Molecular Components and Functions of the Endocannabinoid System in Mouse Prefrontal Cortex

Background. Cannabinoids have deleterious effects on prefrontal cortex (PFC)-mediated functions and multiple evidences link the endogenous cannabinoid (endocannabinoid) system, cannabis use and schizophrenia, a disease in which PFC functions are altered. Nonetheless, the molecular composition and the physiological functions of the endocannabinoid system in the PFC are unknown. Methodology/Principal Findings. Here, using electron microscopy we found that key proteins involved in endocannabinoid signaling are expressed in layers V/VI of the mouse prelimbic area of the PFC: presynaptic cannabinoid CB1 receptors (CB1R) faced postsynaptic mGluR5 while diacylglycerol lipase alpha (DGL-alpha), the enzyme generating the endocannabinoid 2-arachidonoyl-glycerol (2-AG) was expressed in the same dendritic processes as mGluR5. Activation of presynaptic CB1R strongly inhibited evoked excitatory post-synaptic currents. Prolonged synaptic stimulation at 10Hz induced a profound long-term depression (LTD) of layers V/VI excitatory inputs. The endocannabinoid -LTD was presynaptically expressed and depended on the activation of postsynaptic mGluR5, phospholipase C and a rise in postsynaptic Ca2+ as predicted from the localization of the different components of the endocannabinoid system. Blocking the degradation of 2-AG (with URB 602) but not of anandamide (with URB 597) converted subthreshold tetanus to LTD-inducing ones. Moreover, inhibiting the synthesis of 2-AG with Tetrahydrolipstatin, blocked endocannabinoid-mediated LTD. All together, our data show that 2-AG mediates LTD at these synapses. Conclusions/Significance. Our data show that the endocannabinoid -retrograde signaling plays a prominent role in long-term synaptic plasticity at the excitatory synapses of the PFC. Alterations of endocannabinoid -mediated synaptic plasticity may participate to the etiology of PFC-related pathologies

Immunocytochemical localization of mGluR5 and CB1R in the prelimbic prefrontal cortex (prPFC).

<p>(A) Double confocal immunofluorescence showed no colocalization of both proteins. mGluR5 immunoreactivity was distributed in the neuropil throughout cortical layers, but the staining was more evident in apical dendrites of layers V/VI pyramidal neurons heading for the superficial layers. High CB1R immunoreactivity was in layers II/III, deeper part of layer V and throughout layer VI (Á: enlargement of A). The lack of CB1R labeling showed mGluR5 immunoreactivity in layers I, IV and in the upper part of layer V. (B–F). Electron microscopy of the localization of mGluR5 (DAB immunoreaction product) and CB1R (silver-intensified gold particles) in prPFC cortical layers V/VI. mGluR5 immunoreactivity was in dendritic profiles and spines (s). Typically, CB1R immunopositive synaptic terminals (T) made asymmetric synapses with mGluR5-immunoreactive dendritic spines (s). Note metal particles localizing CB1R (arrows) at perisynaptic and extrasynaptic sites relative to presynaptic membrane specializations of axonal synaptic boutons. Scale bars in A,A': 100 µm; B,C,E,F: 0.33 µm; D:0.20 µm.</p

Postsynaptic receptors and transduction pathways involved in eCB-LTD.

<p>(A) eCB-LTD was not affected by a mixture of the NMDAR antagonist MK801 (40 µM), the D1 receptor antagonist SCH23390 (25 µM) and the D2 receptor antagonist sulpiride (25 µM). (B) The mGluR5 antagonist MPEP (10 µM) completely blocked eCB-LTD (C) Bar graph summarizing experiments showing that the non subtype selective group 1mGluR antagonist LY341495 (50 µM) and the Phospholipase C inhibitor U73122 both prevented eCB-LTD induction. 45 min after the end of the tetanus, the fEPSPs was 76.6±2.64% (n = 62) of baseline in control and 95.8±4.85% (n = 12, p = 0.004 t-test) and 96.33±6.4% (n = 11, p = 0.0009 t-test) in LY341495, respectively. (D) eCB-LTD requires postsynaptic Ca²⁺ rise. Time course of all the experiments performed where the recording pipette was filled with BAPTA (20 mM, n = 11) and where eCB-LTD was completely blocked.</p

Pharmacological characterization of presynaptic CB1R at layer V-VI synapses of the PrPFC.

<p>Layer V-VI pyramidal cells were voltage-clamped and held at -70mV. (A) CB1R-mediated inhibition of evoked transmission. The cannabimimetic CP55,940 (10 µM) reduced evoked EPSCs on average to 48±5 % (n = 6) of basal value. Traces represent the average of 10 consecutive EPSCs taken at the times indicated on the time-course graph. (B) The inhibitory effects of CP55,940 on evoked EPSCs were blocked by pre-treatment with the selective CB1R antagonist SR141716A (10 µM, t-test p = 0.0386) in agreement with the involvement of CB1R. (C) Dose response curve measured 20 min after beginning CP55,940 application. Each point is expressed as the percentage of inhibition of its basal value. The EC50 was 195±0.3 nM. (D) The coefficient of variation, expressed as 1/CV² was reduced following the CP55,940 (p = 0.0107 paired t-test). 1/CV² was calculated with 60 sweeps i.e. 10 min before and 20 min after CP55,940.</p

Percentage of presynaptic and postsynaptic elements immunoreactive for CB1R, mGluR5 or DGL-α.

<p>The percentage of CB1R-labeled presynaptic compartments relative to postsynaptic mGluR5 and DGL-α is shown. Prelimbic prefrontal cortical sections used for counting were obtained from 3 adult mice. Electron micrographs were taken at a final magnification of x15,000.</p

Presynaptic CB1R-mediated LTD in the PrPFC.

<p>(A) Left: A 10 min 10 Hz stimulation of layer II-III fibers (arrow) induced a profound long-term depression of evoked EPSCs recorded in patch-clamped layer V-VI pyramidal neurons. The induction of LTD was completely prevented when slices were preincubated and tetanized in the presence of the CB1R antagonist AM251 (4 µM). Traces represent the average of 12 consecutive EPSCs before and 25 minutes after LTD induction in the absence (upper left) or presence (upper right) of AM251. Right: The coefficient of variation, 1/CV², was significantly reduced after induction of eCB-LTD (p = 0.0025 paired t-test). Calibration bars: x: 50 ms, y: 100 pA. (B) Representative continuous 3 seconds sweeps showing the spontaneous EPSCs (sEPSCs) recorded before (left) and after eCB-LTD induction (right). The distribution of sEPSCs inter-event intervals (left panel) but not of their amplitude (right panel) was modified following induction of LTD suggesting a presynaptic modulation (Kolmogorov-Smirnov test: inter event interval p<0.005, amplitude p = 0.507). Calibration bars: x: 100 ms, y: 10 pA. (C) Responses to hyperpolarizing and depolarizing somatic current pulses of a typical pyramidal neuron in the PFC before and after induction of eCB-LTD. Similar I–V curves were obtained suggesting that eCB-LTD induction did not change post-synaptic pyramidal neurons properties. Calibration bars: x: 100 ms, y: 25 mV.</p

Role of 2-AG in eCB-LTD LTD in the PrPFC.

<p>(A) Typical experiment showing that a 5 min stimulation at 10 Hz is sub threshold to induce LTD, even when applied two consecutives times. Calibration bars: x: 10 ms, y: 0.2 mV. (B) Representative experiment showing that 5 min at 10 Hz can induce LTD when applied after bath perfusion with URB 602 (100 µM). Traces were taken at the time indicated on corresponding graph. Calibration bars: x: 10 ms, y: 0.2 mV. (C) Summary bar histogram of all the experiments performed where the first tetanus was given in saline and the second tetanus was given after bath perfusion of URB602. LTD was induced only when URB was present (t-test, p = 0.0271). (D) Averaged time courses of the experiments in which the 5min at 10Hz protocol was given in control ACSF (open circles) of after pre-treatment with URB597 (2 µM, black triangles) or URB602 (black circles).</p

Immunocytochemical localization of mGluR5/DGL-α (A, B) and CB1R/ DGL-α (C, D) in mouse prPFC cortical layers V/VI.

<p>Double preembedding immunogold and immunoperoxidase methods for electron microscopy. (A, B) mGluR5 metal particles (arrows) and DGL-α immunodeposits colocalized in postsynaptic dendrites (den) and dendritic spines (s). mGluR5 labeling was in perisynaptic and extrasynaptic membranes. No mGluR5/DGL-α immunoreactivity was observed in presynaptic terminals (T). (C, D) CB1R immunoparticles were on presynaptic terminals membranes (T) away from synaptic specializations made on postsynaptic DGL-α-immunoreactive dendritic spines (s). Observe that DGL-α-positive spines also received CB1R-immunonegative synaptic terminals, and that a CB1R-labeled presynaptic terminal (thick arrow) probably of inhibitory nature (IT in D) made a synapse with a postsynaptic DGL-α-negative dendritic branchlet. Scale bars: 0.5 µm.</p