Local pretreatment with the cannabinoid CB1 receptor antagonist AM251 attenuates methamphetamine intra-accumbens self-administration.

The endocannabinoid system is a potential target for therapeutic intervention of substance abuse. Cannabinoid CB1 receptor antagonist decreases intravenous methamphetamine self-administration in animal models. This study examined whether the nucleus accumbens (NAcc) is a site of interaction between methamphetamine and the CB1 receptor antagonist AM251. Male Sprague-Dawley rats were trained to lever press and then were surgically implanted with a guide cannula into the right NAcc. Rats were allowed one week to recover and then AM251 (0.1 or 1.0 µg/µL) was reverse dialyzed directly into the NAcc prior to methamphetamine (10 µg/µL) intra-accumbens self-administration. AM251 (1.0 µg/µL) reduced methamphetamine self-administration while AM251 (0.1 µg/µL) had an intermediary effect. The mechanism of self-administration attenuation is not known but could be mediated by AM251 affecting the negative feedback from the NAcc to the ventral tegmental area (VTA). This study provides evidence that the endocannabinoid system is involved with rewarding effects of methamphetamine and suggests a possible therapeutic intervention for methamphetamine abuse.

Cocaine responsiveness or anhedonia in rats treated with methylphenidate during adolescence.

Methylphenidate (MPH) treatment in boys diagnosed with ADHD is reported to decrease the risk of drug abuse in adulthood. Similarly, MPH treatment appears to decrease the cocaine preference of male rats during conditioned place preference (CPP) tests. However, the effects of MPH treatment on later drug use of girls/women or CPP in female rodents have not been fully examined, nor have a clinically-relevant MPH dose and/or administration route been thoroughly studied. Here, Sprague-Dawley rats (n=34/sex/treatment) were treated orally 3x/day on postnatal days (PNDs) 29-50 with water or 3mg MPH/kg, a dose producing serum levels within the human clinical range. CPP assessments to cocaine (10 mg/kg, ip) (PNDs 62-71) indicated MPH-treated rats were less active during pre- and postconditioning sessions (p<.04), but there were no significant MPH-related differences in conditioning strength. Baseline open field activity at PND 84 indicated that MPH-treated females were more active than same-sex controls (p<.05). A cocaine challenge (10 mg/kg, ip) elevated activity similarly in MPH-treated and controls of both sexes. As an anhedonia measure, saccharin solution intake on PNDs 87-90 indicated no significant MPH effects. Estrous cycle phase did not appear to affect cocaine response during CPP or open field assessments. Hormonal levels at PND 90 indicated 63% higher corticosterone levels in MPH-treated females relative to same-sex controls (p<.05), a finding that deserves further investigation. These results address some of the major issues surrounding animal models of MPH treatment and provide additional support for a lack of severe long-term behavioral effects of adolescent MPH.

Altered adult locomotor activity in rats from phencyclidine treatment on postnatal days 7, 9 and 11, but not repeated ketamine treatment on postnatal day 7.

Neonatal ketamine (KET) or phencyclidine (PCP) treatment can trigger apoptotic neurodegeneration in rodents. Previously, we described KET- and PCP-induced altered body weight and home cage, slant board and forelimb hang behaviors in preweaning rats (Boctor et al., 2008). In that study, L-carnitine (LC) attenuated the KET-induced behavioral alterations and body weight decrements. The four subcutaneous (sc) treatment groups were: (1) saline; (2) 10 mg/kg PCP on PNDs 7, 9 and 11; (3) 20 mg/kg KET (6 injections; one every 2h on PND 7); or (4) a regimen of KET and 250 mg/kg LC (KLC) both administered on PND 7, with additional 250 mg/kg doses of LC on PNDs 8-11. A portion of each treatment group was evaluated for postweaning behaviors which included grip strength and motor coordination (postnatal days (PNDs) 22 or 71), locomotor sensitization (PND 42), spatial alternation (PNDs 22-70) and residential running wheel activity (PNDs 72-77). On PND 42 or 78, whole and regional brain weights were measured. Grip strength and motor coordination were unaffected at either age by neonatal treatment. On PND 42, neonatally treated KET- or KLC-treated rats responded to a challenge of 5mg/kg KET with activity similar to controls that received the same challenge. Neonatal PCP treatment, however, induced significant sensitization to a challenge of 3mg/kg PCP on PND 42 relative to controls that received the same challenge, causing increased activity which was especially profound in females. Performance on a continuous spatial alternation task requiring a "win-shift, lose-stay" strategy appeared unaffected by neonatal KET or KLC treatment. PCP treatment, however, caused significantly increased random responding and shorter choice latencies. In addition, neonatal PCP treatment elevated light and dark period running wheel activity and reduced PND 42 and 78 body and whole brain weights. These findings provide further evidence that PCP treatment on PNDs 7, 9, and 11 causes subtle cognitive deficits and long-term alterations in activity that are unrelated to deficits in grip strength or motor coordination. Further, repeated KET treatment on PND 7 does not appear to result in severe behavioral modifications.

Flow cytometric detection of Pig-A mutant red blood cells using an erythroid-specific antibody: application of the method for evaluating the in vivo genotoxicity of methylphenidate in adolescent rats.

A modified flow cytometry assay for Pig-A mutant rat red blood cells (RBCs) was developed using an antibody that positively identifies rat RBCs (monoclonal antibody HIS49). The assay was used in conjunction with a flow cytometric micronucleus (MN) assay to evaluate gene mutation and clastogenicity/aneugenicity in adolescent male and female rats treated with methylphenidate hydrochloride (MPH). Sprague-Dawley rats were treated orally with 3 mg/kg MPH (70/sex) or water (40/sex) 3 x /day on postnatal days (PNDs) 29-50. Eight additional rats (4/sex) were injected i.p. with N-ethyl-N-nitrosourea (ENU) on PND 28. Blood was collected on PNDs 29, 50, and 90, and used for determining serum MPH levels and/or conducting genotoxicity assays. On the first and last days of MPH treatment (PNDs 29 and 50), serum MPH levels averaged 21 pg/microl, well within the clinical treatment range. Relative to our previously published method (Miura et al. [2008]; Environ Mol Mutagen 49: 614-629), the HIS49 Pig-A mutation assay significantly reduced the background RBC mutant frequency; in the experiments with ENU-treated rats, the modification increased the overall sensitivity of the assay 2-3 fold. Even with the increased assay sensitivity, the 21 consecutive days of MPH treatment produced no evidence of Pig-A mutation induction (measured at PND 90); in addition, MPH treatment did not increase MN frequency (measured at PND 50). These results support the consensus view that the genotoxicity of MPH in pediatric patients reported earlier (El-Zein et al. [2005]: Cancer Lett 230: 284-291) cannot be reproduced in animal models, suggesting that MPH at clinically relevant levels may be nongenotoxic in humans.

Use of food wafers for multiple daily oral treatments in young rats.

Many laboratory studies require oral administration of drugs. Dietary administration in food or water is useful, but is not always the best method. Orogastric gavage can be stressful. Here, we describe in detail a relatively stress-free technique that can be applied to multiple daily administrations using a palatable food item. This method was successfully used to administer water or methylphenidate 3 times daily to young pair-housed adolescent rats.

Neonatal NMDA receptor antagonist treatments have no effects on prepulse inhibition of postnatal day 25 Sprague-Dawley rats.

Glutamate activation of the NMDA receptor is essential for neuronal differentiation, migration, and survival. Treatment with NMDA receptor antagonists, such as ketamine (KET) or phencyclidine (PCP), can trigger apoptosis in neonatal rats. However, L-carnitine (LC) treatment appears to prevent glutamate-induced toxicity in the developing CNS. Previously, we described altered preweaning behaviors (i.e., abnormal home cage, slant board and forelimb hang behaviors) resulting from neonatal PCP and KET treatment. Those adverse effects of KET were somewhat ameliorated by LC [Boctor SY, Wang C, Ferguson SA. Neonatal PCP is more potent than ketamine at modifying preweaning behaviors of Sprague-Dawley rats. Toxicol Sci 2008;106:172-9]. Here, a portion of those subjects were evaluated for prepulse inhibition (PPI) of the acoustic startle response at postnatal day (PND) 25 since previous reports described PCP-induced effects on this response. Rats were subcutaneously treated with: saline; 10 mg/kg PCP (1x/day) on PNDs 7, 9 and 11; 20 mg/kg KET (6 injections every 2h on PND 7); or a similar regimen of ketamine and 250 mg/kg LC on PND 7, with a single injection of 250 mg/kg LC on PNDs 8-11 (KLC). Male and female rats were assessed using a standard PPI paradigm with prepulses of 68, 78 and 82 dB. Body weight was decreased 17-21% and whole brain weight was decreased 10% in PCP-treated rats. Specifically, cerebellar weight was significantly less in PCP-treated rats relative to control. Despite the magnitude of those PCP-induced changes, startle response in normal pulse only trials and percent of PPI in PCP-, KET-, and KLC-treated groups were comparable to controls. Average latency to maximum startle was 2.6 ms less in females than males (p<0.007); there were no other significant sex effects. The lack of neonatal PCP treatment on later PPI is similar to that reported by Rasmussen et al. [Rasmussen BA, O'Neil J, Manaye KF, Perry DC, Tizabi Y. Long-term effects of developmental PCP administration on sensorimotor gating in male and female rats. Psychopharmacology (Berl) 2007; 190: 43-9.], and indicates that neonatal PCP-induced effects on PPI [Wang C, McInnis J, Ross-Sanchez M, Shinnick-Gallagher P, Wiley JL, Johnson KM. Long-term behavioral and neurodegenerative effects of perinatal phencyclidine administration: implications for schizophrenia. Neuroscience 2001; 107: 535-50.] appear difficult to replicate.

Neonatal PCP is more potent than ketamine at modifying preweaning behaviors of Sprague-Dawley rats.

Treatment with N-methyl-D-aspartate (NMDA) receptor antagonists, such as ketamine (KET) or phencyclidine (PCP), can trigger apoptotic neurodegeneration in neonatal rodents; however, little is known about the behavioral alterations resulting from such treatment. Here, rats were sc treated with saline; 10 mg/kg PCP on postnatal days (PNDs) 7, 9, and 11; 20 mg/kg KET (six injections every 2 h on PND 7); or a regimen of ketamine and 250 mg/kg L-carnitine (KLC) both administered on PND 7 with additional 250 mg/kg doses of L-carnitine given on PNDs 8-11. Postinjection, the home cage behavior of each pup was categorized on PNDs 7-11. Slant board and forelimb hang behaviors were examined on PNDs 8-11 and 12-16, respectively. The initial KET or KLC injections on PND 7 elevated abnormal home cage activity (i.e., paresis and paddling); however, KLC pup behavior returned to normal by the fourth injection, indicating the protective effects of L-carnitine against NMDA antagonist toxicity. PCP treatment caused substantial abnormal home cage activity on each injection day (PNDs 7, 9, and 11). Latencies to turn on the slant board were significantly longer on PND 8 for KET- and PCP-treated pups and PND 10 for PCP-treated pups. On PND 12, the forelimb hang time of PCP-treated pups was significantly shorter. Body weight was decreased on PNDs 8-18 in PCP-treated pups and PNDs 8-10 in KET-treated pups. These data indicate that developmental NMDA antagonist treatment causes short-term behavioral alterations which appear related to motor coordination and may be cerebellar in nature. Furthermore, single PCP injections appear more potent at altering behavior than multiple injections of KET.

Differences in performance between Sprague-Dawley and Fischer 344 rats in positive reinforcement tasks.

This experimental investigation tested two different strains of rat, Sprague-Dawley (SD) and Fischer 344 (F344), in their ability to learn lever pressing for food (autoshaping) or intracranial self-administration (ICSA) of dextroamphetamine (AMPH) into the nucleus accumbens (NAcc). Additionally, a unique method of intracranial drug delivery was utilized, via reverse dialysis, by the use of a microdiaylsis probe. The experiments revealed definite behavioral differences between SD and F344 animals. The autoshaping data indicated that SD rats, on average, acquired lever pressing for food in fewer training days than F344 rats. Also, the ICSA experiment revealed that SD rats self-administered AMPH at a 30 mug/mul concentration. Lever pressing was significantly greater in those SD rats receiving AMPH than in the F344 drug group. Furthermore, the F344 rats never acquired lever pressing for intra-NAcc delivery of AMPH under our testing regime. These data reveal differences in performance of positively reinforced operant tasks between the inbred F344 rats as compared to the outbred SD strain.

The cannabinoid CB1 receptor antagonist AM251 does not modify methamphetamine reinstatement of responding.

Cannabinoid CB(1) receptor antagonists can decrease methamphetamine self-administration. This study examined whether the CB(1) receptor antagonist AM251 [N-(piperidin-1-yl)-5-(4-indophonyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] modifies reinstatement in rats that previously self-administered methamphetamine. Rats (n=10) self-administered methamphetamine (0.1 mg/kg/infusion) under a fixed ratio 2 schedule. Non-contingent methamphetamine (0.01-1.78 mg/kg, i.v.) yielded responding for saline (reinstatement) that was similar to responding for self-administered methamphetamine. AM251 (0.032-0.32, i.v.) did not affect methamphetamine-induced reinstatement but significantly attenuated Delta(9)-tetrahydrocannabinol (2.0 mg/kg, i.p.)-induced hypothermia. These data fail to support a role for endogenous cannabinoids or cannabinoid CB(1) receptors in reinstatement and, therefore, relapse to stimulant abuse.