Endocannabinoid signaling depends on the spatial pattern of synapse activation

Abstract

The brain's endocannabinoid retrograde messenger system decreases presynaptic transmitter release, but its physiological function is uncertain. We show that endocannabinoid signaling is absent when spatially dispersed synapses are activated on rodent cerebellar Purkinje cells but that it reduces presynaptic glutamate release when nearby synapses are active. This switching of signaling according to the spatial pattern of activity is controlled by postsynaptic type I metabotropic glutamate receptors, which are activated disproportionately when glutamate spillover between synapses produces synaptic crosstalk. When spatially distributed synapses are activated, endocannabinoid inhibition of transmitter release can be rescued by inhibiting glutamate uptake to increase glutamate spillover. Endocannabinoid signaling initiated by type I metabotropic glutamate receptors is a homeostatic mechanism that detects synaptic crosstalk and downregulates glutamate release in order to promote synaptic independence.

Figure 1

Endocannabinoid-mediated plasticity seen for activation of nearby synapses is absent for stimulation of spatially dispersed synapses. (a) Stimulation sites in the molecular layer (ML) and granular layer (GL). (b,c) Effect of a 10 pulse train at 200Hz on the EPSPfast evoked at −70mV in the same rat Purkinje cell by a single stimulus, using ML (b) and GL (c) stimulation. Main traces: response to the train between EPSPfast responses to single stimuli. Insets: EPSPs 2s before and after the train. (d) Suppression of EPSPfast after the train (Ctr) for ML stimulation in 8 cells, and its block by the CB1 antagonist AM251 (5 cells). (e) For GL stimulation the train evokes a small potentiation (11 cells, filled circles) which is unaffected by AM251 (5 cells), and pairing with mock-physiological climbing fibre stimulation (3 pulses delivered 100, 200 and 300msec after the start of GL stimulation) does not affect this (4 cells). (f,g) Effect of the cannabinoid agonist WIN 55,212-2 (5μM) on the EPSC_fast at −70mV evoked by ML (f) or GL stimulation (g). The EPSC_fast was reduced to 15.3 ± 1.4% (s.e.m., 4 cells) and 19.0 ± 2.3% (4 cells) respectively (insignificantly different, P = 0.22). (h,i) Effect of the mGluR1 agonist DHPG (50μM) on the EPSC_fast at −70mV evoked by ML (h) or GL stimulation (i). The EPSC_fast was reduced to 52.6 ± 5.8% and 59.8 ± 4.2% (10 cells) respectively (P = 0.29). DHPG suppression was measured after 1 min exposure to DHPG. All at 33°C.

Figure 2

Absence of endocannabinoid signalling when activating spatially dispersed synapses results from mGluR1 being activated by synaptic crosstalk. (a) Train-evoked EPSC_slow (in NBQX, 27°C, −70mV) for the same EPSC_fast amplitude evoked by interleaved ML and GL stimulation in mouse. (b) Mean EPSC_slow amplitude for 7 cells like a (EPSC_fast amplitude 596 ± 36pA for ML and 572 ± 44pA for GL stimulation; insignificantly different, P = 0.6) and for 7 rat cells (35°C, EPSC_fast amplitude 700 ± 114pA for ML and 667 ± 67pA for GL stimulation, P = 0.73). (c) AMPA receptor mediated EPSC_fast at −70mV in a mouse Purkinje cell evoked by two intensities of ML stimulus (27°C, EPSC amplitudes are in f). Inset: Variance/mean of the EPSC_fast amplitude is independent of stimulus size. (d) In the same cell, with the same stimuli as c, the EPSC_slow evoked (in 25μM NBQX, −70mV) by 10 stimuli at 200Hz increases more with stimulus strength than does the EPSC_fast. (e) Fractional change of EPSC_slow and EPSC_fast. (f) Dependence of EPSC_slow on EPSC_fast amplitude (proportional to number of fibres stimulated). Dotted line predicts relationship if synapses did not interact. (g,h) Fractional increase of EPSC_fast and EPSC_slow with Ca²⁺ buffering increased with EGTA (g, at 27°C) or BAPTA (h, at 35°C), with stimulus adjusted to increase EPSC_fast from 310 ± 20 to 1311 ± 50 pA (g) or from 323 ± 56 to 1164 ± 112 pA (h, in 0.2μM NBQX to improve voltage-uniformity). All data except b from mouse Purkinje cells.

Figure 3

Glutamate spillover mediates synaptic crosstalk detection by mGluR1 receptors. (a) AMPA receptor mediated currents in response to parallel fibre stimulus trains at two intensities (mouse Purkinje cell at 27°C). Inset: traces normalized by the amplitude of the first EPSC_fast of the train, demonstrating that post-train charge entry (shaded: grey, low stim; black, high stim) increases more than the number of fibres stimulated. (b) Dependence of charge transfer after the train on the amplitude of the first EPSC (proportional to number of fibres stimulated). Dotted line extrapolates relation calculated if synapses did not interact (as in Fig 2f). (c) Effect of deleting GLAST (−/−) (WT is wild-type control), and of blocking GLT-1 with dihydrokainate (DHK), on the EPSC_slow evoked in NBQX by trains of parallel fibre stimuli that produce a similar amplitude EPSC_fast (633 ± 48pA in 9 WT cells; 617 ± 36pA in 6 −/− cells; P = 0.8). Insets: response of Purkinje cells at −70mV to 0.5μM AMPA (13 cells each) and 25μM ACPD (15 cells each) is similar in WT and −/− cells (ACPD also activates group II mGluRs, but activating group II mGluRs generates no current in Purkinje cells25). (d) Left: EPSC_slow response (in NBQX) of wild-type and GLAST KO Purkinje cells to trains of 2, 3, 5 or 10 stimuli applied to the parallel fibres at 200Hz. Right: Percentage of 9 wild-type and 10 KO cells that showed a detectable EPSC_slow after different numbers of stimuli.

Figure 4

Endocannabinoid-mediated plasticity is rescued for stimulation of spatially-dispersed synapses by enhancing glutamate spillover, and inhibits glutamate release sufficiently to abolish crosstalk-activation of mGluR1. (a-c) Rescue of plasticity by promoting spillover. (a) Control response to ML and GL stimulus trains in NBQX (−70mV, EPSC_fast amplitude 497 ± 126pA for ML and 560 ± 133pA for GL, P = 0.48; TBOA does not affect EPSC_fast amplitude6). (b) Response to same stimuli with glutamate spillover enhanced by blocking glutamate transporters with TBOA, and with CPCCOEt present to block mGluR1. (c) EPSPfast amplitude after a train for GL stimulation in the absence (Ctr) and presence of 200μM TBOA: enhancing spillover rescues the endocannabinoid-evoked depression (5 cells). AM251 (2μM) blocked the depression rescued in TBOA (8 cells). (d-g) Cannabinoid signalling suppresses glutamate release sufficiently to prevent crosstalk-evoked mGluR1 activation. (d) EPSC_fast and EPSC_slow (in 0.2μM NBQX, to improve voltage uniformity, which reduced EPSC_fast amplitude by 65 ± 4%, 5 cells) evoked by 4 stimuli (200 Hz) to the parallel fibres, 10 sec before (−10s) and after (+10s) a 10 stimulus train (200Hz) in current clamp mode. (e) After the train, suppression of glutamate release by cannabinoid signalling reduced the first EPSC_fast of a 4 pulse train by 44 ± 12%, and reduced the peak EPSC_fast produced by the train by 36 ± 6%, but reduced the EPSC_slow by 93 ± 4% (4 cells). (f,g) In AM251, there is little change in the EPSC_fast or EPSC_slow (10 cells). All in rat, 33°C.