Acute postnatal exposure to di(2-ethylhexyl) phthalate adversely impacts hippocampal development in the male rat.

The distribution of CA3 hippocampal axonal terminal fields undergo a period of widespread connectivity-based changes in the early postnatal stages of life. The purpose of the current study was to examine the effect of acute phthalate exposure during this period of hippocampal development (postnatal days 16-22 (p16-p22)) on morphological outcomes in male and female Long Evans rats. The reproductive toxicity of exposure to phthalates early in life has been well-documented; however, much less is known about the effects of phthalates on brain development. The present research demonstrated that exposure to di(2-ethylhexyl) phthalate (DEHP; 10 mg/kg, i.p.) from p16 to p22 reduced axonal markers in the CA3 distal stratum oriens (SO) and reduced cell density of both immature and mature neurons in the dentate gyrus (DG) and CA3, respectively, in male rats. The same markers in the hippocampus of female rats were similar in saline and DEHP-treated animals. These data suggest that DEHP has a negative impact on the development of the hippocampus in males but not females and recommend more extensive animal studies on phthalate exposure during the vulnerable post-natal developmental period when rapid structural and functional changes are taking place.

Intermittent and continuous enteral nutrition in critically ill dogs: a prospective randomized trial.

BACKGROUND: Malnutrition is a common problem in critically ill dogs and is associated with increased morbidity and mortality in human medicine. Enteral nutrition (EN) delivery methods have been evaluated in humans to determine which is most effective in achieving caloric goals. OBJECTIVES: To compare continuous infusion and intermittent bolus feeding of EN in dogs admitted to a critical care unit. ANIMALS: Fifty-four dogs admitted to the critical care unit and requiring nutritional support with a nasoenteric feeding tube. METHODS: Prospective randomized clinical trial. Dogs were randomized to receive either continuous infusion (Group C) or intermittent bolus feeding (Group I) of liquid EN. The percentage of prescribed nutrition delivered (PPND) was calculated every 24 hours. Frequencies of gastrointestinal (GI), mechanical, and technical complications were recorded and gastric residual volumes (GRVs) were measured. RESULTS: PPND was significantly lower in Group C (98.4%) than Group I (100%). There was no significant difference in GI or mechanical complications, although Group C had a significantly higher rate of technical complications. GRVs did not differ significantly between Group C (3.1 mL/kg) and Group I (6.3 mL/kg) and were not correlated with the incidence of vomiting or regurgitation. CONCLUSIONS AND CLINICAL IMPORTANCE: There was a statistically significant difference in the PPND between continuously and intermittently fed dogs, but this difference is unlikely to be clinically relevant. Critically ill dogs can be successfully supported with either continuous infusion or intermittent bolus feeding of EN with few complications. Increased GRVs may not warrant termination of enteral feeding.

Differential patterns of extracellular signal-regulated kinase-1 and -2 phosphorylation in rat limbic brain regions after short-term and long-term inhibitory avoidance learning.

Activation of the extracellular signal-regulated kinase-1 and -2 has been shown to be required for neural plasticity and memory. Previous pharmacological studies have demonstrated that inhibition of extracellular signal-regulated kinase-1 and -2 blocks inhibitory avoidance retention. The aim of the present study was to investigate the different neural substrates underlying short- and long-term inhibitory avoidance learning and memory in rats using phosphorylated extracellular signal-regulated kinase-1 and -2 labeling as an index of plasticity. Short- and long-term retention tests were given 10 min or 24 h after inhibitory avoidance training. A significant elevation in the number of phosphorylated extracellular signal-regulated kinase-1 and -2-immunoreactive neurons was observed in area 1 of anterior cingulate cortex, the secondary motor cortex, lateral orbital cortex, claustrum, and the medial amygdala nucleus after the short-term inhibitory avoidance test. After the long-term retention test, phosphorylated extracellular signal-regulated kinase-1 and -2-immunoreactive neurons were localized in area 1 of anterior cingulate cortex, prelimbic cortex, and the central nucleus of amygdala. This suggests that phosphorylated extracellular signal-regulated kinase-1 and -2-immunoreactivity may reveal different brain regions involved in the storage of short- and long-term aversive memories.

Simple high-performance liquid chromatographic method to analyze serum creatinine has several advantages over the Jaffé picric acid reaction as demonstrated with a cimetidine dose response in rhesus monkeys.

A simple method for creatinine determination was developed using high-performance liquid chromatography (HPLC) to more accurately monitor serum creatinine levels in experimental animal models when compared to the Jaffé method. The new HPLC procedure will replace the traditional Jaffé method for rhesus monkey kidney function studies. We developed an isocratic method using a polymeric, hydrophilic, silica-based strong cation-exchange bed with a 5.0 mmol/l lithium acetate matrix, pH 4.9, which isolates creatinine with no detectable impurities as determined by three-dimensional ultraviolet-visible spectral analysis. Sample preparation includes deproteination with acetonitrile, evaporation, and resolubilization in mobile phase followed by quantitation with UV detection at 234 nm. Extraction efficiency across the measured range was 96 +/- 2%. From numerous extracted rhesus monkey creatinine curves (n=38) a slope of 251,100 +/- 756 (95% CI) and an intercept of 675.6 +/- 712.7 (95% CI) was calculated. Extraction efficiency and peak purity tests with human plasma were cross-compared with rhesus monkey serum producing equivalent results. An average of 120 samples can be run daily.

A pharmacological analysis of the substrates underlying conditioned feeding induced by repeated opioid stimulation of the nucleus accumbens.

It has previously been demonstrated that stimulation of opiate receptors within the nucleus accumbens results in marked hyperphagia, perhaps reflecting enhancement of taste palatability. Rats that have received multiple morphine treatments also increase feeding in response to environmental stimuli that have been associated with the morphine injections. The present investigation further examined this phenomenon. In Experiment 1, it was shown that induction of conditioned feeding was dose-dependent; significant conditioned feeding was obtained with repeated (n = 5) intra-accumbens injections of 5 or 10 microg/microl morphine but not with saline or 1 microg. The conditioned feeding response was blocked by systemic naltrexone (5 mg/kg). In the second experiment, co-treatment with either a D-1 (SCH 23390, 0.1 mg/kg) or D-2 (haloperidol, 0.25 mg/kg) antagonist did not block the development of conditioned feeding, nor did these drugs block morphine-induced feeding. In Experiment 3, it was found that systemic naltrexone blocked the expression of conditioned feeding (confirming Experiment 1), as did SCH-23390, whereas haloperidol did not affect expression of conditioned feeding. In the fourth experiment, we observed that significant conditioned feeding was induced with repeated treatment with the selective mu agonist D-Ala2, NMe-phe4, Glyol5-enkephalin (DAMGO, 2.5 microg), but not with the delta agonist D-Pen2,5-enkephalin (DPEN, 3.1 microg). The final experiment tested the diurnal variability of the expression of conditioned feeding, and it was found that the magnitude of the effect depended on time of day. In summary, the development of opioid-induced conditioned feeding depends on mu opiate receptor stimulation, but not dopamine receptor stimulation. Its expression, however, involves both opiate and D-1 receptor activation. These findings are considered in terms of putative neural mechanisms governing conditioned meal initiation, and implications for compulsive eating and bulimia are also discussed.

Morphine-associated environmental cues elicit conditioned gene expression.

Drug-associated contextual cues can exert a powerful influence on behavior through associative pairing between the drug and the environment. However, the anatomical and molecular substrates for these effects are not well characterized. Using a drug-conditioning paradigm, we examined the expression of the immediate early gene product, Fos, within specific brain circuits using immunocytochemical detection. Rats were given either morphine (5 mg/ml/kg) or saline once a day for 10 days. The drug administration was always paired with a specific environment (activity monitors) different from the home cage. Following this treatment, the rats were returned to the cages at various times thereafter, with only a mock injection. Conditioned behavioral activation was observed in rats at 3, 5, and 7 days following treatment with morphine. In rats showing the conditioned motor response, several cortical and limbic areas showed substantial increases in the number of Fos positive cells, indicating that these regions were more active during exposure to the drug-paired environment. Areas that were most activated included prefrontal cortex, cingulate cortex, nucleus accumbens, and preoptic area. Further analysis showed that this increase in Fos expression was not directly related to the increase in motor activity, and that the drug-associated conditioning and Fos expression was lessened at 7 days and absent by 14 days post-treatment. These results are discussed in terms of their relevance to the problem of relapse in drug addiction.

N-methyl-D-aspartate receptor-dependent plasticity within a distributed corticostriatal network mediates appetitive instrumental learning.

The effect of microinfusion of the N-methyl-D-aspartate (NMDA) antagonist 2-amino-5-phosphonopentanoic acid (AP-5) into the amygdala, medial prefrontal cortex, and dorsal and ventral subiculum on acquisition of a lever-pressing task for food in rats was examined. Serial transmission between the basolateral amygdala and nucleus accumbens core was also examined in an asymmetric infusion design. AP-5 administered bilaterally into either the amygdala or medial prefrontal cortex markedly impaired learning, whereas administration into the dorsal or ventral subiculum had no effect. Unilateral infusion of AP-5 into either the nucleus accumbens core or amygdala was also sufficient to impair learning. These data provide novel evidence for NMDA receptor-dependent plasticity within corticostriatal networks in the acquisition of appetitive instrumental learning.

Non-peptide GPIIb/IIIa inhibitors. 20. Centrally constrained thienothiophene alpha-sulfonamides are potent, long acting in vivo inhibitors of platelet aggregation.

The synthesis and pharmacology of 4, a potent thienothiophene non-peptide fibrinogen receptor antagonist, are reported. Compound 4 inhibited the aggregation of human gel-filtered platelets with an IC50 of 8 nM and demonstrated an 8-fold improvement in affinity for isolated GPIIb/IIIa receptors over analogues possessing an isoindolinone backbone. Flow cytometry studies revealed that the binding of 4 to resting platelets is a diffusion-controlled process (kon = 3.3 x 10(6) M-1 s-1) and that 4 binds to dog and human platelets with comparable affinity (Kd = 0.04 and 0.07 nM, respectively). Ex vivo platelet aggregation in dogs was completely inhibited by an iv dose of 5 microg/kg [corrected], and an oral dose of 50-90 microg/kg [corrected] followed by low daily doses of 10 microg/kg [corrected] was sufficient to maintain approximately 80% inhibition of ex vivo platelet aggregation over several days. Inhibition of ADP-induced platelet aggregation in anesthetized dogs at 77 +/- 7% resulted in a moderate 2.5-fold increase in bleeding time, while complete inhibition (100%) resulted in an approximately 10-min bleeding time. Additional doses were required to increase the bleeding time to the maximum time allowed in the protocol (15 min), thus indicating a potentially useful and safe separation of efficacy and bleeding time.

Fibrinogen receptor antagonist-induced thrombocytopenia in chimpanzee and rhesus monkey associated with preexisting drug-dependent antibodies to platelet glycoprotein IIb/IIIa.

Most clinical trials with fibrinogen receptor antagonists (FRAs) have been associated with thrombocytopenia. This report describes the occurrence of thrombocytopenia in one chimpanzee and one rhesus monkey upon administration of potent FRAs. Chimpanzee A-264 experienced profound thrombocytopenia on two occasions immediately upon intravenous administration of two different potent FRAs, L-738, 167 and L-739,758. However, an equally efficacious antiaggregatory dose of another potent antagonist, L-734,217, caused no change in platelet count. These compounds did not affect platelet count in five other chimpanzees or numerous other nonhuman primates. Flow cytometric analysis showed drug-dependent antibodies (DDAbs) in the plasma of chimpanzee A-264 that bound to platelets of chimpanzees, humans, and all other primates tested only in the presence of the compounds that induced thrombocytopenia. Rhesus monkey 94-R021 experienced thrombocytopenia upon administration of a different antagonist, L-767,679, and several prodrugs that are converted into the active form, L-767,679, in the blood. More than 20 other FRAs, including those that induced thrombocytopenia in chimpanzee A-264, had no effect on platelet count in this monkey. Flow cytometric measurements again identified DDAbs that reacted with platelets of all primates tested and required the presence of L-767,679. Screening for DDAbs in the plasma of 1,032 human subjects with L-738, 167 and L-739,758 demonstrated that the incidence of these preexisting antibodies in this population was 0.8% +/- 0.6% and 1.1% +/- 0.6%, respectively.

Compatibility of tirofiban HCl with dopamine HCl, famotidine, sodium heparin, lidocaine HCl and potassium chloride during simulated Y-site administration.

OBJECTIVE: To study the compatibility of tirofiban HCl injection 0-05 mg/ml with dopamine HCl, famotidine, sodium heparin, lidocaine HCl and potassium chloride infusion solutions during simulated Y-site administration. METHOD: Tirofiban HCl, dopamine HCl, famotidine, lidocaine HCl and potassium chloride infusions were each prepared from their respective concentrates as per current clinical preparation instructions in both 0.9% sodium chloride and 5% dextrose solutions at both the minimum and maximum concentrations normally administered. Sodium heparin premixed infusion solutions in 5% dextrose and 0-45% sodium chloride were used as-is. Tirofiban HCl solutions were combined 1:1 (simulated Y-site administration) with the dopamine HCl, famotidine, sodium heparin, lidocaine HCl and potassium chloride solutions in separate glass containers and polyvinylchloride Y-site infusion lines. Samples were held for 4 h at room temperature under ambient fluorescent light and were assayed for changes in drug content, degradation, pH, appearance and turbidity. Activity of sodium heparin solutions was measured using an aPTT coagulation assay. RESULTS: All mixtures remained clear and colourless with no visual indication of instability, i.e. precipitation. Clarity of solutions was confirmed by turbidometric analysis. There was no significant loss of drug, increase in known degradates, or appearance of unknown drug-related peaks as determined by HPLC. The activity of heparin in heparin-containing solutions remained unchanged. The pH of all test-solutions remained constant. CONCLUSION: Tirofiban HCl injection 0.05 mg/ml can be co-infused by Y-site administration with dopamine HCl, famotidine, sodium heparin, lidocaine HCl and potassium chloride injection solutions.

Antithrombotic efficacy of thrombin inhibitor L-374,087: intravenous activity in a primate model of venous thrombus extension and oral activity in a canine model of primary venous and coronary artery thrombosis.

The small molecule direct thrombin inhibitor L-374,087 was characterized across species in an in vitro activated partial thromboplastin clotting time (aPTT) assay and in vivo in rhesus monkey and dog thrombosis models. In vitro in rhesus, dog, and human plasma, L-374,087 concentrations eliciting 2-fold increases in aPTT were 0.25, 1.9, and 0.28 microM, respectively. In anesthetized rhesus monkeys, 300 microgram/kg bolus plus 12 microgram/kg/min and 300 microgram/kg bolus plus 30 microgram/kg/min L-374,087 i.v. infusions significantly reduced jugular vein thrombus extension, with both regimens limiting venous thrombus extension to 2-fold that of baseline thrombus mass compared with a 5-fold extension observed in the vehicle control group. Antithrombotic efficacy in the rhesus with the lower-dose regimen was achieved with 2.3- to 2.4-fold increases in aPTT and prothrombin time. In a conscious instrumented dog model of electrolytic vessel injury, the oral administration of two 10 mg/kg L-374,087 doses 12 h apart significantly reduced jugular vein thrombus mass, reduced the incidence of and delayed time to occlusive coronary artery thrombosis, and significantly reduced coronary artery thrombus mass and ensuing posterolateral myocardial infarct size. Antithrombotic efficacy in the dog was achieved with 1.6- to 2.0-fold increases in aPTT at 1 to 6 h after oral dosing with L-374,087. These results indicate significant antithrombotic efficacy against both venous and coronary arterial thrombosis with L-374,087 with only moderate elevations in aPTT or prothrombin time. The oral efficacy of L-374,087 characterizes this compound as a prototype for the further development of orally active direct thrombin inhibitors.

Design and synthesis of a series of potent and orally bioavailable noncovalent thrombin inhibitors that utilize nonbasic groups in the P1 position.

As part of an ongoing effort to prepare therapeutically useful orally active thrombin inhibitors, we have synthesized a series of compounds that utilize nonbasic groups in the P1 position. The work is based on our previously reported lead structure, compound 1, which was discovered via a resin-based approach to varying P1. By minimizing the size and lipophilicity of the P3 group and by incorporating hydrogen-bonding groups on the N-terminus or on the 2-position of the P1 aromatic ring, we have prepared a number of derivatives in this series that exhibit subnanomolar enzyme potency combined with good in vivo antithrombotic and bioavailability profiles. The oxyacetic amide compound 14b exhibited the best overall profile of in vitro and in vivo activity, and crystallographic studies indicate a unique mode of binding in the thrombin active site.

Discovery and development of the novel potent orally active thrombin inhibitor N-(9-hydroxy-9-fluorenecarboxy)prolyl trans-4-aminocyclohexylmethyl amide (L-372,460): coapplication of structure-based design and rapid multiple analogue synthesis on solid support.

Early studies in these laboratories of peptidomimetic structures containing a basic P1 moiety led to the highly potent and selective thrombin inhibitors 2 (Ki = 5.0 nM) and 3 (Ki = 0.1 nM). However, neither attains significant blood levels upon oral administration to rats and dogs. With the aim of improving pharmacokinetic properties via a more diverse database, we devised a resin-based route for the synthesis of analogues of these structures in which the P3 residue is replaced with a range of lipophilic carboxylic amides. Assembly proceeds from the common P2-P1 template 7 linked via an acid-labile carbamate to a polystyrene support. Application of the methodology in a repetitive fashion afforded several interesting analogues out of a collection of some 200 compounds. Among the most potent of the group, N-(9-hydroxy-9-fluorenecarboxy)-prolyl trans-4-aminocyclohexylmethyl amide (L-372,460 8, Ki = 1.5 nM), in addition to being fully efficacious in a rat model of arterial thrombosis at an infusion rate of 10 micrograms/kg/min, exhibits oral bioavailability of 74% in dogs, and oral bioavailability of 39% in monkeys with a serum half-life of just under 4 h. On the basis of its favorable biological properties, inhibitor 8 has been subject to further evaluation as a possible treatment for thrombogenic disorders.

Effects of pentobarbital on pharmacokinetics and pharmacodynamics of a potent fibrinogen receptor antagonist, L-734,217, in dogs.

Effects of pentobarbital on pharmacokinetics and pharmacodynamics of L-734,217, a potent fibrinogen receptor antagonist, were studied in male dogs. L-734,217 was given intravenously at 0.01 mg kg-1, in a cross-over fashion, to conscious dogs or to dogs anesthetized with pentobarbital. Plasma concentrations of L-734,217 were measured using a radioimmunoassay and inhibitory effects on ex vivo platelet aggregation induced by ADP or collagen were determined. In pentobarbital-treated dogs, L-734,217 plasma concentrations during the first 3 h collection period were significantly higher than those in the control animals. Corresponding to the increased plasma levels, the mean ex vivo inhibitory effects on ADP- or collagen-induced platelet aggregation in dogs under anesthesia appeared greater than in those without the anesthetic treatment. Pharmacokinetic analysis revealed a modest, but significant (up to 40%) elevation in the area under the plasma concentration-time curve during 6 h of the drug administration, and a reduction in L-734,217 plasma clearance and volumes of distribution, in the anesthetized dogs. Analysis of pharmacodynamic data indicated that the EC50 and the Hill coefficient of the platelet aggregation response-plasma concentration curve were not altered by pentobarbital treatment. The results are in agreement with the findings that the administration of pentobarbital alone (in the absence of L-734,217) did not affect appreciably the ex vivo platelet aggregatory responses. In a separate group of dogs, L-734,217 was found to be metabolically stable, and was eliminated unchanged renally (64 +/- 4%) and hepatically (32 +/- 6%). In addition, L-734,217 did not bind substantially to canine plasma proteins or blood cellular components. It is possible that alterations of regional hemodynamics, reportedly mediated by pentobarbital, contributed to changes observed in the present study. That is, alterations occurred in L-734,217 elimination and distribution processes which resulted in an increase in drug plasma levels. Since pentobarbital anesthesia influenced only the pharmacokinetics, and not the pharmacodynamics, of L-734,217, the apparent increases in the inhibition of platelet aggregation responses observed following L-734,217 administration to the anesthetized dogs were probably sequential effects of the pharmacokinetic interactions.

Response-reinforcement learning is dependent on N-methyl-D-aspartate receptor activation in the nucleus accumbens core.

The nucleus accumbens, a site within the ventral striatum, is best known for its prominent role in mediating the reinforcing effects of drugs of abuse such as cocaine, alcohol, and nicotine. Indeed, it is generally believed that this structure subserves motivated behaviors, such as feeding, drinking, sexual behavior, and exploratory locomotion, which are elicited by natural rewards or incentive stimuli. A basic rule of positive reinforcement is that motor responses will increase in magnitude and vigor if followed by a rewarding event. It is likely, therefore, that the nucleus accumbens may serve as a substrate for reinforcement learning. However, there is surprisingly little information concerning the neural mechanisms by which appetitive responses are learned. In the present study, we report that treatment of the nucleus accumbens core with the selective competitive N-methyl-D-aspartate (NMDA) antagonist 2-amino-5-phosphonopentanoic acid (AP-5; 5 nmol/0.5 microl bilaterally) impairs response-reinforcement learning in the acquisition of a simple lever-press task to obtain food. Once the rats learned the task, AP-5 had no effect, demonstrating the requirement of NMDA receptor-dependent plasticity in the early stages of learning. Infusion of AP-5 into the accumbens shell produced a much smaller impairment of learning. Additional experiments showed that AP-5 core-treated rats had normal feeding and locomotor responses and were capable of acquiring stimulus-reward associations. We hypothesize that stimulation of NMDA receptors within the accumbens core is a key process through which motor responses become established in response to reinforcing stimuli. Further, this mechanism, may also play a critical role in the motivational and addictive properties of drugs of abuse.

Nonpeptide glycoprotein IIb/IIIa inhibitors: 14: oral antithrombotic efficacy of L-738,167 in a conscious canine model of coronary artery electrolytic injury.

BACKGROUND: A conscious dog model of left circumflex coronary artery electrolytic injury was used to assess the oral antithrombotic efficacy of L-738,167, a potent nonpeptide antagonist of platelet GP IIb/IIIa. L-738,167 was administered either as a single oral pretreatment dose 2 hours before initiation of vessel injury or as two oral doses administered 24 hours apart, 12 hours before and after initiation of vessel injury. METHODS AND RESULTS: In untreated controls, electrolytic coronary injury (50 microA, 3 hours) resulted in thrombotic occlusion and myocardial ischemia in 15 of 16 dogs, with 4 developing lethal arrhythmias. Significant reductions in thrombus mass and complete prevention of myocardial ischemia and infarction were achieved with a single 100- to 300-microg/kg dose of L-738,167 pretreatment and with two 100-microg/kg doses administered 12 hours before and after initiation of vessel injury. Delays and/or reductions in incidence of ischemia, thrombus mass, and infarct sizes also were achieved with 10- to 30-microg/kg pretreatment and with two 30-microg/kg doses administered 12 hours before and after initiation of vessel injury. None of the L-738,167-treated animals developed lethal arrhythmias. A single oral 100-microg/kg dose of L-738,167 achieved >90% inhibitions of ADP (extent)- and collagen (rate)-induced ex vivo platelet aggregation and fivefold to sixfold or greater elevations in bleeding time; a single oral 30-microg/kg dose of L-738,167 achieved sustained 40% to 70% inhibitions of ADP- and collagen-induced ex vivo platelet aggregation and modest twofold to threefold elevations in bleeding time. At 12 to 24 hours after single oral 30- and 100-microg/kg doses of L-738,167, a substantially greater L-738,167 concentration was associated with platelets than free in plasma. CONCLUSIONS: These findings are indicative of potent and sustained oral antithrombotic efficacy and suggest that L-738,167 possesses potential for the oral management of chronic thrombotic occlusive disorders.

Non-peptide glycoprotein IIb/IIIa inhibitors. 17. Design and synthesis of orally active, long-acting non-peptide fibrinogen receptor antagonists.

The synthesis and pharmacological evaluation of 5 (L-738, 167), a potent, selective non-peptide fibrinogen receptor antagonist is reported. Compound 5 inhibited the aggregation of human gel-filtered platelets with an IC50 value of 8 nM and was found to be > 33000-fold less effective at inhibiting the attachment of human endothelial cells to fibrinogen, fibronectin, and vitronectin than it was at inhibiting platelet aggregation. Ex vivo platelet aggregation was inhibited by > 85% 24 h after the oral administration of 5 to dogs at 100 micrograms/kg. The extended pharmacodynamic profile exhibited by 5 appears to be a consequence of its high-affinity binding to GPIIb/IIIa on circulating platelets and suggests that 5 is suitable for once-a-day dosing.

Enhanced reward-related responding following cholera toxin infusion into the nucleus accumbens.

In recent years, considerable focus has been directed to understanding how drugs of abuse affect neuronal function at the molecular level. For example, repeated administration of stimulants or opiates can induce long-lasting alterations in gene expression, transcription factors, and signal transduction pathways. Our laboratory previously showed that intraaccumbens infusion of cholera toxin (CTX), which alters the Gs protein such that production of cyclic Adenosine Monophosphate (AMP) is upregulated, causes pronounced, long-lasting motor activation and sensitization to stimulants. In the present experiments, the effect of intraaccumbens infusion of cholera toxin on reward-related responding was investigated. The conditioned reinforcement (CR) paradigm was employed, which measures an animal's instrumental response to obtain presentation of a stimulus previously paired with a primary reward. When this stimulus supports acquisition of a new operant response (lever-pressing), it is termed a conditioned reinforcer (CR). In the first experiment, the effects of bilateral intraaccumbens infusion of CTX (100 ng/1 microliter) were examined on previously-established responding. CTX treatment resulted in enhanced responding for the CR. This enhancement developed over several days and reached its peak 3 days following infusion. In the second experiment, the influence of CTX was examined on acquisition of responding for the CR. The group treated with CTX (100 ng) discriminated between the CR and control (NCR) lever earlier than the vehicle-infused group, and showed greater levels of responding on the CR lever. In the third experiment, it was determined that infusion of CTX (300 ng bilaterally) into the anterior dorsal striatum did not affect levels of responding, although a later test with cocaine in these animals (25 mg/kg, intraperitoneally) (i.p.) indicated that they were capable of potentiated responding. These data are interpreted as evidence that the G(S) protein-cyclic AMP second messenger system within the nucleus accumbens is directly involved in reward-related behavior.

Nonpeptide glycoprotein IIb/IIIa inhibitors. 15. Antithrombotic efficacy of L-738,167, a long-acting GPIIb/IIIa antagonist, correlates with inhibition of adenosine diphosphate-induced platelet aggregation but not with bleeding time prolongation.

The nonpeptide platelet glycoprotein IIb/IIIa antagonist, L-738, 167, was characterized in dog and nonhuman primate. In an anesthetized canine model of coronary artery electrolytic lesion, L-738,167 elicited dose-dependent (3, 4, 4.5 and 5 micrograms/kg i.v.) decreases in incidence of occlusion, reductions in thrombus mass and elevations in bleeding time. Antithrombotic efficacy correlated with inhibition of adenosine diphosphate-induced platelet aggregation but was dissociated from marked bleeding time elevation. Similarly, suppression of platelet-dependent cyclic flow reductions with L-738,167 in the canine coronary artery (5 micrograms/kg i.v.) and African green monkey carotid artery (10 micrograms/kg i.v.) correlated with inhibition of adenosine diphosphate-induced platelet aggregation but not with inhibition of thrombin-induced platelet aggregation or significant prolongation of bleeding time. In conscious dogs and sedated chimpanzees, single dose intravenous bolus (5-20 micrograms/kg) and oral (25-200 micrograms/kg) administration of L-738,167 exhibited long duration (> or = 8 hr) inhibition of ex vivo platelet aggregation. Once daily oral administration to conscious dogs (10-30 micrograms/kg/day for 15 days) and rhesus monkeys (200-250 micrograms/kg/day for 11 days) maintained significant but submaximal (50-90% inhibition) trough levels of inhibition of adenosine diphosphate-induced ex vivo platelet aggregation. Platelet sensitivity to adenosine diphosphate after multiple days of oral dosing in dogs was similar to pretreatment sensitivity. L-738,167 showed characteristics suitable for chronic oral therapy with a glycoprotein IIb/IIIa inhibitor.

Microinfusion of corticotropin-releasing factor into the nucleus accumbens shell results in increased behavioral arousal and oral motor activity.

Corticotropin-releasing factor (CRF) is a 41 amino acid peptide postulated to be involved in integrating the physiological and behavioral responses to stress. The purpose of this experiment was to determine the effects of CRF microinfused into the nucleus accumbens core (AcbC) and shell (AcbSh) subregions. Rats were tested for general motor activity, cage crossings, and rearing following CRF (0, 125, 250, or 500 ng). Behavioral observations were also made to determine the profile of activity caused by CRF infusion into the Acb. CRF in the AcbSh but not the AcbC regions elicited an increase in general motor activity that lasted approximately 2 h. When compared with ventricular injections, CRF in the AcbSh had greater activating effects. The CRF-induced behavioral profile consisted of increases in grooming, sniffing, and oral behavior. Results are discussed as they pertain to the involvement of the AcbSh in stress, motivated behavior, and drug sensitization.