An animal model of female adolescent cannabinoid exposure elicits a long-lasting deficit in presynaptic long-term plasticity

Abstract

Cannabis continues to be the most accessible and popular illicit recreational drug. Whereas current data link adolescence cannabinoid exposure to increased risk for dependence on other drugs, depression, anxiety disorders and psychosis, the mechanism(s) underlying these adverse effects remains controversial. Here we show in a mouse model of female adolescent cannabinoid exposure deficient endocannabinoid (eCB)-mediated signaling and presynaptic forms of long-term depression at adult central glutamatergic synapses in the prefrontal cortex. Increasing endocannabinoid levels by blockade of monoacylglycerol lipase, the primary enzyme responsible for degrading the endocannabinoid 2-arachidonoylglycerol (2-AG), with the specific inhibitor JZL 184 ameliorates eCB-LTD deficits. The observed deficit in cortical presynaptic signaling may represent a neural maladaptation underlying network instability and abnormal cognitive functioning. Our study suggests that adolescent cannabinoid exposure may permanently impair brain functions, including the brain's intrinsic ability to appropriately adapt to external influences.

Keywords: Adolescent cannabis abuse; CB1R; Cannabinoids; LTD; mGluR2/3.

Figure 1

Female rats receiving adolescent cannabinoids show a deficit in eCB-LTD, but preserved basal synaptic transmission. (A) Injection protocol adapted from previous study (Rubino et al., 2008). Mice were injected twice a day during adolescence (PND35-45) with increasing doses of WIN55 and then allowed to mature to adulthood before experimental procedures. (B) Diagram showing the configuration of the recording and stimulation electrodes within the prelimbic region of the mPFC used in electrophysiological experiments to test synaptic activity at the L2/3→L5 synapse. (C) Application of DNQX shows the glutamatergic component of the fPSP. The red line is an average fPSP of 5 traces with only ACSF in the bath. After 5 min of bath application of 20 mM DNQX, another average of 5 traces was recorded (black line). Note that the fiber volley is spared while the fPSP is abolished under the DNQX condition (no picrotoxin present). (D) I/O curves were determined starting at 20 μA stimulus intensity and increasing stimulation intensity until the amplitude measurements reached a plateau. No differences were detected using independent samples t-tests at any intensity measured (CTRL: n = 10; WIN55-treated: n = 11; all p-values > 0.05). A fitted curve for each group obtained using Boltzman’s sigmoidal equations is also shown. Further detailed analysis of the I/O relationship was performed using different metrics obtained from the Boltzmann sigmoidal analysis to determine whether differences in the shape of the curves were present. A comparison of the means of individual Boltzmann fitted parameters revealed no differences between the WIN55-treated and control mice in the maximum asymptote (A2) p = 0.9130, in the center (x0) p = 0.1676, or in the time constant (dx) p = 0.2390. These thorough analyses of I/O relations at the L2/3→L5 synapse show that there are no differences between control and WIN55 mice in any of the tested characteristics of the I/O curves. (E) The CTRL group showed robust expression of eCB-LTD at the L2/3→L5 synapse in the mPFC. eCB-LTD was induced at time 0 (black arrows) by application of a 10-Hz train for 10 min. LTD magnitude was estimated from fPSPs registered during the period 50-60 min after LTD induction and expressed as a percentage of baseline fPSP amplitudes. Bath application of 5 µM AM 251 completely blocked LTD, indicating that this is an endocannabinoid-dependent form of LTD. (F) eCB-LTD was induced at time 0 (black arrow) with a 10-Hz train applied for 10 min using the previously described protocol (Lafourcade et al., 2007). LTD magnitude was estimated from fPSPs registered during the period 50-60 min after LTD induction as a percentage of baseline fPSP amplitudes. Compared with controls, WIN55-treated mice showed a strong deficit in eCB-LTD expression in response to the 10-Hz stimulation protocol (CTRL: 55.82% ± 4.08%, n = 6; WIN55-treated: 90.54% ± 7.63%, n = 9; t₍₁₃₎ = 3.469, p = 0.004).

Figure 2

Short-term plasticity at the L2/3→L5 synapse is unaffected in WIN55- treated mice. (A-C) Short-term measures of plasticity were measured at 30% stimulus intensity. (A) Paired-pulse ratios (PPR) were measured at the L2/3→L5 synapse at 5 different interstimulus intervals (25, 50, 100, 200, and 300 ms). No differences in mean PPR were observed between CTRL (n = 6) or WIN55-treated (n = 10) animals at any interval tested; all p-values > 0.05. (B) Short-term potentiation (STP) was induced with a brief train of 15 pulses delivered at 50 Hz at time 0 (indicated by a black arrow). RM-ANOVA between WIN55-treated (n = 10) and CTRL (n = 6) samples showed no time × treatment interaction (F_(1.6,21.3) = 1.568, p = 0.232). (C) Short-term depression (STD) was measured during the train used to simulate STP. Each response in the train was compared to the fPSP amplitude at baseline and plotted as a percentage. Depression across the train was observed in all groups, with facilitation being favored in the early portion of the train. RM-ANOVA revealed no significant pulse# × treatment interaction (F_{(2.1, 28.7)} = 0.284, p = 0.760). (D-F) Short-term measures of plasticity were also measured at 70% stimulus intensity. (D) PPRs were measured at 5 different inter-stimulus intervals (25, 50, 100, 200, and 300 ms). No differences in PPR at 70% stimulus intensity were observed between CTRL (n=6) and WIN55-treated (n=10) animals using independent samples t-tests; all p-values > 0.05. (E) RM-ANOVA on STP at 70% stimulus intensity between WIN55-treated (n=10) and CTRL (n=6) samples showed no time × treatment interaction (F_{(2.1, 29.5)} = 1.501, p = 0.239). (F) At 70% stimulus intensity, depression across the train was observed in both groups, with facilitation being favored in the early portion of the train but to a lesser extent than at 30% stimulus intensity. RM-ANOVA revealed no significant pulse# × treatment interaction F_(4,56) = 2.154, p = 0.086.

Figure 3

Inhibition of 2-AG hydrolysis ameliorates the eCB-LTD deficiency at L2/3→L5 synapses. (A) The WIN55-treated group showed no induction of LTD at the L2/3→L5 synapse in the mPFC. However, 10-min stimulation of slices from WIN55-treated mice at 10 Hz in the presence of 1 µM JZL 184 rescued the deficit (Wilcoxon test: WIN55-treated+JZL 184: 69.46% ± 4.90%, Z = −2.366, n = 7 p = 0.018). In addition, an attempt to rescue impaired eCB-LTD in the WIN55-treated mice with 1 µM JZL 184 failed in the presence of selective blocker of CB1R function AM 251 (5 µM). (B) The binned averages of 10 min time segments (calculated based on data in (A) show distinct differences under all experimental conditions. Data were analyzed from the binned average time point in the rectangle marked during the last 10 min of recording. While WIN55-treated slices showed no induction of eCB-LTD, JZL 184 application to WIN55 slices enhanced LTD to control levels (Figure 1E), which failed in the presence of CB1R blocker AM 251 (C) CB1R function is reduced in WIN55- treated mice. The WIN55-treated group shows decreased functionality of CB1R at the L2/3→L5 synapse in the mPFC compared to control mice. After 20 min of baseline recording, continuous application of 1 µM WIN55,212-2, a CB1R agonist, caused a reduction of fPSP amplitude in the control group; this reduction was markedly dampened in the WIN55-treated mice. (D) Analysis of binned averages showed a markedly dampened effect in the WIN55-treated mice.

Figure 4

mGluR2/3-LTD at the L2/3→L5 synapse is suppressed in WIN55-treated mice. mGluR2/3-LTD in the L2/3→L5 pathway was induced at time 0 by a 10-minute bath application of 100 nM LY379268, a potent mGluR2/3 agonist. The CTRL group (A) showed significant expression of LTD, whereas LTD occurred to a lesser extent in the WIN55-treated group (B), suggesting that mGluR2/3-dependent LTD is altered in WIN55-treated mice. (C) Binned averages of activity taken every 10 min across time show significant divergence in mGluR2/3-LTD expression levels in WIN55-treated and control animals at the L2/3→L5 synapse; time × treatment: F_(6,72) = 3.715, p = 0.003. (D) There is a significant difference in expression of mGLuR2/3-LTD in control and WIN55-treated mice at 50-60 min (CTRL: 59.61% ± 3.27%, n = 6; WIN55-treated: 76.62% ± 2.06%, n = 8; t₍₁₂₎ = 4.625, p = 0.001). These results suggest that treatment of mice with WIN55 during adolescence causes a disruption in the mGluR2/3 signaling pathway.

Figure 5

CB1R and mGluR2/3 expression is reduced in WIN55-treated female mice. (A-D) Single optical section images were acquired at 40x magnification and a 1.5x Zoom from cortical layer 5 of the PL mPFC, top panels show images from control mice, middle panels show images from WIN55-treated mice (scale bar = 50 µm) and lower panels give mean intensity of control (open bars) and WIN55-treated (filled bars) groups. (A) Mean intensity of the CB1R fluorescent signal in layer 5 of the PL mPFC of WIN55-treated mice was not significantly different from control mice (p=0.0576). (B) Mean intensity of mGluR2/3 signal in WIN55-treated mice showed a modest decrease in the mPFC (~35%, p = 0.0417). Mean intensity of MAGL (C) and VGluT1 (D) staining in WIN55-treated mice were not different from those found in control animals (p > 0.05). (E) Single optical section images were acquired at 40x magnification and a 7x Zoom from cortical layer 5 of the PL mPFC. The first column (left) in green shows CB1R labeling, second column (middle) in red shows VGluT1, and column 3 (right) shows the merged signals. Top panels show images from control mice and lower panels show images from WIN55-treated mice (scale bar = 10 µm). WIN55-treated animals showed a modest decrease in CB1R co-localized with vGlut1 in L5 in the PL mPFC (~40%, p = 0.0351). White arrows indicate examples of CB1R co-localized with VGluT1.

Figure 6

WIN55 treatment during adolescence impairs performance on a novel object recognition task. WIN55-treated and control mice were tested on a novel object recognition task with a 2-minute delay between the training and the testing phases. (A) Exploration time during training did not differ in the both groups. (B) While the two groups showed no difference in discrimination between objects during training, WIN55-treated mice failed to recognize the familiar object during the test phase. WIN55-treated mice show normal uninduced locomotor activity in the Open Field Activity test: average velocity (C) as well as total distance traveled (D) were unchanged. (E) However, WIN55-treated mice had increased center time in the Open Field Activity Test, consistent with decreased anxiety.