Fig 1.
Expression pattern of LPA1 in the HN supports a role for lysophospholipids in the control of synaptic neurotransmission.
(A) Expression levels of mRNA for indicated LPARs obtained by qRT-PCR of microdissected HNs from neonatal rats (P7) relative to the housekeeping GAPDH. Values were normalized taking mean value for lpa1 as 1. *p < 0.05, one-way ANOVA on Ranks relative to lpa2–6. Plot data can be found in S1 Data. (B–K) Multiple immunolabeling confocal images of the HN from P7 rats showing a close relationship between LPA1-ir patches and structures expressing the nonphosphorylated form of neurofilament H (SMI32), a motoneuron marker (B–F), VGLUT2- (G, H, open arrows), and/or VGAT-ir (I-K) inputs. The 18.8 ± 2.1% (n = 35 HMNs) of VGLUT2-ir and 11.9 ± 1.6% (n = 51 HMNs) of VGAT-ir inputs apposed to HMN somata colocalized with LPA1-ir patches. Note that in 3-D reconstructions (E, F, and Hb), LPA1 staining colocalizes and borders SMI32-ir somata and dendrites (E, F, arrows). The yz- and xz-planes also confirm colocalization of LPA1-ir with excitatory (Ha, open arrows) and inhibitory (K) inputs. Finally, VGLUT2- (Hb) and VGAT-ir (I) inputs appeared apposed on LPA1-containing SMI32-ir dendrites. The xz- and yz-planes are located as indicated by the white dashed lines. Scale bars: B, 50 μm; G, 25 μm; C, D, and J, 10 μm; E and I, 5 μm; and F, H, and K, 2 μm.
Fig 2.
LPA induces STD at excitatory and inhibitory synapses in a dose-dependent manner.
(A) Schematic diagram of the in vitro experimental model used to analyze the effects of LPA on synaptic transmission incoming to HMNs. Whole-cell patch-clamp recordings (Rec) were obtained from somata of HMNs in neonatal brain stem slices. Experiments were performed as in our previous published study [8]. ePSCs were evoked by electrical stimulation (Stim) of the VLRF. A micropipette (arrow) near to a HMN before patch performance is illustrated in the inset. (B) Top, schematic representation of the experimental protocol carried out to study drug effects on ePSCs. Motoneurons were initially allowed to stabilize (Stabil.) with normal artificial cerebrospinal fluid (aCSF) to obtain baseline control recordings. Slices were then superfused for 10 min with aCSF alone (vehicle) or with LPA or s-LPA (drug) before current responses were acquired again. Finally, a last round of acquisition was taken after a 10 min washout with drug-free aCSF. Bottom, examples of eEPSCsAMPA (left panels) and eIPSCsGABAA (middle panels) recorded from HMNs before and following exposure to LPA (2.5 μM) or s-LPA (40 μM) for 10 min and after washing. Right panels, reduction in the eEPSCsAMPA (blue; n > 10 HMNs per drug) or eIPSCsGABAA (green; n = 4 HMNs per drug) amplitude for LPA- or s-LPA-treated groups compared to their respective pretreatment (before) periods. eEPSCsAMPA or eIPSCsGABAA were pharmacologically isolated in the presence of 1 μM strychnine hydrochloride, 30 μM d-tubocurarine, 50 μM (DL)-APV, and 10 μM bicuculline methochloride or NBQX (20 μM), respectively, continuously applied to the bath perfusion. (C) Left panel, examples of eEPSCsAMPA (top) and eIPSCsGABAA (bottom) recorded from two HMNs before and during exposure to the indicated LPA concentrations. This experimental design was carried out in three HMNs per each synaptic category resulting in dose-dependent attenuation of the ePSCs similar to those presented in the plot. Right panel, reduction in eEPSCsAMPA and eIPSCsGABAA amplitude induced by LPA at the indicated concentrations relative to control (before) condition. Each HMN was exposed to a single dose of LPA. Data for each drug concentration were averaged from at least three independent experiments. Mean changes in amplitude obtained after 10 min incubation with vehicle (aCSF, S1 Text) were subtracted from alterations induced by each tested LPA concentration. The study included only those motoneurons that recovered after washing to at least the percentage of change obtained by vehicle perfusion. n ≥ 4 HMNs per each concentration and synaptic signaling system. *p < 0.05, one-way RM-ANOVA relative to ePSCs recorded before lysophospholipid incubation. #p < 0.05, one-way ANOVA on Ranks relative to reduction in amplitude of eEPSCsAMPA measured after the same LPA concentration. Plots data can be found in S1 Data.
Fig 3.
Presynaptic LPA signaling induces excitatory STD.
(A) Current traces of sEPSCsAMPA recorded from a HMN at the indicated conditions. The recording of sEPSCsAMPA was performed under conditions of facilitated synaptic release without TTX in a modified extracellular solution containing high-Ca2+ (4 mM), high-K+ (9 mM), and the receptor antagonists indicated in Fig 2B. (B) Mean sEPSCsAMPA amplitude for LPA-treated group (2.5 μM) compared to their respective pretreatment (before) and washout periods (n = 4 HMNs). (C) Normalized cumulative probability distributions of sEPSCsAMPA amplitude for each condition. Bin width: 2 pA. Note that the cumulative distribution of sEPSCsAMPA amplitude shifted to the left (p < 0.001; Kolmogorov-Smirnov test). (D) Top, eEPSCsAMPA recorded in a HMN at the indicated conditions in response to paired-pulse stimulation of VLRF. The rightmost trace shows the superimposition of the responses scaled to the peak of the first eEPSCsAMPA. Bottom, comparison of PPR measured at specified interpulse intervals for HMNs recorded at the indicated conditions (n = 4 HMNs). Paired-pulse ratio (PPR) was obtained from the amplitude of the first and second eEPSCsAMPA by the formula eEPSCsAMPA2/eEPSCsAMPA1. The stimulus intensity was adjusted so that the eEPSCAMPA1 was approximately 50% of maximal amplitude, then maintained constant throughout the recording period. (E) Top, superimposition of 10 successive eEPSCsAMPA evoked at 0.2 Hz by minimal stimulation of VLRF in HMN before and after treatment with LPA. Characteristically, the intensity of the stimulation was set to elicit eEPSCsAMPA with 30% to 40% failure at the control (before) condition. Bottom, mean eEPSCsAMPA amplitude (left) and failure rate (right) at indicated conditions (n = 4 HMNs). Experiments and analysis described in A–E have been performed as in our previously published study [8]. *p < 0.05, one-way (B, E) or two-way (D) RM-ANOVA relative to control (before) condition. (F) Confocal images of the HN obtained from P7 rats processed by triple immunolabeling for LPA1, VGLUT2 and the presynaptic active zone (a.z.) marker Munc13-1. Note triple colocalizations within the boxed areas. (G) 3-D reconstruction showing LPA1 expression in the presynaptic a.z. of a glutamatergic bouton. Note that LPA-ir colocalizes with Munc13-1 and with a VGLUT2-ir SVs pool in the three planes. The xz- and yz-planes are located as indicated by the white dashed lines. Scale bars: F, 5 μm; G, 2 μm. Plots data can be found in S1 Data.
Fig 4.
LPA modulates AMPAR-mediated neurotransmission via LPA1/Gαi/o-PLC.
(A) Schematic diagram of microinjections performed in the fourth ventricle of neonatal rats at P4. A solution (2 μl) containing the vehicle (RNase-free phosphate-buffered saline [PBS]), control noninterfering RNA (cRNA, 2 μg) or a small interfering RNA directed against lpa1 (siRNAlpa1, 2 μg) was administered by means of a Hamilton syringe. (B) Representative eEPSCsAMPA recorded in HMNs obtained from animals receiving the specified treatments recorded at the indicated conditions. (C, D) Mean eEPSCsAMPA amplitude reduction (C, in percent) and PPR ratio increase (D, in percent) in response to addition to the bath of LPA (2.5 μM) or s-LPA (40 μM) measured at 25 ms interpulse intervals for HMNs recorded under the indicated treatments (control, LPA: n = 13 HMNs, s-LPA: n = 6 HMNs; vehicle, LPA: n = 6 HMNs, s-LPA: n = 4 HMNs; cRNA, LPA: n = 6 HMNs, s-LPA: n = 4 HMNs; siRNA, LPA: n = 9 HMNs, s-LPA: n = 5 HMNs). *p < 0.05, one-way ANOVA relative to control, vehicle and cRNA conditions. (E) Effect of LPA on eEPSCsAMPA from two HMNs in response to paired-pulse stimulation under the presence of the Gαi/o inhibitor pertussis toxin (PTX) (100 ng/ml; left) or the noncatalytic B oligomer of PTX (bPTX) (100 ng/ml; right). Slices were preincubated for 2 h with PTX or bPTX before recordings began and were maintained throughout the experimental procedure. (F) Representative eEPSCsAMPA from 4 HMNs in response to paired-pulse stimulation of VLRF showing the effects of the PLC inhibitor U73122 (1 μM) or its inactive analog U73343 (5 μM) per se (top) or coadded after previous incubation for 10 min with LPA (bottom). (G, H) Mean eEPSCsAMPA amplitude and PPR ratio (25 ms interpulse intervals) under the indicated treatments (PTX and bPTX, n = 6 HMNs; U73122, n = 5 HMNs; LPA+U73122, n = 5 HMNs; U73343, n = 4 HMNs; LPA+U73343, n = 5 HMNs). *p < 0.05, one-way RM-ANOVA relative to control (before) condition. (I) Diagram of the proposed pathway mediating LPA-induced STD at AMPAergic signaling, indicating drug targets. Plots data can be found in S1 Data.
Fig 5.
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.
Fig 6.
LPA induces GABAAergic STD and GABAAγ2 dephosphorylation via postsynaptic LPA1-RhoA/ROCK-CaN signaling.
(A) Spontaneously occurring mIPSCsGABAA recorded from a representative HMN before and after perfusion with the indicated combination of drugs. Bottom, mean mIPSCsGABAA amplitude (left) and frequency (right) at indicated conditions (n = 5 HMNs). mIPSCsGABAA were pharmacologically isolated in the presence of 1 μM tetrodotoxin (TTX), 1 μM strychnine hydrochloride, 30 μM d-tubocurarine, 50 μM (DL)-APV, and NBQX (20 μM) continuously applied to the bath perfusion. *p < 0.05, one-way RM-ANOVA relative to the control (before) condition. (B) Multiple immunolabeling confocal images of the HN from P7 rats showing colocalization between LPA1-ir and gephyrin-ir (top). 3-D reconstruction (bottom) showing that LPA1-ir colocalizes with gephyrin in a SMI32-ir HMN. n, HMN nucleus. The xz- and yz-planes are located as indicated by the white dashed lines. Scale bars: 5 μm. (C) Whole-cell GABAAergic currents evoked by 100 ms pressure pulses of GABA (applied at saturating concentration; 1 mM) in two spinal motoneurons (SMNs) under indicated treatments. Recordings were performed in the presence of TTX in nominally Ca2+-free solution. (D) Western blots of total (T), cytosolic (C), and membrane-associated (M) RhoA in SMNs in untreated and s-LPA incubated (for 10 min) cultures. (E) Whole-cell GABAAergic currents evoked by pulses of GABA in SMNs preincubated with cRNA (top) or siRNAlpa1 (bottom) before and after superfusion with the indicated drugs. (F) Summary data showing the changes in GABAAergic currents measured in SMNs exposed at different treatments (n ≥ 5 SMNs per group). #, *p < 0.05, one-way ANOVA or RM-ANOVA, respectively, relative to s-LPA or LPA treatments of cRNA preincubated SMNs. (G) Western blot (top) and averaged ratio (bottom) of phosphorylated and total GABAAγ2 subunit protein levels (denoted as pGABAAγ2 and GABAAγ2, respectively) in SMNs incubated (10 min) with aCSF alone (control) or supplemented with indicated drugs. β-actin was an internal loading reference. (H) Same as in C under indicated treatments. SMNs were preincubated for 30 min with the calcineurin (CaN) autoinhibitory peptide (Cap; 50 μM). (I) Changes of CaN activity in lysates from cultured SMNs untreated (control) or treated for 10 min with the indicated drugs. *p < 0.05, one-way ANOVA on Ranks relative to control condition. Plots data can be found in S1 Data.
Fig 7.
LPA induces dephosphorylation and internalization of the GABAAγ2 subunit in a ROCK/CaN-dependent manner.
(A, B) Western blot (top) and averaged ratio (bottom) of total (T), cytosolic (C), and membrane-associated (M) GABAAγ2 in cultured SMNs incubated (10 min) with aCSF alone (control) or supplemented with indicated drugs (A). Dynasore (80 μM) or vehicle (0.2% DMSO) were added to the incubation solution 30 min before subsequent s-LPA coaddition for 10 min (B). β-actin was an internal loading reference for T and C fractions and an indicator for fractionation purity. The average densitometric signals for the GABAAγ2 C and M samples were expressed as a fraction of T GABAAγ2 of the same samples and normalized to the corresponding ratio determined for samples representing control conditions. *p < 0.05, one-way ANOVA on Ranks relative to control or vehicle condition. (C) Same as in Fig 6C under indicated treatments. Treatment with dynasore began at least 30 min before patch performance and was present all along the recording protocol. (D) Left, low-magnification photomicrographs showing a group of SMNs at 6 days in vitro treated for 40 min with aCSF alone and stained for GABAAγ2. Right, detail of a SMN exemplifying close association between GABAAγ2- and gephyrin-ir clusters. (E, F) Examples of GABAAγ2- and gephyrin-ir clusters in the surface of neurites obtained from SMNs treated for 40 min with dynasore (E) or 30 min with dynasore plus 10 min with s-LPA+dynasore (F). Scale bars: D, 50 μm; E, F, 5 μm. (G) Normalized mean cluster area (left) and fluorescence intensity (right) of GABAAγ2- and gephyrin-ir clusters analyzed under the indicated treatments (n > 1,200 clusters per condition). *p < 0.005, Student’s t test relative to control or dynasore condition. (H) Diagram of the proposed pathway mediating LPA-induced STD on GABAAR-mediated neurotransmission. Drug targets are also indicated. Plots data can be found in S1 Data.
Fig 8.
Involvement of LPA1 in activity-dependent STD at inhibitory signaling.
(A) Same as in Fig 6E, but GABA pulses were performed before and after 4 min addition to the bath of Glut (30 μM) and Gly (1 μM) and after successive washing (cRNA: n = 4 SMNs; siRNAlpa1: n = 5 SMNs). *p < 0.05, one-way RM-ANOVA relative to the control (before) condition. (B) Same as in Fig 6G but performed from SMNs cultures receiving indicated pretreatments and incubated for 4 min with aCSF alone or with Glut/Gly to stimulate NMDARs. *p < 0.05, one-way ANOVA on Ranks relative to control (untreated) condition. Experiments were carried out in the presence of TTX, d-tubocurarine, strychnine, and NBQX. Plots data can be found in S1 Data.
Fig 9.
Endogenous LPA signaling restrains inspiratory-related baseline activity of HMNs in the adult rat.
(A) Like Fig 1A, but tissue was extracted from adult rats. Inset, comparative expression levels of lpa1 mRNA in HNs from neonatal (P7) and adult rats. *p < 0.05, one-way ANOVA on Ranks relative to lpa2–6 or the adult condition in the inset. (B) Western blot for LPA1 in the HN at P7 and at the adult age. β-actin was an internal loading reference. (C) Low-magnification confocal image taken from a selected region of the HN in an adult rat showing triple immunolabeling for SMI32, VGLUT2, and LPA1. (D) Double immunolabeling noticed LPA1 colocalizing with excitatory terminals (yellow) in the HN. (E, F) Confocal xy-planes showing immune LPA1 staining colocalizing with excitatory terminals (yellow, arrowheads) and SMI32-ir structures (purple). Note in E a VGLUT2-immunopositive input colocalizing with LPA1 that is apposed to the soma of a LPA1-expressing HMN (n, HMN nucleus). (G, H) Images of LPA1 and SMI32 (G) or VGLUT2 (H) are shown in the xy-, xz-, and yz-planes illustrating 3-D reconstructions. The white and yellow crosshairs display locations of xz- and yz-planes. Note that LPA1-ir colocalizes and borders SMI32-ir structures (E–G), supporting cytosolic and membrane localization of the LPAR in HMNs. It also colocalizes with VGLUT2 (E, F, and H), indicating its expression in excitatory terminals. Scale bars: C, 50 μm; D, 20 μm; E–H, 2 μm. (I) Schematic diagram of the in vivo experimental preparation. Unitary discharge activity (Rec) of HMNs was obtained in decerebrated, vagotomized, and artificially ventilated adult rats, which had been injected with a neuromuscular blocking agent. A three-barreled pipette with a barrel for electrophysiological recordings and another for microiontophoretic administration of a drug are illustrated. HMNs were identified by their antidromic activation from the electrode (St.) implanted in the XIIth nerve and by the collision test (top traces) between spontaneous orthodromic (dot) and antidromic (asterisk) evoked action potentials. When the stimulus was triggered by a spontaneous spike at a short latency, the antidromic action potential was occluded (arrowhead). Middle and bottom traces represent the extracellularly recorded spike discharge for an inspiratory HMN and the histogram of the instantaneous firing rate (FR, in spikes (sp)/s), respectively. Mean firing rate (mFR, red dotted line) in each burst was measured and subsequently plotted along time. (J) Instantaneous firing rates (sp/s) of two HMNs in response to microiontophoretic administration of VPC 32183 or vehicle (10% DMSO in PBS, pH 8.0) at the indicated current. During the before condition, a retention current of +5 nA is continuously applied. Note the lack of effect of vehicle and the stimulating effect exerted by the application of the LPA1/3 inhibitor. (K) Time course of the mean FR (mFR, sp/s) per burst in response to microiontophoretic administration (30 s on, 60 s off) of VPC 32183 or vehicle at the indicated applied currents. (L) Mean current-response curves illustrating the effects of microiontophoretically-administered LPA1/3 antagonists VPC 32179 (0.5 mM; n = 7 HMNs), VPC 32183 (1 mM; n = 5 HMNs), Ki16425 (2 mM; n = 8 HMNs) or vehicle (n = 4 HMNs) on motoneuron activity characterized by the change in the mFR per burst. Plots data can be found in S1 Data.
Fig 10.
Proposed mechanism by which a membrane-derived bioactive phospholipid such as LPA regulates synaptic strength.
LPA affects main excitatory and inhibitory synapses by different degrees, loci, and mechanisms of action. At glutamatergic synapses (left diagram), binding of phospholipids to presynaptic Gαi/o-protein-coupled LPA1 indirectly activates MLCK via PLC, promoting MLC phosphorylation and subsequent actomyosin cytoskeleton contraction. This would alter the spatial distribution of SVs within the presynaptic terminal and the RRP size of SVs, which results in rapid and reversible excitatory STD. At the GABAergic synapse (right diagram), LPA—synthesized and/or released in response to Ca2+ entry through NMDAR—interacts with postsynaptic Gα12/13-coupled LPA1, then activates RhoA/ROCK. Subsequently, phosphatase calcineurin acts to dephosphorylate Ser327 of the GABAAγ2 subunit, which in turn undergoes lateral diffusion and internalization by endocytosis. Dephosphorylation of the GABAAγ2 component of GABAARs results in rapid and reversible inhibitory STD.