrated (yellow bars). (M) High-magnification electron microscopy images showing in detail the SVs (membranes in contact with the presynaptic density) docked to the a.z. (arrowheads). Scale bars: G–K, 200 nm; M, 100 nm. (N) Histogram showing the linear density of docked SVs per μm of a.z. under the indicated conditions. Control, n = 133 boutons/a.z.; vehicle, n = 54/104 boutons/a.z.; LPA, n = 102 boutons/a.z.; LPA plus ML-7, n = 102 boutons/a.z. *p < 0.05, one-way ANOVA relative to the control condition. Experiments and analysis were performed as in our previous published study [8]. Plots data can be found in S1 Data.
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pbio.1002153.g005: LPA rearranges SVs at excitatory boutons in a MLCK-dependent manner.(A) Western blot of phosphorylated and total MLC protein levels (denoted as pMLC and MLC, respectively) in the HN of neonatal brain stem slices incubated for 10 min in aCSF alone (control) or supplemented with either LPA (2.5 μM), vehicle (0.2% DMSO), LPA + vehicle, or LPA + ML-7 (10 μM). α-tubulin (α-tub) expression was the internal loading reference. (B) Histogram showing the average ratio of pMLC to total MLC densitometric intensity for the control and treated slices. Ratio values were normalized relative to the control group. Columns represent the average of at least three independent experiments. *p < 0.05, one-way ANOVA on Ranks relative to control condition. (C) eEPSCsAMPA recorded from two HMNs in normal aCSF and after 10 min bath perfusion with the indicated combination of drugs. (D) Average eEPSCAMPA amplitude for the ML-7 (n = 5 HMNs) and LPA+ML-7 (n = 7 HMNs) treated groups of HMNs compared with their respective pretreatment controls (before). (E) eEPSCsAMPA evoked in HMNs by paired-pulse stimulation of VLRF before and following treatment with LPA and finally after coaddition of ML-7. (F) Changes in PPR of eEPSCsAMPA measured in HMNs exposed sequentially to LPA and LPA+ML-7. *p < 0.05, one-way RM-ANOVA relative to the control condition in D and F. (G, H) Electron micrographs of two S-type boutons (containing spherical vesicles) with asymmetric synaptic contacts on the somatic membrane of a HMN depicting details of the procedure used to examine topographically the numerical changes in SVs. The number of SVs was counted in three zones, each 0.1 μm wide, parallel to the membrane of the synaptic cleft and at successively greater distances from the a.z. (G). The first region (red dashed line) encloses an area directly adjacent to the a.z. membrane. The intermediate region (orange dashed line) was located in the interval from 0.1 μm to 0.2 μm away from the a.z. Finally, the more distant region (white dashed line) occupied an area corresponding to the distance interval from 0.2 μm to 0.3 μm. The total number of SVs contained in each bouton section was also quantified (H). (I–K) Electron micrographs of S-type boutons in contact with the somatic membrane of HMNs from neonatal rats following incubation (10 min) of brain stem slices in aCSF alone (control) or supplemented with LPA or LPA+ML-7 at concentrations indicated in A. The boxed region (red dashed line) encloses the area directly adjacent to the a.z. membrane. (L) Quantitative changes in the number of SVs (expressed as percentage change from control) are shown in each spatial compartment. Histogram bins indicate distances from the a.z. as indicated in the legend. Increment in the number of the total pool of SVs per bouton section is also illustrated (yellow bars). (M) High-magnification electron microscopy images showing in detail the SVs (membranes in contact with the presynaptic density) docked to the a.z. (arrowheads). Scale bars: G–K, 200 nm; M, 100 nm. (N) Histogram showing the linear density of docked SVs per μm of a.z. under the indicated conditions. Control, n = 133 boutons/a.z.; vehicle, n = 54/104 boutons/a.z.; LPA, n = 102 boutons/a.z.; LPA plus ML-7, n = 102 boutons/a.z. *p < 0.05, one-way ANOVA relative to the control condition. Experiments and analysis were performed as in our previous published study [8]. Plots data can be found in S1 Data.
Mentions: LPA induces smooth muscle contraction in a PLC-dependent, ROCK-independent manner that involves myosin light chain (MLC) phosphorylation by MLC kinase (MLCK) [31]. These findings point to MLCK as a potential kinase mediating the presynaptic action of LPA on excitatory neurotransmission. Accordingly, LPA increased the p-MLC:MLC ratio in the HN relative to aCSF-incubated brain stem slices, which was fully prevented by coincubation with the specific MLCK inhibitor ML-7 (Fig 5A and 5B). In concordance, though ML-7 per se did not alter the amplitude of eEPSCsAMPA, as we also recently reported [8], it fully suppressed LPA-induced alterations on eEPSCsAMPA amplitude and PPR (Fig 5C–5F). This further supports MLCK as a main molecular substrate activated by LPA signaling within excitatory presynaptic terminals.

Bottom Line: LPA increased myosin light chain phosphorylation, which is known to trigger actomyosin contraction, and reduced the number of synaptic vesicles docked to active zones in excitatory boutons.However, LPA-induced depression of GABAergic transmission was correlated with an endocytosis-independent reduction of GABAA receptors, possibly by GABAAγ2 dephosphorylation and subsequent increased lateral diffusion.We propose that lysophospholipids serve as potential local messengers that tune synaptic strength to precedent activity of the neuron.

View Article: PubMed Central - PubMed

Affiliation: Grupo de Neurodegeneración y Neuroreparación (GRUNEDERE), Área de Fisiología, Facultad de Medicina, Universidad de Cádiz, Cádiz, Spain.

ABSTRACT
Synaptic communication is a dynamic process that is key to the regulation of neuronal excitability and information processing in the brain. To date, however, the molecular signals controlling synaptic dynamics have been poorly understood. Membrane-derived bioactive phospholipids are potential candidates to control short-term tuning of synaptic signaling, a plastic event essential for information processing at both the cellular and neuronal network levels in the brain. Here, we showed that phospholipids affect excitatory and inhibitory neurotransmission by different degrees, loci, and mechanisms of action. Signaling triggered by lysophosphatidic acid (LPA) evoked rapid and reversible depression of excitatory and inhibitory postsynaptic currents. At excitatory synapses, LPA-induced depression depended on LPA1/Gαi/o-protein/phospholipase C/myosin light chain kinase cascade at the presynaptic site. LPA increased myosin light chain phosphorylation, which is known to trigger actomyosin contraction, and reduced the number of synaptic vesicles docked to active zones in excitatory boutons. At inhibitory synapses, postsynaptic LPA signaling led to dephosphorylation, and internalization of the GABAAγ2 subunit through the LPA1/Gα12/13-protein/RhoA/Rho kinase/calcineurin pathway. However, LPA-induced depression of GABAergic transmission was correlated with an endocytosis-independent reduction of GABAA receptors, possibly by GABAAγ2 dephosphorylation and subsequent increased lateral diffusion. Furthermore, endogenous LPA signaling, mainly via LPA1, mediated activity-dependent inhibitory depression in a model of experimental synaptic plasticity. Finally, LPA signaling, most likely restraining the excitatory drive incoming to motoneurons, regulated performance of motor output commands, a basic brain processing task. We propose that lysophospholipids serve as potential local messengers that tune synaptic strength to precedent activity of the neuron.

No MeSH data available.


Related in: MedlinePlus