Developmental alteration of endocannabinoid retrograde signaling in the hippocampus

Abstract

Endocannabinoids are lipid derivatives that mediate paracrine and juxtacrine signaling between cells. In the hippocampal CA1 region, a retrograde endocannabinoid signal suppresses GABA release by acting on presynaptic cannabinoid receptor-1 (CB1) and can be functionally manifested as depolarization-induced suppression of inhibition (DSI). In the present study, whole cell patch-clamp recordings in hippocampal slices were made to examine DSI in rats from P7-P21. Robust DSI develops in rat hippocampus at postnatal ages greater than two weeks, but only modest DSI is observed in P7-9 rat. DSI in neonatal rats can be enhanced by activation of group I metabotropic glutamate receptors (mGluRs) or muscarinic acetylcholine receptors in those neonatal rats. The DSI is also enhanced by sustained low-frequency (1 Hz) stimulation (5 min). This stimulus-enhanced DSI was prevented in the presence of 6-methyl-2-(phenylethynyl)-pyridine (10 microM), a group I mGluR antagonist. WIN55212-2, a synthetic CB1 agonist, produced a similar level of inhibition of GABAergic synaptic transmission at different postnatal time points. Therefore postsynaptic mechanisms appear to be mainly responsible for developmental changes in DSI, although presynaptic mechanisms cannot be ruled out entirely. We have also obtained evidence that tonic endocannabinoid release suppresses GABAergic transmission in the mature but not the neonatal hippocampus. The differential DSI magnitude at different stages of maturation could alter synaptic plasticity and learning and memory during hippocampal development.

Fig. 1.

Developmental changes in depolarization-induced suppression of inhibition (DSI) in hippocampus. A: in CA1 pyramidal neurons, a 4-s depolarizing step resulted in modest suppression of the evoked inhibitory postsynaptic current (eIPSC) at postnatal days 7–8 and 9 but produced a robust suppression at P15 and P19-21. Each inset superimposes the average of 5 consecutive eIPSCs pre-DSI and 3 trials post-DSI. Square step indicates a 4-s depolarization from −60 to 0 mV. B: DSI magnitude was measured as the mean percentage reduction of 3 consecutive eIPSCs after the depolarizing step. Numbers above each bar indicate the number of cells. * P < 0.05 compared with P7-8 rats.

Fig. 2.

The baseline eIPSC amplitude is not correlated with DSI magnitude. A: bar plot of average amplitude of eIPSCs vs. postnatal age. The eIPSC amplitude was significantly larger in >P14 rats than in P7/8 ∼ P13 groups of rat (P < 0.05, ANOVA). There was no significant difference in eIPSC amplitude among the P7-8 ∼ P13 and P14 ∼ P19-21groups (P > 0.05, ANOVA). B: plot of prepulse eIPSC density vs. DSI magnitude from 105 cells (P7–P21). The continuous line is a linear regression to all data points. C: a subset of the data plotted in B (P15–P21). —, a linear regression to all data points, indicating a poor relationship between the eIPSC density and the magnitude of DSI even in these more mature rats (>P14). D: in the presence of SR141716 (2 μM), a 4-s depolarizing step results in little or no suppression of IPSCs (n = 7) in mature rats (P17–P20). Insets: from the same neuron in the absence and presence of SR141716.

Fig. 3.

Presynaptic modulation by cannabinoid receptor-1 (CB1) receptors does not change during development. A: representative experiments from P8 or P18 rat show that the synthetic CB1 agonist WIN55212-2 elicited a robust suppression of eIPSCs and an increase in the amplitude ratio of 2 closely paired eIPSCs. Traces are average eIPSCs from 20 trials before WIN55212-2 and 10 min after drug application. B: WIN55212-2 produced a 52 ± 7% reduction in eIPSC amplitude in P8 rat and a 46 ± 6% reduction in mature rat (P17-20). There was no significant difference between WIN55212-2 effects in young and mature rats (P = 0.51).

Fig. 4.

(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R)-ACPD and carbachol facilitate DSI in neonatal rats (P8-9). A: traces showing DSI are all from the same neuron (top). In the presence of ACPD (10 μM) for 4–8 min, depolarization produced a robust suppression (40 ± 6% reduction of baseline eIPSC amplitude; n = 6; bottom). Prepulse trace is the average of the 5 eIPSCs just prior to a 4-s depolarization, and post pulse trace is the average of 3 eIPSCs after depolarization. B: carbachol (carb) enhances DSI in neonatal rat. Similar arrangement as in A, in the presence of carb (2 μM) for 4–8 min, the same depolarizing step leads to a 48 ± 5% reduction relative to baseline eIPSC amplitude (n = 5).

Fig. 5.

Enhancement of DSI by 1-Hz stimulation. A: DSI was moderate in neonatal rats (P9-11) and was enhanced by low-frequency stimulation (1 Hz for 5 min). Inset: traces show eIPSCs from representative experiments. B: in the presence of 6-methyl-2-(phenylethynyl)-pyridine (MPEP), an mGluR5 antagonist, the same low-frequency stimulation procedure had no effect on DSI in neonatal rats (P10-11). Inset: traces are from the same neuron. C: summary of effect of 1-Hz stimulation on DSI magnitude in the absence and presence of MPEP. * P < 0.05, compared with the control in the absence of MPEP.

Fig. 6.

Ongoing release of endocannabinoids modulates GABAergic transmission in mature rat. A: representative traces are averages of responses before and after 10 min application of SR141716. SR141716 enhanced eIPSCs in mature rat, but not in P8 rat. B: in mature rat, SR141716 (2 μM) caused a 43 ± 9% increase in eIPSC amplitude (P < 0.001), but had no significant effect on the eIPSC (only 7 ± 22% increase, P = 0.71) in P8 rat.