Effects of the endogeneous cannabinoid, anandamide, on neuronal activity in rat hippocampal slices

Abstract

1. The arachidonic acid derivative arachidonylethanolamide (anandamide) is an endogeneous ligand of cannabinoid receptors that induces pharmacological actions similar to those of cannabinoids such as delta9-tetrahydrocannabinol (THC). We examined whether anandamide can influence excessive neuronal activity by investigating stimulation-induced population spikes and epileptiform activity in rat hippocampal slices. For this purpose, the effects of anandamide were compared with those of the synthetic cannabinoid agonist WIN 55,212-2 and its inactive S(-)-enantiomer WIN 55,212-3. 2. Both anandamide (1 and 10 microM) and WIN 55,212-2 (0.1 and 1 microM) decreased the amplitude of the postsynaptic population spike and the slope of the field excitatory postsynaptic potential (field e.p.s.p.) without affecting the presynaptic fibre spike of the afferents. At a concentration of 1 microM, WIN 55,212-2 completely suppressed the postsynaptic spike, whereas the S(-)-enantiomer WIN 55,212-3 produced only a slight depression. The CB1 receptor antagonist SR 141716 blocked the inhibition evoked by the cannabinoids. SR 141716 had a slight facilitatory effect on neuronal excitability by itself. 3. Anandamide shifted the input-output curve of the postsynaptic spike and the field e.p.s.p. to the right and increased the magnitude of paired-pulse facilitation indicating a presynaptic mechanism of action. 4. Anandamide and WIN 55,212-2, but not WIN 55,212-3, attenuated both stimulus-triggered epileptiform activity in CA1 elicited by omission of Mg2+ and spontaneously occurring epileptiform activity in CA3 elicited by omission of Mg2+ and elevation of K+ to 8 mM. The antiepileptiform effect of these cannabinoids was blocked by SR 141716. 5. In conclusion, cannabinoid receptors of the CB1 type as well as their endogeneous ligand, anandamide, are involved in the control of neuronal excitability, thus reducing excitatory neurotransmission at a presynaptic site, a mechanism which might be involved in the prevention of excessive excitability leading to epileptiform activity.

Figure 1

Inhibitory effect of anandamide (AN, 10 μM) on the field e.p.s.p. recorded in the CA1 stratum radiatum. The traces above the graph are the average of five subsequent responses from one representative experiment. The graph shows the time-course of the effect of anandamide on the slope of the field e.p.s.p. The data represent mean values±s.d. of six experiments.

Figure 2

Effect of anandamide on the orthodromically evoked population spike (PS) in the hippocampal CA1 area. (A) Anandamide (AN, 10 μM) reversibly decreased the amplitude of postsynaptic population spike. (B) The anandamide-evoked inhibition was prevented by pretreatment with the CB₁ receptor antagonist, SR 141716 (SR, 2 μM). The traces above the graphs derive from a representative experiment showing the average of five subsequent responses at the end of the control and at the end of drug-application. The graphs show the time-course of the amplitude of the presynaptic fibre spike and the postsynaptic population spike. The data represent mean values±s.d. of 9–12 experiments.

Figure 3

Effect of WIN 55,212-2 on the orthodromically evoked population spike (PS) in the hippocampal CA1 area. (A) WIN 55,212-2 (WIN, 1 μM) completely suppressed the orthodromic response, and this effect was reversed by SR 141716 (1 μM). (B) The suppression of the population spike by WIN 55,212-2 was prevented by pretreatment with the CB₁ receptor antagonist, SR 141716 (SR, 1 μM). The traces above the graphs show the average of five subsequent responses at the end of the control and at the end of drug-application. The graphs show the time-course of the amplitude of the presynaptic fibre spike and the postsynaptic population spike. The data represent mean values±s.d. of 6–10 experiments.

Figure 4

The effects of anandamide and WIN 55,212-2 on the stimulus-response relationship. The slices were stimulated with intensities ranging from subthreshold to maximal. For each response, the amplitude of the presynaptic fibre spike (i), the amplitude of the postsynaptic population spike (ii) and the slope of the field e.p.s.p. (iii) were measured and plotted as function of stimulus-intensity. The data represent mean values±s.d. of 5–6 experiments.

Figure 5

Enhancement of paired-pulse facilitation by anandamide. (A) Average of five consecutive field e.p.s.ps evoked by paired stimuli with an interstimulus interval of 40 ms at control and 60 min after starting the superfusion of anandamide (10 μM). At the end of the anandamide application, the stimulation intensity was increased to counteract a direct depressant effect of anandamide on the first field e.p.s.p. (B) Effects of anandamide (10 μM) on the paired-pulse index (PI) calculated from responses to paired-pulse stimulation of different intervals (20–200 ms) in the CA1 stratum radiatum. Anandamide significantly increased the PI values at all intervals (at least P⩽0.05, n=8). (C) The PI was calculated according to the formula PI=P₂/P₁×100% with P₁ being the average of five responses to the first stimulus and P₂ being the average of five responses to the second stimulus. Data are mean values±s.d.

Figure 6

Effect of the cannabinoids on stimulus-triggered epileptiform activity elicited by omission of Mg²⁺ from the superfusate. (A) Extracellularly recorded population spikes from one representative experiment out of eight similar ones showing the effect of anandamide. (B) Effects on the first, second and third population spike (PS) of the burst by anandamide alone and together with SR 141716, by WIN 55,212-2 and by WIN 55,212-3. The amplitudes of the spikes were normalized with respect to the amplitudes measured during superfusion with the nominal Mg²⁺-free bathing medium. The anandamide-evoked reduction of the spike amplitudes was significant (^*P⩽0.001, n=8). Each column shows the mean±s.d.

Figure 7

Time-course of the effect of the cannabinoids on the frequency of spontaneously occurring epileptiform burst discharges in the stratum pyramidale of the CA3 region. Epileptiform activity was elicited by omission of Mg²⁺ and elevation of K⁺ to 8 mM. (A) Antiepileptic effect of anandamide (10 μM) and blockade of the effect by simultaneous application of SR 141716 (1 μM). (B) Antiepileptiform effect of WIN 55,212-2 (500 nM) and its suppression by SR 141716 (1 μM). Note that the S(−)-enantiomer WIN 55,212-2 failed to alter the frequency of the epileptiform burst discharges. Data points represent the mean±s.d. of 6–9 experiments.