Cannabidiol modulates excitatory-inhibitory ratio to counter hippocampal hyperactivity

Abstract

Cannabidiol (CBD), a non-euphoric component of cannabis, reduces seizures in multiple forms of pediatric epilepsies, but the mechanism(s) of anti-seizure action remain unclear. In one leading model, CBD acts at glutamatergic axon terminals, blocking the pro-excitatory actions of an endogenous membrane phospholipid, lysophosphatidylinositol (LPI), at the G-protein-coupled receptor GPR55. However, the impact of LPI-GPR55 signaling at inhibitory synapses and in epileptogenesis remains underexplored. We found that LPI transiently increased hippocampal CA3-CA1 excitatory presynaptic release probability and evoked synaptic strength in WT mice, while attenuating inhibitory postsynaptic strength by decreasing GABA_ARγ₂ and gephyrin puncta. LPI effects at excitatory and inhibitory synapses were eliminated by CBD pre-treatment and absent after GPR55 deletion. Acute pentylenetrazole-induced seizures elevated GPR55 and LPI levels, and chronic lithium-pilocarpine-induced epileptogenesis potentiated LPI's pro-excitatory effects. We propose that CBD exerts potential anti-seizure effects by blocking LPI's synaptic effects and dampening hyperexcitability.

Keywords: G-protein-coupled receptor; GABA receptors; cannabidiol; cannabinoid; epilepsy; hippocampus; inhibition; lysophosphatidylinositol; neuromodulation; seizure.

Fig. 1:. CBD reduces seizures and LPI-driven CA3→CA1 spike enhancement via GPR55

(A) GPR55 expression in hippocampal slices (10x, 63x resolution) is absent in slices from the GPR55 KO mouse. (B) GPR55 expression (20x) was greater in the pyramidal layer of area CA1 and CA3 than the pyramidal layer of CA2 or granule layer of DG (n_WT=8 slices, p_CA1/CA2=0.033, p_CA1/DG=0.0002, p_CA3/DG=0.0006). GPR55 expression was significantly reduced in all regions in control slices from GPR55 KO littermates (n_KO=3, p<0.0001 for all regions). (C) CBD produced dose-dependent reduction in seizures generated by pentylenetetrazole (PTZ, 105 mg/kg): significant effects at 200 mg/kg, i.p. (n_WT+VEH=18/18 vs n_WT+CBD200=5/15, p<0.0001). However, CBD did not reduce PTZ-induced seizures in GPR55 KO mice (n_KO+VEH =11/13 vs n_KO+CBD200=10/13 KO, p_{KO+VEH/KO+CBD200}>0.9999; p_{WT+CBD200/KO+CBD200}=0.03). (D) CBD decreased mortality in PTZ-induced seizures in both WT (p_{WT+VEH/WT+CBD50}=0.011, p_{WT+VEH/WT+CBD100}=0.0008, p_{WT+VEH/WT+CBD200}<0.0001) and GPR55 KO mice (p_{KO+VEH/KO+CBD100}=0.0028, p_{KO+VEH/KO+CBD200}=0.0001). (E-F) In cell-attached recordings, LPI transiently elevated the probability of evoking an action potential with Schaffer Collateral stimulation (5× 50Hz at 5s intervals) (n=9, p=0.038), an effect that decayed by 20 min following LPI onset (p=0.034 vs peak). The LPI-induced effect was blocked by 1 μM CBD pre-treatment (n_LPI+CBD=7, p_ANOVA=0.017, p_LPI/LPI+CBD=0.025) and absent in GPR55 KO slices (n _{LPI-WT/LPI-KO}=7, p_{LPI-WT/LPI-KO}=0.021). (G) The frequency-current (F-I) relationship and (H) cell-attached spontaneous firing rate of CA1 pyramidal neurons remain unchanged upon LPI (4 μM) addition (n=6, p=0.74). Throughout, symbols and error bars represent mean of independent samples ± standard error of mean; *p≤0.05, **p≤0.01, ***p≤0.001, ****p<0.0001.

Fig. 2:. CBD blocks both pro-excitatory and anti-inhibitory effects of LPI (A_1–2)

Representative electrophysiological traces of spontaneous synaptic transmission. LPI (4 μM) reversibly elevated mEPSC frequency, without changing mEPSC amplitude (A₁, light green traces). Contrarily, LPI diminished both the frequency and the amplitude of mIPSCs, reversed by wash (A₂, pink traces). CBD pre-treatment (1 μM) prevented LPI-mediated increases in mEPSC frequency (A₁, dark green) and LPI-driven decreases in mIPSC amplitude and frequency (A₂, red). Additionally, LPI effects were absent in slices from GPR55 KO mice (A_1–2, black traces). (B_1–2) Time courses of LPI-mediated effects: transient rise in mEPSC frequency (~ 5 min to peak, light green, upper panel, left), and slow, gradual reduction in mIPSC frequency and amplitude (20–30 min, pink, upper panels, right), absent in slices from GPR55 KO mice (black symbols, rear), and prevented by CBD pre-treatment (B₁, dark green, mEPSCs; B₂, mIPSCs red). (C_1–2) Effect of LPI to both elevate mEPSC frequency (left, C₁, n=10, p=0.0001 vs baseline) and lower mIPSC frequency (right, C₂, n=10, p=0.0002) and amplitude (p=0.0003) was absent following CBD application (dark green, n=6, C₁; red, n=5, C₂) and ablated in GPR55 KO mice (black, n_mEPSC=9, n_mIPSC=8). However, effects persisted in the presence of the CB₁R antagonist AM281 (1 μM) (nmEPSC=5, pmEPSC=0.048, nmIPSC=4, p_mIPSC-Freq=0.034; p_mIPSC-Ampl=0.016). LPI-mediated changes also blocked by the synthetic GPR55 inverse agonist CID16020046 (CID, 2.5 μM, n_mEPSC=5, p_mEPSC=0.10; n_mIPSC=4, p_mIPSC-Freq=0.44; p_mIPSC-Ampl=0.52) and the SERCA pump inhibitor thapsigargin (Thapsi, 10 μM, n_mEPSC=3, p_mEPSC=0.76; n_mIPSC=5, p_mIPSC-Freq=0.58; p_mIPSC-Ampl=0.44).

Fig. 3:. LPI regulates synaptically-evoked transmission in the CA3→CA1 microcircuit by augmenting excitation and diminishing disynaptic inhibition. (A₁)

CA1 pyramidal neuron recordings (V_hold=−70 mV), with stimulating electrodes placed in stratum radiatum to activate Schaffer Collateral axons. LPI (4 μM) caused a transient, reversible elevation in synaptically-evoked EPSCs (eEPSCs) in WT (light green) slices, blocked by CBD pre-treatment (dark green), and absent in GPR55 KO slices (black). (A_2–3) LPI elevated eEPSC amplitude (n_LPI =5, p_LPI/baseline=0.021, 5 min window, 5 min post-LPI application), corresponding to a drop in the paired pulse ratio (PPR) of second vs. first events (p_LPI/baseline=0.0092, p_LPI/Wash=0.020). Both LPI-mediated effects were blocked by 1 μM CBD (Ampl: n_CBD+LPI=5, p_ANOVA=0.0078, p_LPI/CBD+LPI=0.0057; PPR: p_ANOVA=0.036, p_LPI/CBD+LPI =0.042), and deficient in GPR55 KO mice (Ampl: n_KO=5, p_WT/KO=0.028; PPR: p_WT/KO=0.048). (B_1–3) Effects on disynaptic inhibitory currents isolated by holding CA1 neurons at 0 mV (~E_Glu), (Suppl. Fig. 4C) (B₁) LPI gradually reduced eIPSC amplitude (pink), an effect prevented by CBD pre-treatment (red), and deficient in GPR55 KO slices (black). (B_2–3) eIPSC amplitude lowered by application of LPI (n=6, p_LPI/baseline=0.0006, 5 min window, 25 min post LPI). LPI reversibly elevated PPR over the same time intervals (p_LPI/baseline=0.039). LPI-mediated effects were prevented by CBD pre-treatment (Ampl: n_CBD+LPI=4, p_ANOVA=0.0022, p_LPI/CBD+LPI=0.014; PPR: p_LPI/CBD+LPI=0.021), and absent in GPR55 KO slices (Ampl: n_KO=5, p_WT/KO=0.0018; PPR: p_WT/KO=0.013).

Fig. 4:. LPI transiently strengthens PV neuron inputs while persistently weakening their inhibitory output.

(A_1–3) Recording in genetically-labeled PV-Cre x Ai9 interneurons. LPI (4 μM)-elevated Schaffer collateral-evoked (e) EPSC amplitude (light green, n=5, p_LPI/baseline=0.0007), reverted during wash (p_LPI/Wash=0.012). Concurrently, LPI reduced PPR (p_LPI/baseline=0.022; p_LPI/Wash=0.031). CBD pre-treatment prevented effects of LPI on amplitude (dark green, n_CBD+LPI=5, p_LPI/CBD+LPI=0.035) and PPR (p_CBD+LPI =0.0020). (B_1–3) LPI reduced amplitude of optogenetically-evoked monosynaptic IPSCs (oIPSCs) in slices from PV-Cre x Ai32/ChR2 mice (pink, n_LPI=6, p_LPI/baseline=0.0052, p_LPI/Wash=0.039), but did not change the PPR (p_LPI/baseline=0.71). CBD blocked effects of LPI on IPSCs (red, p=0.028).

Fig. 5:. LPI mediates a GPR55-dependent, progressive downregulation of GABA_AR γ₂ and gephyrin declustering, via S327 dephosphorylation.

(A) LPI (4 μM) reduced the intensity of GABA_AR γ₂ puncta at 30- and 60-min post-application (n_VEH=10, n_LPI-30=7, n_VEH/LPI-60=7; p_ANOVA<0.0001: p_VEH/LPI-30=0.0001, p_VEH/LPI-60=0.0003). LPI also diminished gephyrin puncta intensity at 30 and 60 min post-LPI (p_ANOVA=0.0016: p_VEH/LPI-30=0.0011, p_VEH/LPI-60=0.032). CBD (1 μM, red) prevented both LPI-mediated decreases in GABA_AR γ₂ (n_CBD+LPI=8, p_LPI/CBD+LPI<0.0001) and gephyrin (p_LPI/CBD+LPI=0.0007) puncta at 30 min. Similarly, lentiviral shRNA-mediated knockdown of GPR55 (black, sh, n_shRNA=5) deterred effects of LPI on GABA_AR γ₂ (p_scr/shRNA=0.0052) and gephyrin (n_scr=5, p_scr/shRNA=0.0064), relative to scrambled shRNA controls (gray, scr, n_scr=5). CBD co-application with LPI produced a higher γ2 level than LPI+shRNA alone (p_{CBD+LPI/LPI-shRNA}=0.017). (B) LPI reduced (γ₂ phospho-S327)/(total γ₂ expression) in whole hippocampal lysates (n=8 for all, p_LPI-30=0.0099; p_LPI-30=0.0029). LPI-mediated S327 dephosphorylation was reversed by inhibitors (n=4 for all) of RhoA-activated protein kinase (ROCK) (10 μM YM-27632, p=0.76), IP₃Rs (1 μM xestospongin C, p=0.41), and calcineurin (10 μM cyclosporin, p=0.22), and by depletion of intracellular calcium stores (10 μM thapsigargin, p=0.76). However, LPI-mediated effects remained intact in the presence of inhibitors of phospholipase C (10 μM U73122, p<0.0001), protein kinase C (0.1 μM bisindolylmaleimide II, p=0.024), and PP1α (5 nm tautomycetin, p=0.0002). Right, biochemical scheme based on literature^, consistent with pharmacological data.

Fig. 6:. Acute seizures elevate GPR55 expression and increase 18:0 LPI level, effects prevented by CBD pre-treatment

(A-B) Seizures induced by PTZ (105 mg/kg, i.p.) drove region-specific elevations in GPR55 immunoreactivity (n=6 for all), in areas CA1 (p_ANOVA=0.014, p_{Non-Sz+VEH/SZ+VEH}=0.031, representative images) and CA3 (p_ANOVA=0.027, p_{Non-Sz+VEH/SZ+VEH}=0.044). The seizure-induced GPR55 elevation was blocked by in vivo pre-treatment with CBD (200 mg/kg, i.p.), 1 h prior to giving PTZ (CA1 p_{SZ+VEH/Sz+CBD}=0.0069, CA3 p_{SZ+VEH/Sz+CBD} =0.034). (C) PTZ seizures increase Gpr55 mRNA expression, prevented by CBD (200 mg/kg, i.p.), 1 h prior to PTZ injection (n_Non-Sz+VEH=7, n_Sz+VEH=7, n_Sz+CBD=4 n, p_ANOVA<0.0001, p_{Non-Sz+VEH/Sz+VEH}=0.021; p _{Sz+VEH/SZ+CBD}<0.0001). CBD-treated animals had a lower GPR55 mRNA expression compared with non-seizure, vehicle controls (p_{Sz+VEH/Sz+CBD}=0.0060). (D) Linear standard control curves for 3 LPI isoforms, 16:0, 18:0, 20:4. (E) PTZ significantly elevated 18:0 LPI, assayed by HPLC-MS (n_Non-Sz+VEH=8, n_Sz+VEH=5, n_Sz+CBD=4; p_ANOVA=0.0086, p_{Non-Sz+VEH/Sz+VEH}=0.031, p_{Sz+VEH/Sz+CBD} =0.0097). (F) Simplified depiction of how CBD might oppose acute seizure generation. In proposed scenario, CBD interrupts an acute hyperactivity-induced LPI-GPR55-mediated positive feedback loop. LPI promotes excitability by a dual mechanism: stimulating transient glutamate release and restructuring the inhibitory post-synapse, downregulating inhibition. This elevates E:I ratio, which likely promotes acute hyperactivity (dotted line), and drives principal cell firing. Hyperactivity in turn upregulates GPR55 and its agonist, LPI. CBD blocks GPR55-dependent pro-excitatory and anti-inhibitory effects of LPI, and also dampens spike firing via direct effects on ion channels^,,– thus inhibiting a positive feedback loop that favors hyperactivity. Italics represent findings from prior published studies as indicated above. (G-K) CBD action on electrographic seizures. Representative EEG traces (G) and quantification demonstrating that CBD (100 mg/kg, i.p.) administered 1 hour prior to PTZ (60 mg/kg, i.p.)-induced seizures reduces electrographic seizure average power (H, p=0.0036), increases latency to first electrographic seizure (I, p=0.040), and produces a non-significant trend towards reduced EEG seizure duration (J, p=0.11). (K) CBD decreases behaviorally-observed PTZ-induced seizures at Racine stages 1–6 relative to matched vehicle-treated control animals (p=0.047).

Fig. 7:. CBD prevents Lithium-Pilocarpine epileptogenesis-induced elevation of GPR55 immunoreactivity and LPI responsiveness.

(A) Experimental paradigm for Li-PLC epileptogenesis (see Methods,^,). (B) Li-PLC epileptogenesis induces a 55±29% elevation in hippocampal Gpr55 mRNA expression without reaching statistical significance (nCTRL=9, n_Li-PLC+VEH=11, n_Li-PLC+CBD=7, p_ANOVA=0.19). (C) Li-PLC epileptogenesis increases hippocampal GPR55 immunoreactivity (IHC, 20x resolution), in area CA1 (CA1 n_CTRL=9, n_Li-PLC-VEH =7; p_ANOVA=0.024: p_{CTRL/Li-PLC+VEH}=0.020), CA3 (n_CTRL=9, n_Li-PLC-VEH =8; p_ANOVA=0.027: p_{CTRL/Li-PLC+VEH}=0.028), and DG (n_CTRL=8, n_Li-PLC-VEH =7; p_ANOVA=0.0084: p_{CTRL/Li-PLC+VEH}=0.014). CBD, administered 200 mg/kg p.o. after the period of epileptogenesis, prevents the rise in GPR55 expression in CA1 (n_Li-PLC+CBD=12, p_{Li-PLC+VEH/Li-PLC+CBD}=0.042), CA3 (n_Li-PLC+CBD=11, p_{Li-PLC+VEH/Li-PLC+CBD}=0.042), and DG (n_Li-PLC+CBD=10, p_{Li-PLC+VEH/Li-PLC+CBD}=0.0096). (D) Li-PLC epileptogenesis increases GPR55 puncta intensity colocalized with Vglut1 at putative presynaptic excitatory terminals in CA1 stratum radiatum (S.R., 63x); effect prevented by chronic p.o. CBD. Left: representative images, Right: (n_CTRL=8, n_Li-PLC-VEH=8, n_Li-PLC+CBD=10; pANOVA=0.0069: p_{CTRL/Li-PLC+VEH}=0.006, p_{Li-PLC+VEH/Li-PLC+CBD}=0.022). (E) GPR55 intensity increases at putative post-synaptic inhibitory sites labeled with gephyrin (S.R., 63x), following Li-PLC-induced epileptogenesis, prevented by CBD treatment after the period of epileptogenesis. Left: representative images, Right: n_CTRL=7, n_Li-PLC-VEH=6, n_Li-PLC-VEH=9; p_ANOVA=p=0.0007: p_{CTRL/Li-PLC+VEH}=0.04, p_{Li-PLC+VEH/Li-PLC+CBD}=0.0004). (F) Pro-excitatory ex vivo effects of the GPR55 agonist LPI were potentiated in slices from rats following Li-PLC-induced epileptogenesis (filled light green circles) in comparison to non-epileptic controls (unfilled light green circles), effects prevented by chronic in vivo treatment with CBD p.o. (filled dark green circles). Vertical axis, normalized values relative to pre-LPI baseline; mEPSC freq: (n_CTRL=9, n_Li-PLC+VEH=9, n_Li-PLC+CBD=7; p_ANOVA=0.0099: p_{CTRL/Li-PLC+VEH}=0.038; p_{Li-PLC+VEH/Li-PLC+CBD}=0.0087). The potentiated effects of LPI also persisted following washout (p_ANOVA=0.01: p_{CTRL/Li-PLC+VEH} p=0.012, p_{Li-PLC+VEH/Li-PLC+CBD} p=0.034) (G) LPI produced a greater anti-inhibitory drop in mIPSC amplitude in epileptic (filled light red circles) vs. non-epileptic controls (unfilled light red circles), effects prevented by in vivo CBD p.o. treatment (filled dark red circles, n_CTRL=6, n_Li-PLC+VEH=10, n_Li-PLC+CBD=8; p_ANOVA=0.0032: p_{CTRL/Li-PLC+VEH} =0.0017; p_{Li-PLC+VEH/Li-PLC+CBD}=0.047). (H-I) Acute CBD (1 μM) blocked LPI effects on both mEPSC frequency (H) and mIPSC amplitude (I) in both Li-PLC slices and non-epileptic controls.

Fig. 8:. CBD decreases “second-dose” kainic acid (KA)-induced seizures.

(A) Gpr55 mRNA expression was significantly increased 48 h following kainic acid injection (KA1, 24 mg/kg s.c.) relative to vehicle-treated, non-seizure controls (n_Non-Sz+Veh=8, n_KA1+VEH=8; p_ANOVA=0.018: p_{Non-Sz+Veh/KA1+Veh}=0.039). However, pre-treatment with CBD (200 mg/kg, s.c.) 1 h prior to KA1 injection prevented the rise in Gpr55 mRNA at 48 h (n_KA1+CBD1=5, p_{KA1+Veh/KA1+CBD1}=0.039). (B) GPR55 immunoreactivity increases in CA1 and CA3 48 h post-KA1, effects prevented by CBD 200 mg/kg 1 hour prior to KA1 (CA1: n=11 for all, p_ANOVA p=0.02: p_{Non-Sz+VEH/KA1+VEH}=0.040, p_{KA1+VEH/KA1+CBD1}=0.048; CA3: n_Non-Sz+VEH=12, n_KA1+VEH=11, n_KA1+CBD1=11, p_ANOVA<0.0001: p_{Non-Sz+VEH/KA1+VEH} =0.0006, p_{KA1+VEH/KA1+CBD1}=0.0001; DG n_Non-Sz+VEH=11, n_KA1+VEH=11, nK_A1+CBD1=10, p_ANOVA=0.09). (C) Effects of 2^nd dose of KA administered 48 h post KA1 (KA2, 24 mg/kg, s.c.). Behavioral seizures assessed via modified Racine Scale, 2 h after KA1 or KA2 (timing of assays, dashed vertical lines), followed by injection with diazepam 10 mg/kg, s.c. While 2/15 animals demonstrated convulsive seizures (as defined as 3–4 on Racine Scale) following KA1, a significantly greater proportion displayed KA-induced seizures after KA2 (14/15, p<0.0001, Fisher’s Exact Test). CBD (200 mg/kg, s.c.) reduced the frequency of convulsive seizures following KA2 when CBD was administered 1 h prior to KA1 injection (CBD1, p=0.05) or 1 h prior to KA2 (CBD2, p=0.0017). There was no significant difference between KA1 seizure incidence with CBD1 or VEH1 administration (p=0.11, data not shown).