Research report
- The Adelson Center for the Biology of Addictive Diseases and The Mauerberger Chair in Neuropharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel
Abstract
Preconditioning, a phenomenon where a minor noxious stimulus protects from a subsequent more severe insult, and post-conditioning, where the protective intervention is applied following the insult, offer new insight into the neuronal mechanism(s) of neuroprotection and may provide new strategies for the prevention and treatment of brain damage.
We have previously reported that a single administration of an extremely low dose of Δ9-tetrahydrocannabinol (THC; the psychoactive ingredient of marijuana) to mice induced minor long-lasting cognitive deficits. In the present study we examined the possibility that such a low dose of THC will protect the mice from more severe cognitive deficits induced by the epileptogenic drug pentylenetetrazole (PTZ).
THC (0.002 mg/kg, a dose that is 3–4 orders of magnitude lower than the doses that induce the conventional effects of THC) was administered 1–7 days before, or 1–3 days after the injection of PTZ (60 mg/kg). The consequences of this treatment were studied 3–7 weeks later by various behavioral tests that evaluated different aspects of memory and learning. We found that a single administration of THC either before or after PTZ abolished the PTZ-induced long-lasting cognitive deficits. Biochemical studies indicated a concomitant reduction in phosphorylated-ERK (extracellular signal-regulated kinase) in the cerebella of mice 7 weeks following the injection of THC.
Our results suggest that a pre- or post-conditioning treatment with extremely low doses of THC, several days before or after brain injury, may provide safe and effective long-term neuroprotection.
Research highlights
► Pentylenetetrazole (PTZ) induces long lasting cognitive deficits in mice. ► An extremely low dose of tetrahydrocannabinol (THC) protects from PTZ-induced cognitive damage. ► An extremely low dose of THC evokes pre- and post-conditioning mechanisms that protect the brain. ► An extremely low dose of THC induces long lasting changes in ERK/MAPK activity.
Keywords
- Neuroprotection;
- Cannabinoid;
- Epileptic seizures;
- Preconditioning;
- Postconditioning;
- Cognitive deficit
Figures and tables from this article:
- Fig. 1. Administration of an ultra low dose of THC 3 days before the injection of PTZ protects the mice from PTZ-induced cognitive deficits, as measured by the oasis maze. (A) The experiment consisted of 4 groups of mice: control, THC (0.002 mg/kg), PTZ (60 mg/kg) and THC + PTZ, 6–8 mice per group. The mice were tested in the oasis maze 3 weeks after the injection of PTZ. In trials 1–3 (training), all the 20 wells were filled with water and the mice learned to find and drink from a well. In trials 4–10, the mice had to locate a single well that was filled with water. The time it took the mouse to reach the well and drink the water was measured (“latency”). A two-way ANOVA for trials 4–10 revealed a significant effect of treatment (F(3, 188) = 2.87, p < 0.05). The PTZ-injected mice needed a significantly longer time than the control mice to find the filled well (Tukey, p < 0.05) while the THC + PTZ group did not differ from the control group (Tukey,p = 0.753) but significantly differed from the PTZ group (Tukey, p < 0.05). (B) The average time it took each group (control (C), THC (T), PTZ (P) and THC + PTZ (P + T)) to find the filled well when the learning curve reached a quasi steady-state (trials 6–10). A one-way ANOVA showed a significant effect of treatment (F(3, 134) = 3.14, p < 0.05) with a significant difference between control and PTZ-treated mice and between PTZ- and THC + PTZ-treated mice, but not between control and THC + PTZ treated mice. Error bars indicate SEM. * indicates a significant difference (Tukey,p < 0.05).
- Fig. 2. Administration of an ultra low dose of THC 1 or 7 days before the injection of PTZ protects the mice from PTZ-induced cognitive deficits, as measured by the oasis maze. The experiments consisted each of 4 groups of mice: control (C), THC (T; 0.002 mg/kg), PTZ (P; 60 mg/kg) and THC + PTZ (P + T), 6–8 mice per group. (A) The mice were injected with THC one day before the administration of PTZ and tested in the oasis maze 3 weeks later. The average time it took each group to find the single well that was filled with water at steady-state (trials 6–10) was calculated. A one-way ANOVA revealed a significant effect of treatment (F(3, 194) = 19.22, p < 0.01). Post hoc analysis showed a significant difference between the PTZ and the control group and between the PTZ and THC + PTZ group. There was no significant difference between the THC + PTZ and the control group. Error bars indicate SEM. * indicates a significant difference (Tukey, p < 0.01). (B) The mice were injected with THC 7 days before the administration of PTZ and tested in the oasis maze 3 weeks later. A one-way ANOVA revealed a significant effect of treatment (F(3, 179) = 3.14, p < 0.05). The PTZ-injected group performed significantly worse than the control group, while pretreatment with THC significantly improved the performance and there was no significant difference between the control and the THC + PTZ groups. Error bars indicate SEM. * indicates a significant difference (Tukey, p < 0.05).
- Fig. 3. Motor activity of mice in an open-field. The experiment consisted of four groups of mice: control (C), THC (T; 0.002 mg/kg), PTZ (P; 60 mg/kg) and THC + PTZ (P + T), 6–8 mice per group (THC was injected 3 days before PTZ). The mice were tested 4 weeks after the injection of PTZ in an open field. The number of squares that the mouse crossed per the 5 min session was recorded. The figure depicts the mean number of crossing of each group of mice. One-way ANOVA showed no difference between the four groups (F(3, 27) = 0.55, p = 0.98). Error bars indicate SEM.
- Fig. 4. Administration of an ultra low dose of THC 3 days before the injection of PTZ protects the mice from PTZ-induced cognitive deficits, as measured by the place recognition test. The experiment consisted of 4 groups of mice: control, THC (0.002 mg/kg), PTZ (60 mg/kg) and THC + PTZ, 6–8 mice per group. The mice were tested in the place recognition test 5 weeks after the injection of PTZ. On day one the mice were introduced to two identical objects located within the arena. On day 2 the mice were introduced to the same arena but one of the objects was placed in a new location. The time the mouse spent in exploring each object was recorded for 5 min in each session. Relative time of exploration was calculated as the time spent exploring the object over total exploration time for both objects. (A) On day 1 the four groups of mice did not differentiate between the two objects. (B) On day 2 the control group explored the object that was placed in a new location for a significantly longer time than the other object (t-test, *p < 0.01), while the THC- and PTZ-treated groups did not dissociate between the two objects. The THC + PTZ treated mice, similar to the control group, spent a significantly longer time exploring the object that was moved to a new location compared to the other object (t-test, *p < 0.01). Error bars indicate SEM.
- Fig. 5. Administration of an ultra low dose of THC 3 days before the injection of PTZ protects the mice from PTZ-induced cognitive deficits, as measured by the object recognition test. The experiment consisted of 4 groups of mice: control, THC (0.002 mg/kg), PTZ (60 mg/kg) and THC + PTZ, 6–8 mice per group. The mice were tested in the object recognition test 5 weeks after the injection of PTZ. On day one the mice were introduced to two identical objects located within the arena. On day 2 one of the objects was replaced with a new object. Relative exploration time was calculated as the time spent exploring the object over total exploration time for both objects. A, On day 1 the four groups of mice did not dissociate between the two objects. B, On day 2 the control group explored the new object for a significantly longer time than the other object (t-test, *p < 0.01), while the THC- and PTZ-treated groups did not dissociate between the two objects. The THC + PTZ treated mice, similar to the control group, spent a significantly longer time exploring the new object compared to the other object (t-test, *p < 0.01). Error bars indicate SEM.
- Fig. 6. Treatment with an ultra low dose of THC 3 days after the injection of PTZ protects the mice from PTZ-induced cognitive deficits, as measured by the oasis maze. A, The experiment consisted of 4 groups of mice: control, THC (0.002 mg/kg), PTZ (60 mg/kg) and PTZ + THC, 7–9 mice per group. The mice were tested in the oasis maze 3 weeks after the injection of THC. In trials 1–3 (training), all the 20 wells were filled with water and the mice learned to find and drink from a well. In trials 4–10, the mice had to locate a single well that was filled with water. The time it took the mouse to reach the well and drink the water was measured (“latency”). A two-way ANOVA for trials 4–10 revealed a significant effect of treatment (F(3, 223) = 4.62, p < 0.01). The PTZ-injected mice needed a significantly longer time than the control mice to find the filled well while the PTZ + THC group did not differ from the control group, indicating the absence of cognitive deficits. B, The average time it took each group (control (C), THC (T), PTZ (P) and PTZ + THC (P + T)) to find the filled well when the learning curve reached a quasi steady-state (trials 6–10). A one-way ANOVA showed a significant effect of treatment (F(3, 159) = 4.92, p < 0.01) with a significant difference between control and PTZ-treated mice and between PTZ- and PTZ + THC-treated mice, but not between control and THC + PTZ treated mice. Error bars indicate SEM. * indicates a significant difference (Tukey, p < 0.01).
- Fig. 7. Treatment with an ultra low dose of THC 3 days after the injection of PTZ protects the mice from PTZ-induced cognitive deficits, as measured by the place- and object-recognition tests. The experiments consisted of 4 groups of mice: control, THC (0.002 mg/kg), PTZ (60 mg/kg) and PTZ + THC, 7–9 mice per group. The mice were tested in the place- and object-recognition tests 5 weeks after the injection of THC (see details in the legends of Fig. 4 and Fig. 5). (A) On day 2 of the place recognition test the control group explored the object that was placed in a new location for a significantly longer time than the other object (t-test, *p < 0.01), while the THC- and PTZ-treated groups did not dissociate between the two objects. The PTZ + THC treated mice, similar to the control group, spent a significantly longer time exploring the object that was moved to a new location compared to the other object (t-test, *p < 0.01). (B) On day 2 of the object recognition test the control group explored the new object for a significantly longer time than the other object (t-test, *p < 0.01), while the THC- and PTZ-treated groups did not dissociate between the two objects. The PTZ + THC treated mice, similar to the control group, spent a significantly longer time exploring the new object compared to the other object (t-test, *p < 0.01). Error bars indicate SEM.
- Fig. 8. An ultra-low dose of THC induces a long term reduction in phosphorylated ERK. Mice (n = 7–8 per group) were injected with either 0.002 mg/kg THC or vehicle and the levels of pERK2 and total ERK2 in their cerebella were determined by densitometric analysis of western blots 7 weeks following the treatment. The figure presents the cumulative results from three separate experiments. A significant reduction in phosphorylated ERK2 (pERK2), but not in total ERK2 (T-ERK), was found in THC-treated mice compared to vehicle-treated mice (t-test, *p < 0.01), hence pERK2/total ERK2 (P/T-ERK) significantly decreased by 34% in THC-injected mice compared to control mice (t-test, *p < 0.01).
- Corresponding author at: Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel. Tel.: +972 3 6406078; fax: +972 3 6409113.
Copyright © 2011 Elsevier B.V. All rights reserved.
