Purine receptor-mediated endocannabinoid production and retrograde synaptic signalling in the cerebellar cortex

Abstract

Background and purpose: Presynaptic CB₁ cannabinoid receptors can be activated by endogenous cannabinoids (endocannabinoids) synthesized by postsynaptic neurones. The hypothesis of the present work was that activation of calcium-permeable transmitter-gated ion channels in postsynaptic neurones, specifically of P2X purine receptors, can lead to endocannabinoid production and retrograde synaptic signalling.

Experimental approach: GABAergic inhibitory postsynaptic currents (IPSCs) were recorded with patch-clamp techniques in Purkinje cells in mouse cerebellar slices. Purine receptors on Purkinje cells were activated by pressure ejection of ATP from a pipette.

Key results: ATP evoked an inward current in Purkinje cells, most likely due to P2X receptor activation. The ATP-evoked currents were accompanied by currents via voltage-gated calcium channels. ATP suppressed electrical stimulation-evoked IPSCs and miniature IPSCs (mIPSCs) recorded in the presence of tetrodotoxin, and these effects were prevented by the CB₁ antagonist rimonabant and the calcium chelator BAPTA (applied into the Purkinje cell). ATP also suppressed mIPSCs when voltage-gated calcium channels were blocked by cadmium, and intracellular calcium stores were depleted by thapsigargin. However, ATP failed to suppress mIPSCs when the extracellular calcium concentration was zero.

Conclusions and implications: ATP elicits CB₁ receptor-dependent retrograde synaptic suppression, which is probably mediated by an endocannabinod released by the postsynaptic neurone. An increase in intracellular calcium concentration in the postsynaptic neurone is necessary for this retrograde signalling. We propose that ATP increases the calcium concentration by two mechanisms: calcium enters into the neurone via the P2X receptor ion channel and the ATP-evoked depolarization triggers voltage-gated calcium channels.

Figure 1

ATP responses in Purkinje cells. Glutamatergic and GABAergic synaptic input to Purkinje cells was blocked by DNQX, AP5 and bicuculline, and the patch-clamp pipette contained the calcium-sensitive fluorescent dye Oregon green 488 BAPTA-5N. (A1) Pressure ejection of ATP from a pipette elicited an inward current (‘purinergic current’) which was accompanied by several calcium spikes (a calcium spike is shown with higher temporal resolution). (A2) The fluorescent images and the (A3) statistical evaluation of calcium concentration changes indicate that ATP increased the calcium concentration in the soma and the dendrites. (B1-B3) ATP responses of the neurones shown in (A1-A3) during cadmium (10⁻⁴ M) superfusion. Means ± standard error of the mean of five experiments. Currents and calcium concentrations were recorded in the same neurones. * Indicates significant difference versus dendrite (P < 0.05). # Indicates significant difference (P < 0.05) versus ΔF/F₀ in the absence of cadmium (shown in A3).

Figure 2

ATP acts itself and A1 adenosine receptors are not involved in the effects of ATP. In addition to DNQX, AP5 and bicuculline, cadmium was added to the superfusion ACSF to block voltage-gated calcium channels. ATP was pressure ejected from a pipette four times. Amplitudes of ATP-evoked currents were expressed as percentages of the initial reference value PRE. Shown are means ± standard error of the mean. (A) After the second ATP application, solvent (SOL; n= 3) or the ecto-nucleotidase inhibitor ARL67156 (n= 8) was superfused. The original tracings were recorded in a slice with ARL67156 superfusion at time points 1 and 2. (B) After the second ATP application, solvent (SOL; n= 3) or the A1 receptor antagonist DPCPX (n= 4) was superfused. The original tracings were recorded in a slice with DPCPX superfusion at time points 1 and 2.

Figure 3

The stable ATP analogue GTPγS does not affect the pattern of ATP-evoked currents. In addition to DNQX, AP5 and bicuculline, cadmium was added to the superfusion ACSF to block voltage-gated calcium channels. The patch clamp pipette contained GTP or GTPγS. (A) The mGluR agonist DHPG was pressure ejected from a pipette three times. In the presence of GTP in the patch-clamp pipette (n= 7), the DHPG-evoked charge transfer remained constant (charge transfer was calculated for the 50-s period following DHPG application). GTPγS in the patch-clamp pipette (n= 7) prolonged the effect of the first DHPG application (thereby increasing charge transfer), and diminished the effects of the further DHPG applications. * Indicates significant difference from GTP (P < 0.05). (A2-A3) Original tracings of DHPG-evoked currents in the presence of GTP or GTPγS. (B) ATP was pressure ejected from a pipette three times. In the presence of GTP in the patch-clamp pipette (n= 5), the ATP-evoked charge transfer remained constant (charge transfer was calculated for the 50-s period following ATP application). The ATP effect was similar in experiments with GTPγS in the patch-clamp pipette (n= 5). (B2-B3) Original tracings of purinergic currents in the presence of GTP or GTPγS.

Figure 4

Ivermectin potentiates ATP-evoked currents. In addition to DNQX, AP5 and bicuculline, cadmium was added to the superfusion ACSF to block voltage-gated calcium channels. ATP was pressure ejected from a pipette four times. After the second ejection, solvent (SOL) or ivermectin (IVM) was superfused. ATP-evoked charge transfer values were expressed as percentages of the initial reference value PRE. Means ± standard error of the mean of seven (SOL), three (IVM 10⁻⁶ M), three (IVM 10⁻⁵ M), six (IVM 5 × 10⁻⁵ M) and four (IVM 10⁻⁴ M) experiments. * Indicates significant difference from SOL (P < 0.05). (A2) The original tracings were recorded in a slice with IVM 10⁻⁴ M superfusion at time points 1 and 2 (indicated in A1).

Figure 5

ATP suppresses electrically evoked inhibitory postsynaptic currents (eIPSCs). DNQX and AP5 were present in the superfusion ACSF. eIPSCs were elicited every 20 s by electrical stimulation in the molecular layer. Every three eIPSCs were averaged and expressed as percentages of eIPSCs during the initial reference period PRE. Pressure ejection of ATP evoked purinergic currents which were accompanied by calcium spikes (not shown). The points after ATP application are the averages of three eIPSCs elicited 5, 25 and 45 s after the 5-s ATP application. Means ± standard error of the mean of 9 (SOL) and 10 (RIM) experiments. Filled symbols indicate significant difference versus the time point preceding ATP ejection (P < 0.05); * Indicates significant difference from SOL (P < 0.05). (A2) The original tracings were recorded at time points 1 and 2 (indicated in A1) in the presence of solvent or rimonabant.

Figure 6

ATP suppresses miniature inhibitory postsynaptic currents (mIPSCs) recorded in the presence of tetrodotoxin. In addition to DNQX and AP5, tetrodotoxin (3 × 10⁻⁷ M) was present in the superfusion ACSF to block voltage-gated sodium channels. (A–C) The frequency, amplitude and cumulative amplitude of mIPSCs were evaluated in 10-s periods and expressed as percentages of values during the initial reference period PRE. Pressure ejection of ATP evoked purinergic currents which were accompanied by calcium spikes (see D). Means ± standard error of the mean of 16 (SOL) and 18 (RIM) experiments. Filled symbols indicate significant difference versus PRE (P < 0.05); * indicates significant difference from SOL (P < 0.05). (D) An original tracing recorded in the presence of solvent (the period between the dashed lines was not evaluated in A-C).

Figure 7

Superfusion with calcium-free artificial cerebrospinal fluid (ACSF) diminishes the ATP-evoked current and the increase in intracellular calcium concentration. Glutamatergic and GABAergic synaptic input to Purkinje cells was blocked by DNQX, AP5 and bicuculline, and the patch-clamp pipette contained the calcium-sensitive fluorescent dye Oregon green 488 BAPTA-5N. The entire experiment was performed in the presence of cadmium (10⁻⁴ M) and thapsigargin (10⁻⁵ M). (A1) Pressure ejection of ATP from a pipette elicited a strong purinergic current. (A2) The fluorescent images and the (A3) statistical evaluation of calcium concentration changes indicate that ATP increased the calcium concentration in the soma and the dendrites. (B1-B3) ATP responses of the neurones shown in A1–A3 during superfusion with calcium free ACSF (Ca_o²⁺= 0 mM). Means ± standard error of the mean of four experiments. Currents and calcium concentrations were recorded in the same neurones. * Indicates significant difference versus dendrite (P < 0.05). # Indicates significant difference (P < 0.05) versus ΔF/F₀ in the presence of calcium in the ACSF (shown in A3).

Figure 8

ATP suppresses sIPSCs recorded in the presence of cadmium and thapsigargin. In addition to DNQX and AP5, cadmium (10⁻⁴ M) and thapsigargin (10⁻⁵ M) were present in the superfusion ACSF. The experiments in Figures 8 and 9 were performed on the same neurones. Figure 8 shows the first part of the experiment, in which an ACSF with normal calcium concentration was superfused. Figure 9 shows the second part of the experiments, during which calcium-free ACSF was superfused. (A–C) The frequency, amplitude and cumulative amplitude of cadmium sIPSCs were evaluated in 10-s periods and expressed as percentages of values during the initial reference period PRE. Pressure ejection of ATP evoked purinergic currents, but calcium spikes were prevented by cadmium in the ACSF (see D). Means ± standard error of the mean of 12 (SOL) and 10 (RIM) experiments. Filled symbols indicate significant difference versus PRE (P < 0.05); * indicates significant difference from SOL (P < 0.05). (D) An original tracing recorded in the presence of solvent (the period between the dashed lines was not evaluated in A–C).

Figure 9

ATP does not suppress sIPSCs recorded in the presence of cadmium and thapsigargin and calcium-free ACSF. In addition to DNQX and AP5, cadmium (10⁻⁴ M) and thapsigargin (10⁻⁵ M) were present in the calcium-free superfusion ACSF. Figure 8 shows the first part of the experiment, during which ACSF with normal calcium concentration was superfused. (A–C) The frequency, amplitude and cumulative amplitude of cadmium sIPSCs were evaluated in 10-s periods and expressed as percentages of values during the initial reference period PRE. Means ± standard error of the mean of 12 (SOL) and 10 (RIM) experiments. (D) An original tracing recorded in the presence of solvent (the period between the dashed lines was not evaluated in A–C).