Increased mortality, hypoactivity, and hypoalgesia in cannabinoid CB1 receptor knockout mice

Abstract

Delta9-Tetrahydrocannabinol (Delta9-THC), the major psychoactive ingredient in preparations of Cannabis sativa (marijuana, hashish), elicits central nervous system (CNS) responses, including cognitive alterations and euphoria. These responses account for the abuse potential of cannabis, while other effects such as analgesia suggest potential medicinal applications. To study the role of the major known target of cannabinoids in the CNS, the CB1 cannabinoid receptor, we have produced a mouse strain with a disrupted CB1 gene. CB1 knockout mice appeared healthy and fertile, but they had a significantly increased mortality rate. They also displayed reduced locomotor activity, increased ring catalepsy, and hypoalgesia in hotplate and formalin tests. Delta9-THC-induced ring-catalepsy, hypomobility, and hypothermia were completely absent in CB1 mutant mice. In contrast, we still found Delta9-THC-induced analgesia in the tail-flick test and other behavioral (licking of the abdomen) and physiological (diarrhea) responses after Delta9-THC administration. Thus, most, but not all, CNS effects of Delta9-THC are mediated by the CB1 receptor.

Figure 1

Targeting the CB1 gene. The CB1 gene was mutated by replacing the coding region, which is confined to a single exon (hatched box), between amino acids 32 and 448 with PGK-neo.

Figure 2

Comparison of the specific binding of [³H]CP55,940 to cannabinoid receptors in CB1^+/+ and CB1^−/− mice. The distribution of [³H]CP55,940 binding sites in sagittal brain sections (A and B) and transverse spleen sections (E and F) are shown in CB1^+/+ (Left) and CB1^−/− (Right) mice. Nonspecific binding, determined in the presence of 10 μM CP55,244, is shown in brain (C and D) and spleen (G and H). [³H]CP55,940 binding in sagittal brain sections in CB1^−/− mice did not differ from nonspecific binding in CB1^+/+ mice. [³H]CP55,940 binding in spleen was comparable in CB1^+/+ and CB1^−/− mice. (Bar equals 1 mm.)

Figure 3

Mortality rate in offspring from heterozygous (CB1^+/− × CB1^+/−) matings. Note that more than 30% of all CB1 knockout mice under 24 weeks of age die of natural causes. In contrast, less than 5% of the heterozygous and wild-type littermates die during the same time span.

Figure 4

Baseline physiological and behavioral responses in CB1^+/+ and CB1^−/− mice. (A) Body temperature was similar in both genotypes. CB1^−/− mice showed (B) increased immobility in the ring catalepsy test, (C) decreased activity in the open field, (D) normal responses in the tail-flick test, and (E) hypoalgesia in the hotplate and (F) formalin tests. (G) Locomotor activity of knockout and wild-type animals was similar during the hotplate test. The number of animals studied in each test is shown at the bottom of each bar. ∗ denotes P < 0.05; ∗∗ denotes P < 0.005, n = 8–12 for each genotype.

Figure 5

HU210 effects. (A) Chemical structure of HU210. (B) Body temperature was measured by using a rectal probe immediately before and 50 min after the drug injection. HU210 produces a pronounced hypothermia in CB1^+/+ mice, but not in CB1^−/− mice. (C) HU210 produces a robust analgesia in the tail-flick test in CB1^+/+ mice, but not in CB1^−/− mice. Note that only the response of a single CB1^+/+ animal is shown for comparison after injection of 5 mg/kg and 10 mg/kg HU210, respectively. ∗∗ denotes P < 0.005; ∗∗∗ denotes P < 0.0005; n = 10 for each genotype.

Figure 6

Δ9-THC effects. (A) Chemical structure of Δ9-THC. (B) Wild-type CB1^+/+ animals displayed significant dose-dependent hypothermia. In contrast, CB1^−/− mice showed no alteration in body temperature. (C) CB1^+/+ mice, but not CB1^−/− mice, displayed a profound Δ9-THC-induced catalepsy in the Pertwee ring test (25). As the baseline responses differed somewhat between the two genotypes (Fig. 3B), the drug-induced scores are expressed relative to values of vehicle injected mice. (D) Δ9-THC-analgesia determined with the tail-flick test. Note that the analgesic effects of Δ9-THC are similar in CB1^+/+ and CB1^−/− mice at doses up to 50 mg/kg. A dose of 100 mg/kg did not increase tail-flick latencies, but produced other behavioral and physiological responses (see text for details). In the hotplate test, Δ9-THC induced (E) analgesia and (F) hypoactivity in CB1^+/+ but not CB1^−/− mice. ∗ denotes P < 0.05; ∗∗ denotes P < 0.005; ∗∗∗ denotes P < 0.0005; n = 8–12 for each genotype.