Discontinuation of methylphenidate after long-term exposure in nonhuman primates.

Attention-deficit hyperactivity disorder (ADHD) is a common human neurobehavioral disorder that usually begins in early childhood. Methylphenidate (MPH) has been used extensively as a first-line medicine for the treatment of ADHD. Since ADHD is often diagnosed in early childhood and can persist for the entire lifespan, individuals may take MPH for many years. Given that in the course of one's lifetime a person may stop taking MPH for periods of time, or may implement lifestyle changes that may reduce the need for MPH entirely, it is important to understand how cessation of MPH affects the adult brain following long-term use of MPH. The blockage of the dopamine transporter (DAT) and the norepinephrine transporter (NET) by MPH may help with ADHD symptoms by boosting monoamine levels in the synapse. In the present study, microPET/CT was used to investigate possible neurochemical alterations in the cerebral dopamine system after cessation of long-term MPH administration in nonhuman primates. MicroPET/CT images were collected from adult male rhesus monkeys 6 months after they stopped receiving vehicle or MPH following 12 years of chronic treatment. The neurochemical status of brain dopaminergic systems was evaluated using the vesicular monoamine transporter 2 (VMAT2) ligand [18F]-AV-133 and a tracer for imaging dopamine subtype 2 (D2) and serotonin subfamily 2 (5HT2) receptors, [18F]-FESP. Each tracer was injected intravenously and ten minutes later microPET/CT images were obtained over 120 min. The binding potential (BP) of each tracer in the striatum was obtained using the Logan reference tissue model with the cerebellar cortex time activity curve (TAC) as an input function. Brain metabolism was also evaluated using microPET/CT images of [18F]-FDG. [18F]-FDG was injected intravenously, and ten minutes later, microPET/CT images were obtained over 120 min. Radiolabeled tracer accumulation in regions of interest (ROIs) in the prefrontal cortex, temporal cortex, striatum, and cerebellum were converted into standard uptake values (SUVs). Compared to the vehicle control group, the BPs of [18F] AV-133 and [18F]-FESP in the striatum were not significantly altered in MPH treated groups. Additionally, no significant differences were detected in the SUVs of [18F]-FDG in the MPH treated group compared with control. This study demonstrates that 6 months after cessation of long-term, chronic MPH treatment, there are no significant neurochemical or neural metabolic changes in the central nervous system (CNS) of non-human primates (NHPs) and suggests that microPET imaging is helpful in assessing the status of biomarkers of neurochemical processes linked to chronic CNS drug exposure. (Supported by NCTR).

MicroPET/CT assessment of neurochemical effects in the brain after long-term methylphenidate treatment in nonhuman primates.

Methylphenidate (MPH) is a psychostimulant approved by the FDA to treatment Attention-Deficit Hyperactivity Disorder (ADHD). MPH is believed to exert its pharmacological effects via preferential blockade of the dopamine transporter (DAT) and the norepinephrine transporter (NET), resulting in increased monoamine levels in the synapse. We used a quantitative non-invasive PET imaging technique to study the effects of long-term methylphenidate use on the central nervous system (CNS). We conducted microPET/CT scans on young adult male rhesus monkeys to monitor changes in the dopaminergic system. We used [18F] AV-133, a ligand for the vesicular monoamine transporter 2 (VMAT2), and [18F]FESP a ligand for the D2 and 5HT2 receptors. In this study we evaluated the effects if chronic MPH treatment in the nonhuman primates (NHP). Two-year-old, male rhesus monkeys were orally administered MPH diluted in the electrolyte replenisher, Prang, twice a day, five days per week (M-F) over an 8-year period. The dose of MPH was gradually escalated from 0.15 mg/kg initially to 2.5 mg/kg/dose for the low dose group, and 1.5 mg/kg to 12.5 mg/kg/dose for the high dose group (Rodriguez et al., 2010). Scans were performed on Mondays, about 60 h after their last treatment, to avoid the acute effects of MPH. Tracers were injected intravenously ten minutes before microPET/CT scanning. Sessions lasted about 120 min. The Logan reference tissue model was used to determine the Binding Potential (BP) of each tracer in the striatum with the cerebellar cortex time activity curve as an input function. Both MP treatment groups had a lower [18F] AV-133 BP, although this failed to reach statistical significance. MPH treatment did not have a significant effect on The BP of [18F] FESP in the striatum. Long-term administration of MPH did not significant change any of the marker of monoamine function used here. These data suggest that, despite lingering concerns, long-term use of methylphenidate does not negatively impact monoamine function. This study also demonstrates that microPET imaging can distinguish differences in binding potentials of a variety of radiotracers in the CNS of NHPs. This approach may provide minimally-invasive biomarkers of neurochemical processes associated with chronic exposure to CNS medications. (Supported by NCTR).

Minimally invasive biomarkers of general anesthetic-induced developmental neurotoxicity.

The association of general anesthesia with developmental neurotoxicity, while nearly impossible to study in pediatric populations, is clearly demonstrable in a variety of animal models from rodents to nonhuman primates. Nearly all general anesthetics tested have been shown to cause abnormal brain cell death in animals when administered during periods of rapid brain growth. The ability to repeatedly assess in the same subjects adverse effects induced by general anesthetics provides significant power to address the time course of important events associated with exposures. Minimally-invasive procedures provide the opportunity to bridge the preclinical/clinical gap by providing the means to more easily translate findings from the animal laboratory to the human clinic. Positron Emission Tomography or PET is a tool with great promise for realizing this goal. PET for small animals (microPET) is providing valuable data on the life cycle of general anesthetic induced neurotoxicity. PET radioligands (annexin V and DFNSH) targeting apoptotic processes have demonstrated that a single bout of general anesthesia effected during a vulnerable period of CNS development can result in prolonged apoptotic signals lasting for several weeks in the rat. A marker of cellular proliferation (FLT) has demonstrated in rodents that general anesthesia-induced inhibition of neural progenitor cell proliferation is evident when assessed a full 2weeks after exposure. Activated glia express Translocator Protein (TSPO) which can be used as a marker of presumed neuroinflammatory processes and a PET ligand for the TSPO (FEPPA) has been used to track this process in both rat and nonhuman primate models. It has been shown that single bouts of general anesthesia can result in elevated TSPO expression lasting for over a week. These examples demonstrate the utility of specific PET tracers to inform, in a minimally-invasive fashion, processes associated with general anesthesia-induced developmental neurotoxicity. The fact that PET procedures are also used clinically suggests an opportunity to confirm in humans what has been repeatedly observed in animals.

MicroPET/CT assessment of FDG uptake in brain after long-term methylphenidate treatment in nonhuman primates.

Methylphenidate (MPH) is a psychostimulant commonly used for the treatment of Attention-Deficit Hyperactivity Disorder (ADHD). Since the long-term effects of this drug on the central nervous system (CNS) are not well understood, we conducted microPET/CT scans on young adult male rhesus monkeys (n=4/group) to gather information on brain metabolism using the uptake of [(18)F]Fluoro-2-deoxy-2-d-glucose (FDG) as a marker. Approximately two-year old, male rhesus monkeys were treated orally with MPH twice per day, five days per week (M-F) over a 6-year period. Subjects received MPH at either 2.5 or 12.5mg/kg/dose or vehicle (Prang). To minimize the acute effects of MPH on FDG uptake, microPET/CT scans were scheduled on Mondays before their first daily dosing of the week (approximately 68h since their last treatment). FDG (370±8.88MBq) was injected intravenously and 30min later microPET/CT images were obtained over 60min. Radiolabeled tracer accumulation in regions of interest (ROIs) in the prefrontal cortex, temporal cortex, striatum and cerebellum were converted into Standard Uptake Values (SUVs). Compared to the control group, the uptake of FDG in the cerebellum was significantly decreased in both the low and high dose groups. These preliminary data demonstrate that microPET imaging is capable of distinguishing differences in retention of FDG in the brains of NHPs treated chronically with MPH and suggests that this approach may provide a minimally invasive biomarker for exploring the effects of chronic MPH treatment on aspects of brain function.

Ketamine anesthesia during the first week of life can cause long-lasting cognitive deficits in rhesus monkeys.

Previously our laboratory has shown that ketamine exposure (24h of clinically relevant anesthesia) causes significant increases in neuronal cell death in perinatal rhesus monkeys. Sensitivity to this ketamine-induced neurotoxicity was observed on gestational days 120-123 (in utero exposure via maternal anesthesia) and on postnatal days (PNDs) 5-6, but not on PNDs 35-37. In the present study, six monkeys were exposed on PND 5 or 6 to intravenous ketamine anesthesia to maintain a light surgical plane for 24h and six control animals were unexposed. At 7 months of age all animals were weaned and began training to perform a series of cognitive function tasks as part of the National Center for Toxicological Research (NCTR) Operant Test Battery (OTB). The OTB tasks used here included those for assessing aspects of learning, motivation, color discrimination, and short-term memory. Subjects responded for banana-flavored food pellets by pressing response levers and press-plates during daily (M-F) test sessions (50 min) and were assigned training scores based upon their individual performance. As reported earlier (Paule et al., 2009) beginning around 10 months of age, control animals significantly outperformed (had higher training scores than) ketamine-exposed animals for approximately the next 10 months. For animals now over 3 and one-half years of age, the cognitive impairments continue to manifest in the ketamine-exposed group as poorer performance in the OTB learning and color and position discrimination tasks, as deficits in accuracy of task performance, but also in response speed. There are also apparent differences in the motivation of these animals which may be impacting OTB performance. These observations demonstrate that a single 24-h episode of ketamine anesthesia, occurring during a sensitive period of brain development, results in very long-lasting deficits in brain function in primates and provide proof-of-concept that general anesthesia during critical periods of brain development can result in subsequent functional deficits. Supported by NICHD, CDER/FDA and NCTR/FDA.

MicroPET imaging of ketamine-induced neuronal apoptosis with radiolabeled DFNSH.

Recent reports indicate that 6-12 h of ketamine anesthesia can trigger neuronal apoptosis in postnatal day (PND) 7 rats. In vitro, ex vivo, and confocal fluorescent imaging studies suggest that dansyl compounds can accumulate within the cytoplasm of the apoptotic cell. High-resolution positron emission tomography (microPET) imaging has been proposed as a minimally invasive method for detecting apoptosis in the rat brain. Compared with [(18)F]-labeled annexin V, which binds to externalized phosphatidylserine (PS) on the outer membrane of apoptotic cells, intracellular uptake of the dansylhydrazone of p-fluorobenzaldehyde (DFNSH) may lead to improved target-to-background contrast ratios. In this study, the effect of ketamine on the uptake and retention of [(18)F]-DFNSH in the rat brain was investigated using microPET imaging. On PND 7, rat pups in the experimental group were exposed, at 2-h intervals, to six subcutaneous injections of ketamine (20 mg/kg) and control rat pups received six injections of saline. On PND 35, [(18)F]-DFNSH (37 MBq) was injected into the tail vein of rats and microPET images were obtained over 2 h following the injection. Radiolabeled tracer accumulation in the region of interest (ROI) in the frontal cortex was converted into standard uptake values (SUVs). The radiotracer was quickly distributed into the brains of both ketamine- and saline-treated rats. Compared with the control group, the uptake of [(18)F]-DFNSH was significantly increased in the ROI, frontal cortex area of ketamine-treated rats. In addition, the wash-out duration of the tracer was prolonged in the ketamine-treated animals. This study demonstrates that microPET imaging is capable of distinguishing differences in retention of [(18)F]-DFNSH in ROI and suggests that this compound may serve as a minimally invasive biomarker of neuronal apoptosis in rodents.

Changes in gene expression after phencyclidine administration in developing rats: a potential animal model for schizophrenia.

Repeated administration of phencyclidine (PCP), an N-methyl-d-aspartate (NMDA) receptor antagonist, during development, may result in neuronal damage that leads to behavioral deficits in adulthood. The present study examined the potential neurotoxic effects of PCP exposure (10mg/kg) in rats on postnatal days (PNDs) 7, 9 and 11 and the possible underlying mechanism(s) for neurotoxicity. Brain tissue was harvested for RNA extraction and morphological assessments. RNA was collected from the frontal cortex for DNA microarray analysis and quantitative RT-PCR. Gene expression profiling was determined using Illumina Rat Ref-12 Expression BeadChips containing 22,226 probes. Based on criteria of a fold-change greater than 1.4 and a P-value less than 0.05, 19 genes including NMDAR1 (N-methyl-d-aspartate receptor) and four pro-apoptotic genes were up-regulated, and 25 genes including four anti-apoptotic genes were down-regulated, in the PCP-treated group. In addition, the schizophrenia-relevant genes, Bdnf (Brain-derived neurotrophic factor) and Bhlhb2 (basic helix-loop-helix domain containing, class B, 2), were significantly different between the PCP and the control groups. Quantitative RT-PCR confirmed the microarray results. Elevated neuronal cell death was further confirmed using Fluoro-Jade C staining. These findings support the hypothesis that neurodegeneration caused by PCP occurs, at least in part, through the up-regulation of NMDA receptors, which makes neurons possessing these receptors more vulnerable to endogenous glutamate. The changes in schizophrenia-relevant genes after repeated PCP exposure during development may provide important information concerning the validation of an animal model for this disorder.

Gene expression profiling in the developing rat brain exposed to ketamine.

Ketamine, a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist, is associated with accelerated neuronal apoptosis in the developing rodent brain. In this study, postnatal day (PND) 7 rats were treated with 20 mg/kg ketamine or saline in six successive doses (s.c.) at 2-h intervals. Brain frontal cortical areas were collected 6 h after the last dose and RNA isolated and hybridized to Illumina Rat Ref-12 Expression BeadChips containing 22,226 probes. Many of the differentially expressed genes were associated with cell death or differentiation and receptor activity. Ingenuity Pathway Analysis software identified perturbations in NMDA-type glutamate, GABA and dopamine receptor signaling. Quantitative polymerase chain reaction (Q-PCR) confirmed that NMDA receptor subunits were significantly up-regulated. Up-regulation of NMDA receptor mRNA signaling was further confirmed by in situ hybridization. These observations support our working hypothesis that prolonged ketamine exposure produces up-regulation of NMDA receptors and subsequent over-stimulation of the glutamatergic system by endogenous glutamate, triggering enhanced apoptosis in developing neurons.

The effects of chronic methylphenidate administration on operant test battery performance in juvenile rhesus monkeys.

Methylphenidate (MPH) is an amphetamine derivative widely prescribed for the treatment of attention deficit-hyperactivity disorder. Recent concern over its genotoxic potential in children [11] spurred a study on the effects of chronic MPH treatment in a nonhuman primate population and the studies reported here were conducted in conjunction with that study in the same animals. Here, the focus was on the ability of juvenile rhesus monkeys to learn how to perform a battery of operant behavioral tasks while being treated chronically with MPH. Performance of the National Center for Toxicological Research (NCTR) Operant Test Battery (OTB) was used to quantify the learning of tasks thought to model specific aspects of cognitive function including learning, motivation, color and position discrimination, and short-term memory. The OTB tasks designed to assess these specific behaviors included Incremental Repeated Acquisition (IRA), Progressive Ratio (PR), Conditioned Position Responding (CPR), and Delayed Matching-to-Sample (DMTS), respectively. Juvenile males (n=10/group) pressed levers and press-plates for banana-flavored food pellets. Subjects were treated orally, twice a day, five days per week (M-F) for 66 weeks with escalating doses (0.15 mg/kg initially, increased to 2.5 mg/kg for the low dose group and to 12.5 mg/kg for the high dose group) and tested in OTB tasks 30 to 60 min after the morning dose. The findings indicate that MPH at doses up to 2.5 mg/kg twice per day were well tolerated (performance was no different than controls) but at doses of 12.5 mg/kg twice per day there was a significant decrement in OTB performance, characterized by decreases in both percent task completed and response rates for all tasks. These effects of MPH seem primarily due to decreases in motivation to perform for food, which is not surprising given the well known appetite suppressing effects of amphetamine-like stimulants. Thus, the current data do not strongly suggest cognitive impairments following chronic MPH administration.

WITHDRAWN: Effects of Reinforcer Type on Performance of Psychological Tasks.

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The effects of L-carnitine on the combination of, inhalation anesthetic-induced developmental, neuronal apoptosis in the rat frontal cortex.

The anesthetic gas nitrous oxide (N2O) and the volatile anesthetic isoflurane (ISO) are commonly used in surgical procedures for human infants and in veterinary and laboratory animal practice to produce loss of consciousness and analgesia. Recent reports indicate that exposure of the developing brain to general anesthetics that block N-methyl-D-aspartate (NMDA) glutamate receptors or potentiate GABA(A) receptors can trigger widespread apoptotic neurodegeneration. In the present study, the question arises whether a relatively low dose of ISO alone or its combination with N2O entails significant risk of inducing enhanced apoptosis. In addition, the role of L-carnitine to attenuate these effects was also examined. Postnatal day 7 (PND-7) rat pups were exposed to N2O (75%) or a low dose of ISO (0.55%) alone, or N2O plus ISO for 2, 4, 6 or 8 h with or without L-carnitine. The neurotoxic effects were evaluated 6 h after completion of anesthetic administration. No significant neurotoxic effects were observed for the animals exposed to N2O or ISO alone. However, enhanced apoptotic cell death was apparent when N2O was combined with ISO at exposure durations of 6 h or more. Co-administration of L-carnitine (300 or 500 mg/kg, i.p.) effectively protected neurons from the anesthetic-induced damage. These data indicate that 6 h or more of inhaled anesthetic exposure consisting of a combination of N2O and ISO results in enhanced neuronal apoptosis, and L-carnitine effectively blocks the neuronal apoptosis caused by inhalation anesthetics in the developing rat brain.

Effect of chronic MK-801 and/or phenytoin on the acquisition of complex behaviors in rats.

The purpose of the present experiment was to assess the effects of chronic MK-801 (an N-methyl-D-aspartate receptor antagonist) and/or phenytoin (a sodium channel blocker) treatment on behavioral acquisition and performance in rats. Learning, audio/visual discrimination and motivation were modeled using incremental repeated acquisition (IRA), audio/visual discrimination (AVD) and progressive ratio (PR) tasks, respectively. MK-801 and/or phenytoin were administered daily, 7 days/week by orogastric gavage beginning just after weaning on postnatal day (PND) 23 and continuing until PND 306. Monday through Friday behavioral assessments began on PND 27 and continued until PND 430. Throughout treatment, subjects in the high dose MK-801 (1.0 mg/kg/day) and the high dose drug combination (1.0 mg/kg/day MK-801+150 mg/kg/day phenytoin) groups exhibited decreased body weight gains compared to control subjects. For these two affected groups, response rates were also decreased in all tasks. Task acquisition, as evidenced by an increase in response accuracy, was decreased for both these groups in the AVD task, but only for the high dose MK-801 group in the IRA task. The data suggest that chronic MK-801 treatment adversely affects the acquisition of IRA and AVD task performance and that the inclusion of phenytoin in the MK-801 dosing regimen blocks some of the adverse effects of chronic MK-801 treatment on IRA task acquisition. These findings are in marked contrast with those observed in nonhuman primates and suggest important species differences associated with chronic exposure to compounds that block NMDA receptors and/or sodium channels.

Response sequence difficulty in an incremental repeated acquisition (learning) procedure.

Incremental repeated acquisition (IRA) procedures require subjects to learn a different sequence of behavioral responses during each experimental session with required response sequences increasing incrementally in length as subjects demonstrate mastery of shorter response chains. The purpose of this study was to investigate whether some response sequences are more or less difficult to acquire than others. If true, then sequence difficulty must be considered as a potential confound when attempting to assess the effects of drugs and other experimental manipulations on learning. Accuracy for each response sequence was assessed using control data from two large rodent studies, and each sequence was classified as easy, moderate or hard. Sequences that required responses on adjacent levers were easier (characterized by higher accuracies) to acquire than those that required responses on non-adjacent levers. In addition, sequences that required responses on only two of three response levers were easier to acquire than those that required responses on all three levers. These results provide strong evidence for differing levels of response sequence difficulty in IRA procedures with sequence difficulty seeming to be dependent on whether or not responses are required on adjacent levers.

Behavioral effects associated with chronic ketamine or remacemide exposure in rats.

The effects of chronic exposure to ketamine or remacemide on the acquisition and performance of food-reinforced operant behaviors was assessed in female Sprague-Dawley rats. Ketamine is an anesthetic N-methyl-D-aspartate (NMDA) receptor antagonist, whereas remacemide is an active central nervous system compound with both NMDA receptor antagonist and sodium channel blocking properties. Learning, audio/visual discrimination and motivation were modeled using incremental repeated acquisition (IRA), audio/visual discrimination (AVD) and progressive ratio (PR) tasks, respectively. Ketamine (10 or 100 mg/kg/day), remacemide (100 or 150 mg/kg/day) or water was administered daily (7 days/week) via orogastric gavage beginning on postnatal day (PND) 23 and continuing until PND 257. Monday through Friday behavioral assessments began on PND 27 and continued until PND 383. Chronic treatment with the high dose of ketamine decreased response rate in all tasks suggesting decreased motivation or motoric capabilities. Chronic treatment with ketamine or remacemide had no effect on the acquisition of IRA task performance at any dose tested. While chronic treatment with either high-dose ketamine or low-dose remacemide only delayed the acquisition of AVD task performance for a brief period midway through treatment, chronic treatment with high-dose remacemide delayed the acquisition of AVD task performance until late in treatment. The findings for ketamine are quite different from those of MK-801 (the prototypic NMDA receptor antagonist) in a previous rat study in which MK-801 severely disrupted the acquisition of both IRA and AVD task performances. These observations suggest important differences in the mechanism of action between ketamine and MK-801. For example, ketamine has a much lower binding affinity than MK-801 for the NMDA receptor, the dopamine transporter and the dopamine D2 receptor. In addition, the findings for remacemide observed in rats are in marked contrast with those seen in monkeys where chronic remacemide had profound disruptive effects on the acquisition of both IRA and AVD task performances and suggest important species differences.

L-carnitine protects neurons from 1-methyl-4-phenylpyridinium-induced neuronal apoptosis in rat forebrain culture.

1-Methyl-4-phenylpyridinium ion (MPP+), an inhibitor of mitochondrial complex I, has been widely used as a neurotoxin because it elicits a severe Parkinson's disease-like syndrome with an elevation of intracellular reactive oxygen species (ROS) and apoptosis. L-carnitine plays an integral role in attenuating the brain injury associated with mitochondrial neurodegenerative disorders. The present study investigates the effects of L-carnitine against the toxicity of MPP+ in rat forebrain primary cultures. Cells in culture were treated for 24 h with 100, 250, 500 and 1000 microM MPP+ alone or co-incubated with L-carnitine. MPP+ produced a dose-related increase in DNA fragmentation as measured by cell death ELISA (enzyme-linked immunosorbent assay), an increase in the number of TUNEL (terminal dUTP nick-end labeling)-positive cells and a reduction in the mitochondrial metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). No significant effect was observed with the release of lactate dehydrogenase (LDH), indicating that cell death presumably occurred via apoptotic mechanisms. Co-incubation of MPP+ with L-carnitine significantly reduced MPP+-induced apoptosis. Western blot analyses showed that neurotoxic concentrations of MPP+ decreased the ratio of BCL-X(L) to Bax and decreased the protein levels of polysialic acid neural cell adhesion molecules (PSA-NCAM), a neuron specific marker. L-carnitine blocked these effects of MPP+ suggesting its potential therapeutic utility in degenerative disorders such as Parkinson's disease, Alzheimer's disease, ornithine transcarbamylase deficiency and other mitochondrial diseases.

Assessing the potential toxicity of MK-801 and remacemide: chronic exposure in juvenile rhesus monkeys.

The present experiment examined the effects of chronic exposure to either 0.1 or 1.0 mg/kg MK-801 [a selective N-methyl-D-aspartate (NMDA) receptor antagonist] or 20.0 or 50.0 mg/kg remacemide (an NMDA receptor antagonist which also blocks fast sodium channels) in juvenile rhesus monkeys. Endpoints were monitored to provide a general index of subjects' health and included measures of clinical chemistry, hematology, ophthalmology, spontaneous home-cage behavior, and peak drug plasma levels. In general, both drugs were well tolerated and produced no treatment-related effects during 2 years of dosing and assessment. Periodic plasma drug level determinations provided limited evidence that both compounds may induce their own metabolism. The present results contrast sharply with previously reported effects of long-lasting impairments in the acquisition of incremental learning and in the development of color and position discrimination in these same subjects. These observations highlight the importance of collecting a broad range of toxicology data, including tests of cognitive function, to make comprehensive assessments of new drug safety. In the present case, the less obvious effects of these drugs on cognition defined the toxicologic response.

No alterations in the performance of two interval timing operant tasks after alpha-difluoromethylornithine (DFMO)-induced cerebellar stunting.

The cerebellum is critically involved in temporal processes in the millisecond range and may be involved in longer time estimations (i.e. in the seconds range). Estimates in the millisecond range are impaired after developmentally induced cerebellar alterations, however, little is known about the effects of similar alterations on longer timing performance. Appropriately timed DFMO treatment reliably causes cerebellar stunting in rats, however, its effects on temporal estimation performance are unknown. Here, male and female Sprague-Dawley rats were treated with subcutaneous injections of 500 mg/kg DFMO on postnatal days 5-12, causing a 10% cerebellar weight reduction at adulthood. As adults, subjects were tested under one of two paradigms - a differential reinforcement of low response rate (DRL) task requiring that subjects withhold a lever press response for 10-14 s or a temporal response differentiation (TRD) task requiring that subjects maintain a lever press response for 10-14 s. Training and steady-state performance of the DRL and TRD tasks were not significantly altered by DFMO treatment. Performance after acute challenges with two dopaminergic agonists (2.00-7.50 mg/kg methylphenidate and 0.10-1.00 mg/kg d-amphetamine) was measured after which all subjects underwent behavioral extinction. Generally, performance after methylphenidate and d-amphetamine was similar in control and DFMO-treated rats and DFMO treatment had no differential effects on performance during extinction. These results support findings from an earlier study [Ferguson SA, Paule MG, Holson RR. Neonatal dexamethasoneon day 7 in rats causes behavioral alterations reflective of hippocampal, but not cerebellar, deficits. Neurotoxicol Teratol, 2001; 23:57-69] indicating that developmental cerebellar stunting has few effects on time estimation within the range of seconds.

Differential effects of two NMDA receptor antagonists on cognitive-behavioral development in nonhuman primates I.

The present experiment examined effects of chronic exposure to remacemide (an N-methyl-D-aspartate [NMDA] antagonist which also blocks fast sodium channels) or MK-801 (which blocks NMDA receptors, exclusively) on learning and motivation in young rhesus monkeys. Remacemide (20 or 50 mg/kg/day) or MK-801 (0.1 or 1.0 mg/kg/day) was administered every day to separate groups of animals via orogastric gavage for up to 2 years. Immediately prior to dosing, 5 days per week (M--F), throughout the 2-year dosing period, an incremental repeated acquisition (IRA) task was used to assess learning and a progressive ratio (PR) task was used to assess motivation. The results indicate an effect of 50 mg/kg/day remacemide to impair learning (IRA) which persisted even after drug treatment was discontinued. MK-801 had no effect on learning but transiently increased motivation. Because the effects of remacemide occurred independently of changes in motivation or response rates, they are likely due to specific cognitive impairments and are not due to an inability of subjects to fulfill the motoric requirements of the task. The fact that MK-801 did not alter learning suggests that NMDA antagonism alone may be insufficient to produce learning deficits in young monkeys and that such deficits may rely on the ancillary blockade of fast sodium channels.

Differential effects of two NMDA receptor antagonists on cognitive--behavioral performance in young nonhuman primates II.

The present experiment examined the effects of chronic exposure to remacemide (an NMDA antagonist that also blocks fast sodium channels) or MK-801 (which blocks NMDA receptors more selectively) on the acquisition of color and position discrimination and short-term memory behavior in juvenile rhesus monkeys. Throughout the 2-year dosing period, a conditioned position responding (CPR) task was used to assess color and position discrimination and a delayed matching-to-sample (DMTS) task was used to assess memory. Chronic exposure to high doses of either drug delayed the acquisition of accurate color and position discrimination without altering response rates. In the case of MK-801, these effects abated within 6 months of the start of treatment. In the case of remacemide, the effects persisted for 17 months of dosing. Neither compound significantly altered performance of the short-term memory task at any time point or at any dose tested. The fact that the effects of remacemide on behavioral performance were more persistent than those seen for MK-801 suggests that tolerance may develop to the behavioral effects of MK-801, which does not develop to the effects of remacemide. Alternatively, these results may suggest that the concurrent antagonism of NMDA receptors and fast sodium channels may have more profound consequences for behavior than does the antagonism of NMDA receptors alone.

Neonatal dexamethasone on day 7 in rats causes behavioral alterations reflective of hippocampal, but not cerebellar, deficits.

Developmental glucocorticoid treatment in rats has been shown to cause body and brain weight decrements concurrent with behavioral alterations. Here, Sprague-Dawley rats were treated with the synthetic glucocorticoid, dexamethasone (DEX), on postnatal day (PND) 7 (1.5 mg/kg, sc, injected in the morning and afternoon). Behavioral assessments of negative geotaxis, locomotor activity (open field, maze exploration, residential running wheel, residential figure 8 maze), open-field activity response to amphetamine, acoustic startle, prepulse inhibition (PPI) of acoustic startle, juvenile play behavior, anxiety (emergence tests), motor coordination (rotarod performance), spatial learning (Morris water maze and food-reinforced complex maze), and operant performance (time estimation and response inhibition) were assessed in male rats. Body weight was decreased beginning at PND 43 until sacrifice on PND 127. Whole and regional brain weights were less, especially hippocampus, cerebellum, brainstem, and cortical remnant. Indications of delayed development were apparent; specifically, DEX-treated rats took significantly longer to turn on PND 8, but not PND 9, in the negative geotaxis test. DEX treatment induced deficits in the Morris water maze that were similar to hippocampal deficits. Open-field activity changes were inconsistent; however, DEX-treated rats were hyperactive during the dark period in running wheel tests. There were no indications of changes in reactivity or emotionality.