Presynaptic CB1 receptors regulate synaptic plasticity at cerebellar parallel fiber synapses

Abstract

Endocannabinoids are potent regulators of synaptic strength. They are generally thought to modify neurotransmitter release through retrograde activation of presynaptic type 1 cannabinoid receptors (CB1Rs). In the cerebellar cortex, CB1Rs regulate several forms of synaptic plasticity at synapses onto Purkinje cells, including presynaptically expressed short-term plasticity and, somewhat paradoxically, a postsynaptic form of long-term depression (LTD). Here we have generated mice in which CB1Rs were selectively eliminated from cerebellar granule cells, whose axons form parallel fibers. We find that in these mice, endocannabinoid-dependent short-term plasticity is eliminated at parallel fiber, but not inhibitory interneuron, synapses onto Purkinje cells. Further, parallel fiber LTD is not observed in these mice, indicating that presynaptic CB1Rs regulate long-term plasticity at this synapse.

Fig. 1.

Immunostaining reveals selective elimination of CB1Rs from cerebellar granule cells in CB1α6⁻ animals. Sagittal sections (50 μm thick, ≥4 per animal) from 2-mo-old mice were stained with a rabbit polyclonal antibody raised against the last 15 amino acids of the CB1R C-terminus (Bodor et al. 2005; Nyiri et al. 2005) and imaged at ×40. Images (z-projections of 11 images taken at 1-μm intervals) of the cerebellar cortex are shown for a representative experiment of control (A), CB1α6⁻ (B), and global CB1 knockout (C) animals (n = 5 each). The molecular layer (ML), the Purkinje cell layer (PL), and the granular layer (GL) are indicated in C.

Fig. 2.

CB1R function is eliminated from parallel fibers in a neuron and age-specific manner. Depolarization-induced suppression of excitatory and inhibitory synapses (DSE/DSI) was examined in control and CB1α6 ^– mice. Purkinje cells were depolarized to 0 mV for 2 s and the effect on PF-excitatory postsynaptic currents (EPSCs, A and B), interneuron IPSCs (C), and climbing fiber EPSCs (D) were assessed. Experiments were conducted in p18 animals (B–D) and in p13 animals before full Cre expression (A). A–D, left: traces from representative experiments are shown for control (black) and CB1α6^– (red) mice with EPSCs prior to (thin traces) and following depolarization (thick traces) superimposed. A–D, right: summaries of the average amplitudes of synaptic responses (±SE) are shown for control and CB1α6^– mice (n = 4–10 neurons per condition). For the electrophysiological traces, vertical scale bars correspond to 200 pA in A–C and 300 pA in D, and horizontal scale bars correspond to 5 ms in A and B, 10 ms in C, and 20 ms in D.

Fig. 3.

Elimination of presynaptic CB1Rs alters short-term synaptic plasticity at PF-PC synapses. PF-excitatory postsynaptic potentials (EPSPs) were measured before and after the presentation of a conditioning train consisting of 3–20 PF stimuli at 100 Hz. Traces from representative experiments are shown for a control (A) and a CB1α6^– mouse (B). Typical responses to conditioning trains with 10 PF stimuli are shown for each experiment, and insets show the average PF-EPSPs measured before (thin) and 2–4 s after (thick) the train. C: the short-term plasticity in Purkinje cells recorded from control (n = 7, black) and CB1α6^– mice (n = 4, red) are summarized as a function of the number of stimuli in the conditioning train. Error bars = SE. (*, P < 0.05; **, P < 0.01; t-test).

Fig. 4.

Presynaptic PF-LTP is unchanged in CB1α6^– mice. PF-EPSCs were recorded from Purkinje cells in response to test pulses delivered at 0.05 Hz before and after a burst of PF stimuli (15 s at 8 Hz) (Salin et al. 1996; van Beugen et al. 2006). Average EPSCs recorded in the 5 min prior to (thin traces) and 15–20 min after the conditioning train (thick traces), are shown for representative experiments for control animals (A) and CB1α6^– animals (B). C: the average EPSC amplitude (±SE) is plotted as a function of time for control (n = 9) and CB1α6^– (n = 10) animals. D: the cumulative distribution of the amplitudes of long term plasticity (the average EPSP amplitude 15–20 min after the conditioning train/the average EPSP amplitude for the 5 min prior conditioning train) are plotted for the experiments summarized in C. The conditioning train resulted in LTP of PF-EPSCs for both control and CB1α6^– animals, and there was no statistically significant difference in the extent of potentiation (25 and 22%, respectively, P = 0.81, t-test).

Fig. 5.

PF-LTD is eliminated in CB1α6^– mice. PF-long-term depression (LTD) was induced with a conditioning train consisting of 10 PF stimuli at 100 Hz (thin blue bars in A and B) followed by 2 CF stimuli at 20 Hz (thick blue bars in A and B), repeated every 10 s for 5 min. Traces from representative experiments are shown for a control (A) and a CB1α6^– mouse (B). Typical responses to conditioning stimuli are shown for each experiment. Insets: average PF-EPSPs measured for the 5 min before (thin) and 15–20 min after (thick) the induction protocol. C: summary of the average normalized EPSP amplitude (±SE) is plotted as a function of time recorded for control (n = 10, black) and CB1α6^– (n = 10, red) mice. D: the cumulative distributions of the normalized EPSP amplitudes 15–20 min after the conditioning train are plotted for the experiments summarized in C.