Altering cannabinoid signaling during development disrupts neuronal activity

Abstract

In adult cortical tissue, recruitment of GABAergic inhibition prevents the progression of synchronous population discharges to epileptic activity. However, at early developmental stages, GABA is excitatory and thus unable to fulfill this role. Here, we report that retrograde signaling involving endocannabinoids is responsible for the homeostatic control of synaptic transmission and the resulting network patterns in the immature hippocampus. Blockade of cannabinoid type 1 (CB1) receptor led to epileptic discharges, whereas overactivation of CB1 reduced network activity in vivo. Endocannabinoid signaling thus is able to keep population discharge patterns within a narrow physiological time window, balancing between epilepsy on one side and sparse activity on the other, which may result in impaired developmental plasticity. Disturbing this delicate balance during pregnancy in either direction, e.g., with marijuana as a CB1 agonist or with an antagonist marketed as an antiobesity drug, can have profound consequences for brain maturation even in human embryos.

Fig. 1.

CB1 receptors are present and can be activated by endocannabinoids at IN–PC connections. (A–E) Subcellular distribution of CB1 in the hippocampus. Electron micrographs of the CA1 strata radiatum (A–C) and pyramidale (D and E) at P4. (A–C) CB1 receptors and GABA are labeled with diffuse immunoperoxidase–diaminobenzidine staining and immunogold particles (15 nm), respectively. B and C are serial sections of the same bouton. Double-immunolabeled (CB1 and GABA) synaptic boutons form a symmetric synapse (arrowheads in A–C) on the GABA-negative dendrite. Two other GABA-positive profiles (but CB1-negative) are marked with asterisks. Several axonal profiles producing asymmetric (presumed glutamatergic) synapses (arrows) are GABA-negative. (D and E) CB1 receptors are labeled with preembedding ultra-small immunogold particles with silver amplification. CB1 receptors are expressed only on the plasma membrane of the axon terminal that forms a symmetric synapse on a PC body (arrowhead in E). (Scale bar: 0.5 μm.) (F)Ina P3 PC, a 5-s depolarizing step resulted in a robust decrease of evoked GABA PSCs (gray traces). DSG was blocked 15 min after application of the CB1 receptor antagonists AM251. Note that control GABA PSCs were increased after AM251 (black traces) suggesting that CB1 receptors are tonically activated and decrease GABA release at IN–PC connections. Application of the CB1 receptor agonist WIN55212-2 resulted in a decrease of evoked GABA PSC and blocked DSG by occlusion. Endocannabinoid accumulation in the presence of AM404 also reduced DSG by occlusion. (G) Summary of all experiments showing DSG blockade by AM251. (H) Effect of WIN55212-2, AM251, SR141716A, and AM404 on DSG measured 15 min after drug application.

Fig. 2.

CB1 receptors are present and can be activated by endocannabinoids at IN–IN connections. (A) Electron micrograph of the CA1 stratum radiatum at P4. CB1 receptors and GABA are labeled with diffuse immunoperoxidase–diaminobenzidine staining and immunogold particles (15 nm), respectively. Double-immunolabeled (CB1 and GABA) synaptic bouton forms a symmetric synapse (arrowhead) on the GABA-positive dendrite. One GABA-positive profile (but CB1-negative) is marked with an asterisk. (Scale bar: 0.5 μm.) (B) In a P2 stratum radiatum IN, a 5-s depolarizing step resulted in a decrease of GABA PSCs, which was blocked after 15-min application of AM251. Note that control GABA PSCs were increased after AM251 (black traces) suggesting that CB1 receptors are tonically activated and decrease GABA release at IN–IN connections. (Upper) Example of 17 superimposed traces before and after the depolarizing step in control conditions. (Lower) The black trace represents the average of the five GABA PSCs evoked before the depolarizing step. The gray trace represents the average of the three GABA PSCs evoked after the depolarizing step. (C) Summary of seven experiments showing the time course of DSG blockade in INs by AM251.

Fig. 3.

Ongoing GABA_A receptor-dependent release of endocannabinoids in vitro.(A) The amplitude of pharmacologically isolated GABA PSCs (WC, whole-cell recording) was increased after application of AM251 (3 μM) in a PC (P3, Upper, first image from left) and in a stratum radiatum IN (P3, Lower, first image from left). Note the appearance of large-amplitude GABA PSCs in the presence of AM251 and of spontaneous pure GABA_A receptor-mediated bursts of activity (traces 1 and 2, shown on a faster time scale). On the far right are summaries of the effect of AM251 on GABA PSC amplitude in PCs and INs. (B) The frequency and amplitude of GABA PSCs was decreased after AM404 (10 μM) in a P3 PC (Top) and in a P2 IN (Middle). The graphs on the far right show a summary of the effects of AM404 on GABA PSC frequency and amplitude in PCs and INs. The spontaneous firing frequency of INs was decreased the presence of AM404 (P2; CA, cell-attached recording; Bottom). On the right is a summary of the effect of AM404 on IN spontaneous firing frequency.

Fig. 4.

Resting activation of cannabinoid receptors limits network excitability in vitro. (A) Application of SR141716A (3 μM) resulted in an increase in GDP (*) frequency and amplitude in a PC (P0). On the right are histograms showing the effects of SR141716 on GDP frequency and charge transfer. (B) (Left and Center) Simultaneous recording of two stratum oriens INs (P3) showing IN1 firing during a GDP and the intracellular compound glutamate/GABA synaptic drive received by IN2. Firing (IN1) and GDP (IN2) charge transfer were increased in the presence of SR141716A (3 μM). (Right) Histogram summarizing the effect of SR141716A on IN firing frequency during GDPs. (C) Application of AM404 (10 μM) abolished GDP activity in a PC (P5), demonstrating that endocannabinoids are released in these conditions. (D) Direct activation of CB1 receptors with WIN55212-2 (3 μM) also abolished GDP activity in a PC (P4).

Fig. 5.

Ongoing activation of CB1 receptors prevents the occurrence of epileptic activity in vivo. (A) (Left Upper) In control conditions, most of the spontaneous cortical activity was composed of biphasic sharp waves (SPWs). The frequency and amplitude of SPWs ranged from 1.9 to 4 events per min and from 23 to 90 μV, respectively (n = 26 animals). Average duration was 468 ± 98 ms (event 1). (Right Upper) Injection of SR141716A (10 mg/kg) induced two patterns of hyperactivity. SPWs of larger amplitude (100–400 μV) occurred in bursts of three to five events (event 2) and lasting 5 ± 2 s, n = 9. In six of nine animals, this epileptic activity was followed (onset 13 ± 2.5 min) by ictal-like events (ILEs, event 3) lasting 6 ± 2 s. (Left Lower) SPW (event 1) and ILE (event 3) shown on a faster time scale. (Right Lower) Summary of all experiments in which ILEs were recorded (n = 6). (B) (Upper) Injection of AM404 (10 mg/kg) reduced both the frequency and amplitude of SPWs. (Lower) Summary of the effect of AM404 and WIN55212-2. The frequency and amplitude of SPWs were measured over a 5-min period 15–20 min after drug administration. AM404 (n = 6) reduces significantly these parameters to 62 ± 12% (*, P < 0.002) and 36 ± 3% (P < 0.006) of control, respectively. Injection of WIN55212-2 (10 mg/kg, n = 5) also significantly decreased the frequency and amplitude of SPWs to 54 ± 10% (*, P < 0.001) and 38 ± 8% (P < 0.007) of control, respectively.