Hyperlocomotion and paw tremors are two highly quantifiable signs of SR141716-precipitated withdrawal from delta9-tetrahydrocannabinol in C57BL/6 mice

Increasing evidence suggests that marijuana abstinence leads to clinically significant withdrawal symptoms in humans. In mouse models, following chronic treatment with delta9-tetrahydrocannabinol (THC), administration of the selective cannabinoid CB1 receptor antagonist SR141716 (rimonabant) elicited varying behavioral responses, depending on mouse strain and dosing regimen. In the present study, C57BL/6 mice were injected s.c. with THC (25 mg/kg) or vehicle twice daily for 4.5 days. SR141716 (15 mg/kg) was administrated i.p. 4 h following the last THC treatment. During a 2-h observation period immediately following the SR141716 challenge, the total locomotor, ambulatory and stereotypic activities of THC-treated mice were 4.1, 3.3, and 3.8 times those of vehicle-treated mice, respectively. The number of paw tremors elicited in THC-treated mice was 111 ± 11 during the 45 min immediately following SR141716, whereas only 1.1 ± 0.4 was associated with vehicle-treated animals. In contrast, the number of scratching bouts was higher in vehicle-treated (182 ± 20) vs THC-treated (17 ± 4) mice. The present study is the first to demonstrate hyperlocomotion as an explicit sign of THC abstinence in mice. Together with paw tremors, the two unambiguous withdrawal signs may permit highly quantitative investigation of THC abstinence in C57BL/6 mice and may facilitate investigation of the mechanisms involved via both pharmacological and genetic manipulations, and ultimately potential treatments for cannabis dependence.     

The cannabinoid antagonist SR141716A facilitates memory acquisition and consolidation in the mouse elevated T-maze

Δ⁹-THC and synthetic cannabinoids produce memory impairment in humans as well as in laboratory animals. The high concentration of cannabinoid CB1 receptors and the presence of endocannabinoids in the hippocampus suggest that a cannabinoid neurochemical system may play a role in learning and memory processes. Thus, the objective of the present work was to study the effect of the cannabinoid antagonist SR141716A (SR) on memory acquisition, consolidation and retrieval in a recently developed elevated T-maze (ETM) model of anxiety and memory. In addition, we investigated whether pre-training SR administration was capable of reversing scopolamine-induced memory impairment. Adult male mice were exposed to the closed arm as many times as necessary for the animals to reach the avoidance criterion of remaining in the closed arm for 300 s; they were then tested (exposed to the closed arm) 24 h and 7 days after the training. SR (0.5, 1.0 or 2.0 mg/kg) was administered i.p. 20 min before the training, immediately after training or 20 min before the test in the mice. The elevated plus-maze (EPM) was used to investigate a possible influence of SR on locomotion and on the anxiety-related behavior. SR provoked memory improvement, which was observed when the drug was administered before (effect on memory acquisition/consolidation) or immediately after the training (effect on memory consolidation), but not when the drug was administered before the test (effect on memory retrieval). Also, SR administration reversed scopolamine-induced amnesia. These effects were observed in the absence of changes in locomotion or anxiety levels. Our results demonstrate that the blockade of cannabinoid receptors may improve memory acquisition and consolidation in the ETM model.     

Effects of apomorphine on rat behavior in the elevated plus-maze

It has been reported that novelty may evoke both an exploratory and a fear drive, thus generating behavior responding to an approach/avoidance conflict. However, not much is known about the approach component. Whereas there exists abundant evidence referring to the avoidance component as the main target for the anxiolytic action of benzodiazepines, the involvement of dopaminergic mechanisms in fear and anxiety is controversial. The present study examined the effects of the dopaminergic agonist apomorphine, the D₂ dopaminergic antagonist sulpiride and the combined treatment sulpiride plus apomorphine on conventional and non-conventional measures of the behavior of rats exposed to an elevated plus-maze. Systemic injection of apomorphine (0.25, 0.5 and 1.0 mg/kg) caused a selective increase in the time spent in the open arms and in the open arm extremities. Pre-treatment with sulpiride blocked these effects while this dopaminergic antagonist had no effect by its own. Apomorphine produced no significant effects on stretching, flat-back-approach or scanning. Therefore, apomorphine increased the behavioral response linked to the approach component of the conflict without affecting risk assessment behaviors. These findings suggest that dopaminergic mechanisms, probably through D₂ receptors, may also be involved in the mediation of the conflict derived from the need of gathering information for confirming, identifying and localizing danger and take the appropriate action for avoiding the threatening stimuli of the elevated plus-maze. A role for dopaminergic mechanisms in the setting up of adaptive responses in a fear-inducing environment is discussed.     

H1 but not H2 histamine antagonist receptors mediate anxiety-related behaviors and emotional memory deficit in mice subjected to elevated plus-maze testing

This study investigated the role of H₁ and H₂ receptors in anxiety and the retrieval of emotional memory using a Trial 1/Trial 2 (T1/T2) protocol in an elevated plus-maze (EPM). Tests were performed on 2 consecutive days, designated T1 and T2. Before T1, the mice received intraperitoneal injections of saline (SAL), 20 mg/kg zolantidine (ZOL, an H₂ receptor antagonist), or 8.0 or 16 mg/kg chlorpheniramine (CPA, an H₁ receptor antagonist). After 40 min, they were subjected to the EPM test. In T2 (24 h later), each group was subdivided into two additional groups, and the animals from each group were re-injected with SAL or one of the drugs. In T1, the Student t-test showed no difference between the SAL and ZOL or 8 mg/kg CPA groups with respect to the percentages of open arm entries (%OAE) and open arm time (%OAT). However, administration of CPA at the highest dose of 16 mg/kg decreased %OAE and %OAT, but not locomotor activity, indicating anxiogenic-like behavior. Emotional memory, as revealed by a reduction in open arm exploration between the two trials, was observed in all experimental groups, indicating that ZOL and 8 mg/kg CPA did not affect emotional memory, whereas CPA at the highest dose affected acquisition and consolidation, but not retrieval of memory. Taken together, these results suggest that H₁ receptor, but not H₂, is implicated in anxiety-like behavior and in emotional memory acquisition and consolidation deficits in mice subjected to EPM testing.     

氯化锂对FMR1基因敲除小鼠的高架十字迷宫行为的干预作用

目的 探讨糖原合成酶激酶3（GSK3）抑制剂氯化锂对FMR1基因敲除（KO）小鼠的高架十字迷宫行为的干预作用及机制。方法 给90只30日龄FMR1基因敲除小鼠连续5d腹腔注射不同剂量的氯化锂,用药第6天进行高架十字迷宫行为学实验,通过录像机录像,然后用Smart软件分析录像,观察能否改善KO鼠的高架十字迷宫的表型;同时通过免疫印迹技术检测KO及野生型（WT）鼠的海马和皮层中GSK3β和磷酸化GSK3β（p-GSK3β）的变化。结果 在高架十字迷宫实验中,与WT组小鼠比较,KO组小鼠的运动性、兴奋性、探索性明显增强,且KO组小鼠在开放区域的活动时间、次数以及路程均明显高于WT组。KO鼠用氯化锂后,在开放区域的活动时间、次数以及路程均减低,差异具有统计学意义（P ＜0.05）。免疫印迹实验结果显示,KO鼠p GSK3β表达比WT鼠少;用氯化锂后,KO鼠p-GSK3β表达增加。WT鼠用氯化锂后,开放区域活动时间、次数以及路程有较少改善,p-GSK3β表达也有增加。未使用氯化锂的KO鼠与WT鼠相比,总GSK3β表达无明显差异,KO鼠和WT鼠使用氯化锂后,总GSK3β无明显改变,P ＞0.05。结论 GSK3β的抑制剂氯化锂能改善KO鼠的高架十字迷宫表型,对KO鼠有治疗作用,其机制可能与氯化锂导致的p-GSK3β表达增加有关。     

Longitudinal study of daily variation of rats' behavior in the elevated plus-maze

We conducted a longitudinal study about daily variation of Wistar male rats' behavior in the elevated plus-maze (EPM) evaluated in the 1st, 2nd, 3rd, 6th, 12th, and 18th months of life. Animals were submitted to the plus-maze in 12 sessions at 2-h intervals (n=72, 6 per time point). Spontaneous rest-activity rhythm of four animals was assessed by observation of 24-h videotape records. Time series were analyzed by Cosinor method. Behavioral rates on the six occasions and in light and dark phases were compared by means of two-way ANOVA with repeated measures. Exploratory behavior in EPM was smaller in the light phase and in older animals. Higher values of open and closed arms exploration were observed in the first and third months of the dark phase, and in the first month of the light phase. Adjustment to the 24-h period was significant at all stages for rest-activity data, number of entries in closed arms, and time on center, and for three to five stages for open-arm exploration. In general, 24 h variability was more pronounced in younger animals compared with older ones. The present study showed that: (1). a significant amount of total variability of the behavioral indexes analyzed could be attributed to 24 h variation, (2). light/dark phases differences in EPM exploration were present at all developmental stages, (3). older Wistar rats explored less the EPM and were less active in their home cage compared with younger ones, and (4). behavioral indexes (EPM) decrease was phase related and partially related to a reorganization of rest-activity rhythm.     

Effect of different illumination levels on rat behavior in the elevated plus-maze

The present study addressed the role of environmental light intensity on the exploratory behavior of rats in the elevated plus-maze, with the specific goal of determining the light intensity threshold for triggering the aversion to the open arms. Male Wistar-derived rats were tested in the elevated plus-maze under different illumination levels: 0, 1, 3, 10, 30, 100 and 300 lx. Exploratory behavior occurring in the open arms (e.g., entries and time spent in these arms) was more intense under 0 and 1 lx than under the other illumination levels, which did not differ among themselves; on the other hand, locomotor behavior (as indicated by frequency of entries and distance run in the closed arms) was not altered under all illumination conditions. The data indicated that vision is important in triggering aversion to the open arms of the elevated plus-maze. They also indicated that the threshold of such aversion was found between 1 and 3 lx environmental illumination and that the phenomena is not intensity-dependent but rather of an all-or-none type. It should be emphasized that these conclusions only stand for unfamiliar environments. The role of light in familiar environments is currently under investigation in our laboratory.     

Low-dose thioperamide injected into the cerebellar vermis of mice immediately after exposure to the elevated plus-maze impairs their avoidance behavior on re-exposure to the apparatus

The present study investigated the effect of thioperamide (THIO), an H₃ histaminergic receptor antagonist, microinjected into the cerebellar vermis on emotional memory consolidation in male Swiss albino mice re-exposed to the elevated plus-maze (EPM). We implanted a guide cannula into the cerebellar vermis using stereotactic surgery. On the third day after surgery, we performed behavioral tests for two consecutive days. On the first day (exposure), the mice (n=10/group) were exposed to the EPM and received THIO (0.06, 0.3, or 1.5 ng/0.1 µL) immediately after the end of the session. Twenty-four hours later, the mice were re-exposed to the EPM under the same experimental conditions, but without drug injection. A reduction in the exploration of the open arms upon re-exposure to the EPM (percentage of number of entries and time spent in open arms) compared with the initial exposure was used as an indicator of learning and memory. One-way analysis of variance (ANOVA) followed by the Duncan post hoc test was used to analyze the data. Upon re-exposure, exploratory activity in the open arms was reduced in the control group, and with the two highest THIO doses: 0.3 and 1.5 ng/0.1 µL. No reduction was seen with the lowest THIO dose (0.06 ng/0.1 µL), indicating inhibition of the consolidation of emotional memory. None of the doses interfered with the animals'' locomotor activity. We conclude that THIO at the lowest dose (0.06 ng/0.1 µL) microinjected into the cerebellum impaired emotional memory consolidation in mice.     

The effects of nicotine on nitric oxide induced anxiogenic-like behaviors in the dorsal hippocampus

Nicotine, an active tobacco derived alkaloid, regulates the activity of the neuronal nitric oxide synthase (nNOS) as well as the release of nitric oxide (NO) in the nervous system. Nicotinic acetylcholine receptors and nNOS are abundantly co-expressed in the hippocampal neurons and are found to alter anxiety-like behaviors in rodents. Dorsal hippocampus may be a site for modulation of anxiety. Therefore, in this study, we investigated the possible interactions between nicotine and NO systems of the dorsal hippocampus and the resultant effect on anxiety-like behaviors. The elevated plus-maze (EPM) test has been used to test the anxiety. Intraperitoneal administration of nicotine (0.5 mg/kg) decreased the open arm time percentage (%OAT) and open arm entries percentage (%OAE) but not the locomotor activity, indicating an anxiogenic-like response. Intra-CA1 injection of l-arginine (a NO precursor) or l-NAME (a NOS inhibitor) also caused anxiogenic-like effects. On the other hand, injection of the low dose nicotine before different doses of l-arginine or l-NAME blocked the anxiogenic-like response induced by the drugs. Our results suggested that, both NO and nicotinic cholinergic systems not only play a part in the modulation of the anxiety in mice dorsal hippocampus, but also demonstrate a complex interaction in this respect.     

Anxiolytic effects of repeated treatment with an essential oil from Lippia alba and (R)-(-)-carvone in the elevated T-maze

Lippia alba (Mill.) N.E. Brown (Verbenaceae) is widely used in different regions of Central and South America as a tranquilizer. The plant''s anxiolytic properties, however, merit investigation. The present study evaluated the effects of repeated daily (14 days) intraperitoneal (ip) treatment with an essential oil (EO) from a chemotype of L. alba (LA, chemotype II, 12.5 and 25 mg/kg; N = 6-8) and (R)-(-)-carvone (25 mg/kg; N = 8-12), the main constituent of this chemotype, on male Wistar rats (weighing 250 g at the beginning of the experiments) submitted to the elevated T-maze (ETM). The ETM allows the measurement of two defensive responses: inhibitory avoidance and one-way escape. In terms of psychopathology, these responses have been related to generalized anxiety and panic disorder, respectively. Treatment with the EO impaired ETM avoidance latencies, without altering escape, in a way similar to the reference drug diazepam (P < 0.05) (avoidance 2: control = 84.6 ± 35.2; EO 12.5 mg/kg = 11.8 ± 3.8; EO 25 mg/kg = 14.6 ± 2.7; diazepam = 7 ± 2.1). (R)-(-)-carvone also significantly altered this same response (P < 0.05; avoidance 1: control = 91.9 ± 31.5; carvone = 11.6 ± 1.8; diazepam = 8.1 ± 3.3). These results were not due to motor changes since no significant effects were detected in an open field. These observations suggest that LA exerts anxiolytic-like effects on a specific subset of defensive behaviors that have been implicated in generalized anxiety disorder, and suggest that carvone is one of the constituents of LA responsible for its action as a tranquilizer.