CB₂ cannabinoid receptors inhibit synaptic transmission when expressed in cultured autaptic neurons

Abstract

The role of CB₂ in the central nervous system, particularly in neurons, has generated much controversy. Fueling the controversy are imperfect tools, which have made conclusive identification of CB₂ expressing neurons problematic. Imprecise localization of CB₂ has made it difficult to determine its function in neurons. Here we avoid the localization controversy and directly address the question if CB₂ can modulate neurotransmission. CB₂ was expressed in excitatory hippocampal autaptic neurons obtained from CB₁ null mice. Whole-cell patch clamp recordings were made from these neurons to determine the effects of CB₂ on short-term synaptic plasticity. CB₂ expression restored depolarization induced suppression of excitation to these neurons, which was lost following genetic ablation of CB₁. The endocannabinoid 2-arachidonylglycerol (2-AG) mimicked the effects of depolarization in CB₂ expressing neurons. Interestingly, ongoing basal production of 2-AG resulted in constitutive activation of CB₂, causing a tonic inhibition of neurotransmission that was relieved by the CB₂ antagonist AM630 or the diacylglycerol lipase inhibitor RHC80267. Through immunocytochemistry and analysis of spontaneous EPSCs, paired pulse ratios and coefficients of variation we determined that CB₂ exerts its function at a presynaptic site of action, likely through inhibition of voltage gated calcium channels. Therefore CB₂ expressed in neurons effectively mimics the actions of CB₁. Thus neuronal CB₂ is well suited to integrate into conventional neuronal endocannabinoid signaling processes, with its specific role determined by its unique and highly inducible expression profile.

Fig. 1. CB₂ is trafficked to both the axonal and somatodendritic compartments of transfected CB₁ null neurons

A) Micrograph shows composite (top) and component panels (below) for axons from a mCB₂-HA/mCherry transfected neuron. mCherry shows the full outline of the transfected processes, HA staining identifies the mCB₂ HA tag, and 2H3 labels axons. Arrows indicate examples of overlap between the HA and 2H3 immunoreactivity. B) A neuron transfected with HA-mCB₂ and mCherry but stained for HA and the dendritic marker MAP2. Arrows indicate examples of overlap between the HA and MAP2 immunoreactivity. Scale bars: 5 μm and 20 μm for panels A and B, respectively.

Fig. 2. CB₂ expression restores DSE to CB₁ null neurons

(A) Time course of EPSCs recorded before and after a depolarizing step to 0 mV from a holding potential of −70 mV (arrow at 20 sec) in wild type (CB₁+/+, n=6), CB₁ null (CB₁−/−, n=6) and CB₂ transfected CB₁ null neurons (CB₁−/− + CB₂, n=12). Scale bars = 1 nA, 10 ms. (B) Summary of the maximal inhibition of neurotransmission achieved by 3 seconds of depolarization in each class of neuron. Data analyzed using one way ANOVA with Bonferroni multiple comparison test. (C) Plot of the effect of increasing lengths of depolarization from 50 ms up to 10 s on EPSC magnitude for CB₂ transfected CB₁ null neurons (n=3–11 for each time point). (D) 1 μM AM630 prevents DSE following a 3 depolarizing stimulus in CB₂ transfected CB₁ null neurons that previously exhibited DSE, suggesting the rescue of DSE is due to the expression of functional CB₂ receptors (n=4). Data analyzed using paired Student's t-test. (E) 1 μM AM630 did not prevent DSE following a 3 depolarizing stimulus in wild type neurons that previously exhibited DSE (n=3). Scale bars in (D) and (E) = 1 nA, 5 ms. Data in (D) and (E) analyzed using paired Student's t-test. *: p<0.05, ***: p<0.001.

Fig. 3. 5 μM 2-AG mimics the effects of depolarization in CB₂ neurons

(A) Representative time course showing that treatment with 5 μM 2-AG suppresses neurotransmission and the inhibition can be reversed/blocked by 1 μM AM630. Inset shows individual traces for indicated time points. Scale bar = 1 nA, 10 ms (B) 5 μM 2-AG suppresses EPSCs (n=9) to a similar extent as 3 s depolarizing stimulus (n=11). This suppression is blocked by 1 μM AM630 (n=5). 2-AG has no effect on neurotransmission in non-transfected CB₁ null neurons (n=4). Data analyzed using oneway ANOVA with Bonferroni's multiple comparison test. ***: p<0.001 vs. basal. ††: p<0.01 vs. 2-AG, †††: p<0.001 vs. 2-AG.

Fig. 4. CB₂ activation decreases sEPSC frequency, but not amplitude suggesting a presynaptic site of action

Spontaneous EPSCs (sEPSCs) were recorded from CB₂ expressing neurons. Representative traces of sEPSCs recorded under (A) basal conditions, during 5 μM 2-AG treatment and during 5 μM 2-AG treatment with 1 μM AM630 present. Scale bars = 10 pA, 100 ms (B) Cumulative probability histogram of sEPSC frequency shows that the inter-event interval increases following 2-AG treatment and returns back to baseline when AM630 is applied. (C) Summary data for sEPSC frequency. (D) Cumulative probability histogram of sEPSC amplitude shows that 2-AG and AM630 do not alter the sEPSC amplitude. (E) Summary data for sEPSC amplitude. n=8 for all treatments. Data in (C) and (E) analyzed using one way ANOVA with Bonferroni's multiple comparison test. *: p<0.05 vs. 2-AG.

Fig. 5. CB₂ activation increases the paired pulse ratio and coefficient of variation suggesting a presynaptic site of action

(A) 5 μM 2-AG increases the paired pulse ratio (PPR) in CB₁ null neurons expressing CB₂ (n=6). (B) A 3 sec depolarization (DSE) also increases the PPR (n=6). The increases in PPR suggest a presynaptic site of action. (C) 5 μM 2-AG increases the coefficient of variation (CV) in CB₁ null neurons expressing CB₂. (D) Analysis of CV for 2-AG treatment demonstrates that r < π < 1 (n=8). (E) DSE also increases the CV (n=11). (F) Analysis of CV for DSE demonstrates that r < π < 1. The increases in CV as well as the measurements of r and π all point to a presynaptic site for CB₂ activation resulting in synaptic depression. Data analyzed using paired Student's t-test. * p < 0.05 vs. basal. ***: p<0.001 vs. basal.

Fig. 6. CB2 activation by 2-AG does not alter GIRK or outward currents

A) 2-AG (5 μM) did not alter peak inwardly rectifying potassium currents observed while hyperpolarizing CB₂-transfected neurons to −130mV after a brief depolarization to −50mV. These currents are reliably elicited by postsynaptic GABA_B (25μM baclofen, n=5) and adenosine A1 (200 nM N6-cyclopentyladenosine, n=4) receptor activation in wild type neurons. Top trace: representative 2-AG treatment of CB₂ expressing neuron. Bottom trace: representative trace for a baclofen treated neuron. Scale bars: 500pA, 50msec. B) Current-Voltage plot shows that peak outward currents in response to successive voltage steps are unaltered by application of 2-AG (5μM) in CB₂-expressing neurons. p<0.05 for GABA_B and Adenosine A1 vs. CB₂ using one way ANOVA with Dunnett's posthoc test.

Fig. 7. CB₂ expressed in neurons displays constitutive activity

(A) CB₂ expressing CB₁ null neurons have smaller EPSCs than untransfected CB₁ null neurons from the same coverslip (n=5). (B) Treatment with 1 μM AM630 increases the sizes of EPSCs in CB₂ expressing neurons (n=8). (C) AM630 decreases the PPR in CB₂ neurons, suggesting a presynaptic site of action (n=7). (D) Cumulative probability histogram of sEPSC frequency shows that the inter-event interval decreases following 1 μM AM630 treatment (n=8). (E) Summary data for sEPSC frequency. (F) Representative time course of neuron treated with 10 μM RHC80267, a DGL inhibitor and 1 μM AM630. (G) Summary of data from neurons treated with RHC80267 (n=9 for RHC80267 alone and n=4 for RHC80267 + AM630). RHC80267 increases the relative size of EPSCs and occludes the AM630 increase seen in (B) suggesting CB₂ constitutive activity is due to ongoing 2-AG synthesis. Data in (A) and (G) analyzed using unpaired Student's t-test. Data in (B),(C), and (E) analyzed using paired Student's t-test. *: p<0.05, ***: p<0.001.

Fig. 8. CB₂ inhibits neurotransmission through inhibition of voltage gated calcium channels

(A,B) In CB₂ expressing CB₁ null neurons, exchanging the external medium with medium containing 0.2 mM CaCl₂ (low Ca²⁺) increases the inter-event interval of sEPSCs (n=9). The low Ca²⁺ medium occludes the inverse agonist effect of AM630 observed in Fig. 7. (C) Exchanging the medium with low Ca²⁺ does not alter sEPSC amplitude. AM630 also has no further effect on sEPSC amplitude (n=9). Data in (B) and (C) analyzed using one-way ANOVA with Bonferroni's multiple comparison test.