Disruption of endocannabinoid release and striatal long-term depression by postsynaptic blockade of endocannabinoid membrane transport

Abstract

Activation of the CB1 cannabinoid receptor inhibits neurotransmission at numerous synapses in the brain. Indeed, CB1 is essential for certain types of both short- and long-term synaptic depression. It was demonstrated recently that CB1 is critical for activity-dependent long-term depression (LTD) at glutamatergic corticostriatal synapses in acute brain slice preparations. Here, we show that CB1 activation is necessary, but not solely sufficient, for induction of LTD and that the requisite signaling by endocannabinoids (eCBs) occurs during a time window limited to the first few minutes after high-frequency stimulation delivery. In addition, we have applied intracellularly anandamide membrane transporter inhibitors to provide novel evidence that postsynaptic transport mechanisms are responsible for the release of eCBs from striatal medium spiny neurons. These findings shed new light on the mechanisms by which transient eCB formation participates in the induction of long-lasting changes in synaptic efficacy that could contribute to brain information storage.

Figure 1.

CB1 activation is required for induction, but not maintenance, of striatal LTD. After obtaining a stable EPSC for at least 10 min, HFS was paired with a postsynaptic depolarization (black arrow). This stimulation protocol resulted in a long-lasting decrease in EPSC amplitude that was unchanged when the CB1 antagonist SR141716A (3 μm) was bath applied 10 min after HFS (♦). LTD was blocked when SR141716A was applied 1 min after HFS (•), whereas variable results were obtained when SR141716A was applied ∼3 min after HFS (□). Points are the averaged values over 1 min. Bottom, Thirty trace averages of EPSCs recorded 10 min before (left) and 20-30 min after (right) HFS. Left, SR141716A applied 1 min after HFS. Right, SR141716A applied 10 min after HFS. Calibration: 100 pA, 25 msec.

Figure 2.

Intracellular, but not extracellular, application of AMT inhibitors blocks striatal LTD. a, Including the AMT inhibitor VDM11 (10 μm) intracellularly via the patch pipette blocked LTD (•), whereas a similarly included vehicle (0.02% DMSO) had no effect (○). Top, Sample traces for VDM11 inside (top) and vehicle inside (bottom); traces are as in Figure 1. b, LTD was also blocked when AM404 (2 μm) was included intracellularly. Top, Traces are as in Figure 1. c, Slices pretreated extracellularly with VDM11 (10 μm) expressed LTD. Top, Traces are as in Figure 1. Calibration: 100 pA, 25 msec. d, PPR of EPSCs 20-30 min after HFS relative to baseline (percentage). Vehicle (Veh)-loaded cells and cells pretreated extracellularly with VDM11 (VDM11 out) exhibited enhanced PPR after HFS. Cells loaded intracellularly with either VDM11 (VDM11 in) or AM404 (AM404 in) did not exhibit this PPR increase.

Figure 5.

Effects of intracellular loading with AEA or 2-AG are blocked with coloading of an AMT inhibitor. a, b, Including AEA or 2-AG in the postsynaptic neuron was associated with an increase in the PPR. This increase was blocked when VDM11 (10 μm) was also included in the pipette but not when VDM11 was applied extracellularly. Top, Ten trace average of EPSCs recorded when AEA (a) or 2-AG (b) was coloaded with VDM11 (left) or pretreated extracellularly with VDM11 (right). Calibration: 100 pA, 25 msec. c, Intracellular or extracellular application of VDM11 alone had no effect on the PPR relative to vehicle-loaded cells. This was also true for extracellular application of the VDM11 vehicle Tocrisolve100. d, When stimulus intensities were identical across experiments, the PPR was enhanced in cells loaded with 50 mm AEA, and this increase was blocked with extracellular application of SR141716A and cointracellular loading with AM404 (10 mm). The number of cells is indicated above each bar. ^*p < 0.05, relative to vehicle-loaded cells.

Figure 3.

PPR increases in cells loaded intracellularly with AEA or 2-AG. Vehicle-loaded cells (Veh; 0.2% DMSO) had a low PPR, consistent with PPR values obtained previously from MSNs at this age (Choi and Lovinger, 1997A). Intracellular loading with AEA or 2-AG (50 μm) was associated with a significant increase in the PPR, whereas similarly loaded cells in slices pretreated with the CB1 antagonist SR141716A (3 μm) did not exhibit this PPR increase. SR141716A alone did not significantly affect the PPR. The number of cells is indicated above each bar. ^*p < 0.05, relative to vehicle. Calibration: 100 pA, 25 msec.

Figure 4.

CB1 activation alone is not sufficient to induce striatal LTD. a, Extracellular application of the CB1 agonist WIN55,212-2 (WIN; 3 μm) caused a significant decrease in PS amplitude. Subsequent bath application of SR141716A (SR; 3 μm) reversed this inhibition. Calibration: 0.2 mV, 10 msec. b, Extracellular bath application of SR141716A subsequent to intracellular AEA (50 μm) loading caused an increase in EPSC amplitude (•). SR141716A alone had no effect on baseline EPSC amplitude (○). Calibration: 200 pA, 25 msec.