Effects of chronic treatment with delta9-tetrahydrocannabinol on cannabinoid-stimulated [35S]GTPgammaS autoradiography in rat brain

Abstract

Chronic Delta9-tetrahydrocannabinol (Delta9-THC) administration produces tolerance to cannabinoid effects, but alterations in signal transduction that mediate these changes are not yet known. The present study uses in vitro autoradiography of agonist-stimulated [35S]GTPgammaS binding to localize cannabinoid receptor-activated G-proteins after chronic Delta9-THC treatment. Cannabinoid (WIN 55212-2)-stimulated [35S]GTPgammaS binding was performed in brain sections from rats treated chronically with 10 mg/kg Delta9-THC for 21 d. Control animals received saline or an acute injection of Delta9-THC. Acute Delta9-THC treatment had no effect on basal or WIN 55212-2-stimulated [35S]GTPgammaS binding. After chronic Delta9-THC treatment, net WIN 55212-2-stimulated [35S]GTPgammaS binding was reduced significantly (up to 70%) in most brain regions, including the hippocampus, caudate-putamen, perirhinal and entorhinal cortex, globus pallidus, substantia nigra, and cerebellum. In contrast, chronic Delta9-THC treatment had no effect on GABAB-stimulated [35S]GTPgammaS binding. In membranes and brain sections, Delta9-THC was a partial agonist, stimulating [35S]GTPgammaS by only 20% of the level stimulated by WIN 55212-2 and inhibiting WIN 55212-2-stimulated [35S]GTPgammaS at high concentrations. Because the EC50 of WIN 55212-2-stimulated [35S]GTPgammaS binding and the KD of cannabinoid receptor binding were unchanged by chronic Delta9-THC treatment, the partial agonist actions of Delta9-THC did not produce the decrease in cannabinoid-stimulated [35S]GTPgammaS binding. These results suggest that profound desensitization of cannabinoid-activated signal transduction mechanisms occurs after chronic Delta9-THC treatment.

Fig. 1.

Effect of Δ⁹-THC and WIN 55212-2 on [³⁵S]GTPγS binding in rat cerebellar membranes (A) and rat brain sections (B). Membranes (A) were incubated with 0.05 nm[³⁵S]GTPγS and 20 μm GDP, as described in Materials and Methods, with various concentrations of either Δ⁹-THC or WIN 55212-2 alone (closed symbols) or with various concentrations of Δ⁹-THC in the presence of either 1 μm or 10 μm WIN 55212-2 (open symbols). Data are expressed as percentage basal [³⁵S]GTPγS binding and represent mean values ± SE from three separate experiments. Rat brain sections (B) were incubated with 0.04 nm[³⁵S]GTPγS and 2 mm GDP, as described in Materials and Methods, and represent basal [³⁵S]GTPγS binding, 10 μm Δ⁹-THC alone, 10 μm WIN 55212-2 alone, and 10 μmWIN 55212-2 + 10 μm Δ⁹-THC.

Fig. 2.

Autoradiograms of brain sections comparing basal and cannabinoid-stimulated [³⁵S]GTPγS binding in control and chronic Δ⁹-THC-treated rats. Sections were incubated with 2 mm GDP and then with [³⁵S]GTPγS (0.04 nm) and 2 mmGDP in the presence and absence of 10 μm WIN 55212-2. Basal binding (assessed in the absence of agonist) is shown on theleft column. Sections from control (middle column) and chronic Δ⁹-THC-treated (right column) rats are shown at the appropriate levels to show (1) caudate-putamen and PAG (top row), (2) caudate-putamen and globus pallidus (middle row), and (3) substantia nigra (bottom row). Cannabinoid-stimulated [³⁵S]GTPγS binding in the cortex and hippocampus is seen in sections at all three levels.

Fig. 3.

Net cannabinoid-stimulated [³⁵S]GTPγS binding in brain regions from chronic (A) and acute (B) Δ⁹-THC-treated and control rats. Sections were incubated with 2 mm GDP, and then with [³⁵S]GTPγS (0.04 nm) and 2 mm GDP in the presence and absence of 10 μm WIN 55212-2. Net stimulated [³⁵S]GTPγS binding was determined by subtracting basal [³⁵S]GTPγS binding from WIN 55212-2-stimulated [³⁵S]GTPγS binding. The levels of sections correspond to the images shown in Figure 2: dorsal, Figure 2, top; middle, Figure 2, middle; and ventral, Figure 2, bottom. *p < 0.005, **p < 0.05.CBLM, Cerebellum; CPU, caudate-putamen;ENT, entorhinal cortex; HIP, hippocampus;PAG, periaqueductal gray; PRH, perirhinal cortex; SEP, septum.

Fig. 4.

Net cannabinoid-stimulated [³⁵S]GTPγS binding in the globus pallidus and substantia nigra. Sections were incubated with 2 mm GDP, and then with [³⁵S]GTPγS (0.04 nm) and 2 mm GDP in the presence and absence of 10 μmWIN 55212-2. Net stimulated binding was determined by subtracting basal [³⁵S]GTPγS binding from WIN 55212-2-stimulated [³⁵S]GTPγS binding. *p < 0.005.

Fig. 5.

Net GABA_B-stimulated [³⁵S]GTPγS binding in brain regions from chronic Δ⁹-THC-treated and control rats. Sections were incubated with 2 mm GDP, and then with [³⁵S]GTPγS (0.04 nm) and 2 mm GDP in the presence and absence of 300 μm baclofen. Net stimulated binding was determined by subtracting basal [³⁵S]GTPγS binding from baclofen-stimulated [³⁵S]GTPγS binding.CBLM, Cerebellum; ENT, entorhinal cortex;HIP, hippocampus; PRH, perirhinal cortex;PRL, prelimbic cortex.