Modulation of Excitatory Synaptic Transmission During Cannabinoid Receptor Activation

Abstract

The present research has reported that cannabinoid receptor 1 (CB1) agonist, delta-(9)-tetrahydrocannabinol (THC) modulates synaptogenesis during overexcitation. Microtubule and synaptic distribution, poly(ADP)-ribose (PAR) accumulation were estimated during overexcitation and in the presence of THC. Low concentration of THC (10 nM) increased synaptophysin expression and neurite length, while high concentration of THC (1 µM) induced neurotoxicity. Glutamate caused the loss of neurons, reducing the number and the length of neurites. The high concentration of THC in the presence of glutamate caused the PAR accumulation in the condensed nuclei. Glutamate upregulated genes that are involved in synaptogenesis and excitatory signal cascade. Glutamate downregulated transcription of beta3 tubulin and microtubule-associated protein 2. THC partially regulated gene expression that is implicated in the neurogenesis and excitatory pathways. This suggests that CB1 receptors play a role in neurite growth and the low concentration of THC protects neurons during overexcitation, whereas the high concentration of THC enhances the neurotoxicity.

Keywords: BDNF; Beta tubulin; Cannabinoid receptor; Glutamate; Neurons; Synapses.

Fig. 1

The effect of THC on neuronal differentiation and synaptic distribution. Synaptic distribution labeled with synaptophysin (SYN, red) and neurons and their neurites labeled with beta3 tubulin (TUB, green) after 24 h of treatment: (a) control, (b) 5 nM THC, (c) 10 nM THC, and (d) 1 µM THC; average of 20 cells imaged per condition. Nuclei were labeled with Hoechst (blue). Zoomed images on the right (fifth column), and in the right top corner of each image with lower magnification. Scale bar in (a) corresponds to 50 µm, in zoomed image a scale bar is 2 µm

Fig. 2

The excitotoxicity of neurons induced by glutamate and partial neuronal protection with low concentration, but not high concentration of THC for 24 h: (a) 200 µM glutamate, (b) 10 nM THC + G (200 µM glutamate), (c) 1 µM THC + G (200 µM glutamate). Synaptic distribution labeled with synaptophysin (SYN, red) and neurons and their neurites labeled with beta3 tubulin (TUB, green). Nuclei were labeled with Hoechst (blue). Zoomed images on the right (fifth column), and in the right top corner of each image with lower magnification. Scale bar in (a) corresponds to 50 µm, in zoomed image a scale bar is 2 µm

Fig. 3

The overexcitation of neurons induced by glutamate and the effect of THC with different concentrations on neuronal differentiation and synaptic distribution. (a) the frequency distribution of neurite number per neuron; average of 20 cells imaged per condition; (b) an average number of beta3 tubulin-labeled neurites per neuron; (c) the frequency distribution of beta3 tubulin-labeled length; (d) an average number of axonal length in 20 neurons measured per condition; (e) an average number of synaptophysin fluorescent intensity (optic density, OD); (f) an average number of beta3 tubulin fluorescent intensity, OD; (g) an average size of synaptophysin-labeled puncta per µm2; (h) an average number of synaptophysin fluorescent intensity in puncta. Control (CON), glutamate (GLU). Control vs. *p < 0.05, **p < 0.005; 10 nM THC vs. @p < 0.05; 1 µM THC vs. #p < 0.05, ##p < 0.005; GLU vs. $p < 0.05, $$p < 0.005; 10nMTHC + GLU vs. &p < 0.05. (Data are mean ± SEM, n = 6. One-way ANOVA, Tukey’s multiple comparison test)

Fig. 4

The effect of THC on the PARylation of proteins in neurons and glial cells. The distribution of PAR (red) in beta3 tubulin-labeled neurons (TUB, green) and in beta3 tubulin-negative glial cells after THC treatment. (a) Control; (b) 5 nM THC, (c) 10 nM THC (d) 1 µM THC. The nuclei labeled with Hoechst (blue). Single short arrow shows the distribution of PAR in cytoplasm and processes of beta3 tubulin-positive cell or single long arrow shows the distribution of PAR in cytoplasm and processes of beta3 tubulin-negative cell. Double arrow shows the PAR accumulation in nuclei of apoptotic cell. Scale bar corresponds to 50 µm

Fig. 5

The excitation of neurons induces PARylation of proteins in neurons and glial cells, and the low concentration of THC partially prevents the overexcitation in neurons. The distribution of PAR (red) in beta3 tubulin-labeled neurons (green) and in beta3 tubulin-negative glial cells in (a) control, (b) 200 µM glutamate (GLU), (c) 10 nM THC + GLU, (d) 1 µM THC + GLU. The nuclei labeled with Hoechst (blue). Single arrow shows the distribution of PAR in cytoplasm and processes of beta3 tubulin-positive cell. Double arrow shows the PAR accumulation in nuclei of apoptotic cell. Scale bar corresponds to 50 µm

Fig. 6

The effect of THC in dose-dependent manner on the PARylation of proteins with or without excitation of neurons by glutamate. The cells were imaged and quantified: (a) an average number of 80 PAR-labeled cells counted per condition, (b) an average number of 80 beta3 tubulin-labeled neurons counted per condition; (c) an average PAR fluorescent intensity (optic density, OD) evaluated in average of 130 cells per condition. Control vs. **p < 0.005, ****p < 0.0001; 10 nM THC vs. @p < 0.05, @@p < 0.005, @@@@p < 0.0001; 1 µM THC vs. #p < 0.05, ##p < 0.005; glutamate vs. $p < 0.05. (Data are mean ± SEM, n = 6. One-way ANOVA, Tukey’s multiple comparison test)

Fig. 7

The PARylation of proteins in neuronal nuclei after excitation of neurons with glutamate and/or THC treatment. The distribution of PAR (red) in nuclei of beta3 tubulin-labeled neurons (TUB, green) in (a) control, (b) 5 nM THC, (c) 10 nM THC (d) 1 µM THC, (e) control, (f) 200 µM glutamate (GLU), (g) 10 nM T (THC) + G (glutamate), (h) 1 µM THC + G (glutamate). The nuclei labeled with Hoechst (blue). Scale bar corresponds to 10 µm. An average area of nuclei in 50 cells was imaged and quantified per condition. The quantitative analysis of an average area of neuronal nuclei (i) and PAR fluorescent intensity in neuronal nuclei (j). Control vs. *p < 0.05, **p < 0.005, ****p < 0.0001; 5 nM THC vs ^^^p < 0.0005, ^^^^p < 0.0001; 1 µM THC vs. ####p < 0.0001. (Data are mean ± SEM, n = 6. One-way ANOVA, Tukey’s multiple comparison test)

Fig. 8

Effects of THC and/or glutamate (GLU) on gene expression. Primary neurons were treated with 100 nM THC and/or 200 µM GLU for 24 h, and then levels of indicated mRNAs measured by qRT-PCR, normalized to values for GAPDH in the same samples. Control (CON), 100 nM THC, 100 nM THC + GLU, or GLU alone. (a) BDNFIV, (b) SOX2, (c) DLX, (d) TUB3, (e) MAP2, (f) CaMKII, (g) ADORA2A, (h) D2R, (i) DAT, (j) GAD1, (k) SEMA3A mRNAs. Control vs. *p < 0.05, **p < 0.005, ****p < 0.0001; 1 µM THC vs. #p < 0.05, ##p < 0.005, ####p < 0.0001, glutamate vs. $p < 0.05, $$$$p < 0.0001. (Data are mean ± SEM, n = 6. One-way ANOVA, Tukey’s multiple comparison test)