Improved models for pharmacological null experiments: calculation of drug efficacy at recombinant D1A dopamine receptors stably expressed in clonal cell lines.

Modern drug discovery demands accurate knowledge of the drug properties of affinity and efficacy at specific receptor proteins. Furthermore, drugs with well defined properties make better tools with which to explore and understand receptor regulation. The use of clonal cell lines stably expressing a given recombinant receptor may provide a highly useful model in which drug effects may be studied on one receptor subtype at a time. The present report was designed to evaluate the utility of a general method in which a clonal cell line stably expressing a recombinant D1A dopamine receptor was used as a model system for studying drug actions by null models. The null model for receptor occlusion (to calculate agonist Ka) and the null model for relative efficacy (to calculate test agonist affinity and epsilon r) were evaluated in these studies. To initiate these studies, rat C6 glioma cells that do not normally express DA receptors have been modified by stable transfection with the primate D1A DA receptor [Machida et al., 1992 (Molec. Pharmacol. 41: 652-659)] to a density of approximately equal to fmol/mg protein. The recombinant receptors show robust stimulation of cAMP in the stably transfected C6 cells. Calculation of agonist Ka from dose-response data requires that a portion of the cell's receptors be occluded in the absence of changes in post-receptor events leading to the response. Receptor reserve is typically reduced by alkylation, thereby lowering maximal response. Unfortunately, most of the currently available alkylating agents are not selective either for a particular receptor or for receptors vs other proteins within a signaling pathway. Short-term agonist treatment offers a possible complement to the use of non-selective or poorly characterized alkylating drugs for reducing maximum response in appropriate cell systems. The null method of receptor occlusion was used to determine the Ka for dopamine when maximum response was decreased by alkylation vs short-term agonist treatment. Direct non-linear curve fitting was used to analyze the data. In addition to DA, two other compounds were used to reduce receptor reserve to validate the method: fenoldopam (relatively high efficacy) and SKF38393 (low efficacy). Analyses indicated that the affinity of DA was similar whether calculated by alkylation (1.1 +/- 0.58 microM), 75 min DA treatment (0.57 +/- 0.16 microM) or 45 min treatment with DA (0.86 +/- 0.11 microM). Short-term agonist treatment experiments using multiple concentrations of DA, fenoldopam, or SKF38393 to decrease receptor reserve provided additional support for the validity of the Ka determinations using this procedure. Other experiments were conducted according to the null model for relative efficacy in which the affinity for DA is calculated by comparing the DA response before and after receptor occlusion, and the affinity and relative intrinsic efficacy of the test agonist are determined as a function of its actions relative to DA. We used the following four test drugs: + Br-APB, a novel agent with potential dopamine agonist properties, and three high-affinity DA agonists, fenoldopam, R-(-)-apomorphine (APO), and SKF38393. Intrinsic efficacy values relative to that of DA (1.0) were as follows: fenoldopam, 0.46 +/- 0.11; APO, 0.19 +/- 0.13; SKF38393, 0.07 +/- 0.01; and +Br-APB, 0.26 +/- 0.40. The agonist affinities (Ka) were: fenoldopam, 0.018 +/- 0.008 microM; APO, 0.80 +/- 0.18 microM; SKF38393, 0.16 +/- 0.04 microM; BR-APB, 0.43 +/- 0.29 microM; and DA, 0.58 +/- 0.17 microM. EC50/Ka ratios were consistent with relative intrinsic efficacies and Ka values were similar to KL values reported for membrane binding studies. Finally, Monte Carlo simulations were conducted to determine the precision of the parameter estimates...

Comparative molecular field analysis-based prediction of drug affinities at recombinant D1A dopamine receptors.

Determination of quantitative structure-activity relationship (QSAR) for affinity at particular dopamine (DA) receptors has become an even greater priority with the cloning of five DA receptor subtypes. The use of agonist affinity at recombinant receptors selectively expressed in clonal cells as the dependent variable in QSAR presents a unique opportunity for accuracy and precision in measurement of biological values. Bound conformations of 11 agonists (for which both affinity data at the recombinant D1A DA receptor and stereochemical configurations were available) were determined by alignment with a template compound, SKF38393, which shows high affinity and selectivity for D1A receptors and is fairly rigid in structure. These aligned structures suggested a 3-point pharmacophore map (one cationic nitrogen and two electronegative centers) of the D1A DA receptor. This map shows both similarities and differences when compared with a previously reported D2 DA receptor pharmacophore map based on biological data from rat brain and with a recently published map of the native D1 DA receptor using several semirigid compounds. Log(1/K(d)) values at recombinant D1A DA receptors were used as the target property for a CoMFA (comparative molecular field analysis) of the 11 aligned structures. The resulting CoMFA model yielded a cross-validated r(2)(q(2)) value of 0.829 and a simple r(2) = 0.96. In contrast, when a CoMFA model was developed for 10 of these compounds using striatal D1 K(d) values, the q(2) value was reduced to 0.178. These results are consistent with the idea that drug affinity data obtained from clonal cells expressing recombinant receptors may be superior to that obtained using heterogeneous mixtures of native receptors prepared from brain membranes. The predictive utility of the CoMFA model was evaluated using several high-affinity dopamine agonists and m- and p-tyramine, two compounds with a single hydroxyl group on the aromatic ring. Predictions were fairly accurate for all compounds but the two tyramines.

Interactions of apomorphine with serum and tissue proteins.

Physical and covalent interactions of apomorphine with serum and tissue proteins could influence the drug's disposition and pharmacological activities in mammals. Ultrafiltration, equilibrium dialysis, and ultraviolet spectrophotometric methods have been used to study the reversible binding of apomorphine to bovine, human, rat, and swine plasma proteins. The degree of binding was generally greater than 90%, but variations were noted in some instances on the basis of drug concentrations and pH over the range of 6.8-7.8. Incubation of [8,9-3H2]apomorphine with bovine serum albumin led to retention of radioactivity and a stoichiometrically controlled released of tritium which arose from the reaction of an electrophilic drug oxidation product and protein, producing drug-protein conjugates. In vitro experiments with mouse striatal brain preparations indicated parallel covalent binding reactions. In vivo experiments in mice indicated accumulation of radioactivity in brain regions and other tissues following daily injections of [8,9-3H2]apomorphine for 14 days. The physical and covalent interactions of apomorphine with mammalian tissue proteins could be the cause of longer disposition half-lives in mammals than those previously reported. The covalent interactions, in particular, may be important in elucidating the mechanism of apomorphine-induced behavioral effects in mice.

CoMFA-based prediction of agonist affinities at recombinant D1 vs D2 dopamine receptors.

We have previously shown that using agonist affinity at recombinant receptors selectively expressed in clonal cells as the dependent variable in three-dimensional quantitative structure-activity relationship studies (3D-QSAR) presents a unique opportunity for accuracy and precision in measurement. Thus, a comparison of affinity's structural determinants for a set of compounds at two different recombinant dopamine receptors represents an attainable goal for 3D-QSAR. A molecular database of bound conformations of 16 structurally diverse agonists was established by alignment with a high-affinity template compound for the D1 receptor, 3-allyl-6-bromo-7,8-dihydroxy-1-phenyl-2,3,4, 5-tetrahydro-1H-benzazepin. A second molecular database of the bound conformations of the same compounds was established against a second template for the D2 receptor, bromocriptine. These aligned structures suggested three-point pharmacophore maps (one cationic nitrogen and two electronegative centers) for the two dopamine receptors, which differed primarily in the height of the nitrogen above the plane of the catechol ring and in the nature of the hydrogen-bonding region. The ln(1/KL) values for the low-affinity agonist binding conformation at recombinant D1 and D2 dopamine receptors stably expressed in C6 glioma cells were used as the target property for the CoMFA (comparative molecular field analysis) of the 16 aligned structures. The resulting CoMFA models yielded cross-validated R2 (q2) values (standard error of prediction) of 0. 879 (1.471, with five principal components) and 0.834 (1.652, with five principal components) for D1 and D2 affinity, respectively. The simple R2 values (standard error of the estimate) were 0.994 (0.323) and 0.999 (0.116), respectively, for D1 and D2 receptor. F values were 341 and 2465 for D1 and D2 models, respectively, with 5 and 10 df. The predictive utility of the CoMFA model was evaluated at both receptors using the dopamine agonists, apomorphine and 7-OH-DPAT. Predictions of KL were accurate at both receptors. Flexible 3D searches of several chemical databases (NCI, MDDR, CMC, ACD, and Maybridge) were done using basic pharmacophore models at each receptor to determine the similarity of hit lists between the two models. The D1 and D2 models yielded different lists of lead compounds. Several of the lead compounds closely resembled high-affinity training set compounds. Finally, homology modeling of agonist binding to the D2 receptor revealed some consistencies and inconsistencies with the CoMFA-derived D2 model and provided a possible rationale for features of the D2 CoMFA contour map. Together these results suggest that CoMFA-homology based models may provide useful insights concerning differential agonist-receptor interactions at related receptors. The results also suggest that comparisons of CoMFA models for two structurally related receptors may be a fruitful approach for differential QSAR.

CoMFA-based prediction of agonist affinities at recombinant wild type versus serine to alanine point mutated D2 dopamine receptors.

Agonist affinity changes dramatically as a result of serine to alanine mutations (S193A, S194A, and S197A) within the fifth transmembrane region of D2 dopamine receptors and other receptors for monoamine neurotransmitters. However, agonist 2D-structure does not predict which drugs will be sensitive to which point mutations. Modeling drug-receptor interactions at the 3D level offers considerably more promise in this regard. In particular, a comparison of the same test set of agonists across receptors differing minimally (point mutations) offers promise to enhance the understanding of the structural bases for drug-receptor interactions. We have previously shown that comparative molecular field analysis (CoMFA) can be applied to comparisons of affinity at recombinant D1 and D2 dopamine receptors for the same set of agonists, a differential QSAR. Here, we predicted agonist K(L) for the same set of agonists at wild type D2 vs S193A, S194A, and S197A receptors using CoMFA. Each model used bromocriptine as the template. ln(1/K(L)) values for the low-affinity agonist binding conformation at recombinant wild type and mutant D2 dopamine receptors stably expressed in C6 glioma cells were used as the target property for the CoMFA of the 16 aligned agonist structures. The resulting CoMFA models yielded cross-validated R(2) (q(2)) values ranging from 0.835 to 0.864 and simple R(2) values ranging from 0.999 to 1.000. Predictions of test compound affinities at WT and each mutant receptor were close to measured affinity values. This finding confirmed the predictive ability of the models and their differences from one another. The results strongly support the idea that CoMFA models of the same training set of compounds applied to WT vs mutant receptors can accurately predict differences in drug affinity at each. Furthermore, in a "proof of principle", two different templates were used to derive the CoMFA model for the WT and S193A mutant receptors. Pergolide was chosen as an alternate template because it showed a significant increase in affinity as a result of the S193A mutation. In this instance both the bromocriptine- and pergolide-based CoMFA models were similar to one another but different from those for the WT receptor using bromocriptine- or pergolide- as templates. The pergolide-based S193A model was more strikingly different from that of the WT receptor than was the bromocriptine-based S193A model. This suggests that a "dual-template" approach to differential CoMFA may have special value in elucidating key differences across related receptor types and in determining important elements of the drug-receptor interaction.

Apomorphine doses impair the reaction time of fast reacting but not slow reacting rats.

Previously, several studies in our laboratories have linked the ability to initiate movement rapidly (reactive capacity) in several rat strains to markers of nigrostriatal dopamine function. In the present investigation we wished to determine the extent to which fast-reacting (FR) and slow-reacting (SR) rats responded differentially to the effects of dopamine agonist (apomorphine) administration. Fisher 344 rats were operantly conditioned on a shock-avoidance, reactive capacity task which requires extremely fast, ballistic, lever release responses. In FR, but not in SR rats, significant dose-dependent decreases in the per cent of successful avoidance trials were observed in response to apomorphine doses. Moreover, apomorphine brought responding in the FR animals to levels below that of the SR rats. Performance of the SR rats was unaffected by drug treatments. A number of interpretations are consistent with these preliminary data, including the idea that basal differences in speed of reaction and differential responses to a dopamine agonist, antiparkinsonian agent may both be associated with a similar mechanism: differential activation of compartmentalized striatal efferent systems.

Effects of multiple pretreatment with apomorphine and amphetamine on amphetamine-induced locomotor activity and its inhibition by apomorphine.

Mice were given a saline preinjection and habituation to the testing environment followed by injection of amphetamine (0.675-5.0 mg/kg IP) and apomorphine (AP, 15-80 micrograms/kg SC) 15 min later. AP produced a dose-dependent inhibition of the amphetamine-induced locomotor activity. A dose of 40 micrograms/kg AP increased approximately threefold the amphetamine dose required to induce the same increase in activity. Repeated administration of AP (30 mg/kg IP once daily for 14 days) resulted in an enhanced response (in the early portion of the time response) to amphetamine challenge, while the ability of subsequent microgram challenge doses of AP to reduce the response were unaffected. Similarly, repeated administration (twice-daily IP injections for 5 days) of amphetamine (5.0 mg/kg) resulted in an enhanced locomotor response to amphetamine challenge and no change in the ability of AP to inhibit the response. These results suggest that repeated administrations of dopamine agonists, although acting through different mechanisms (i.e., indirect versus direct), increase the initial release of neurotransmitter. However, the repeated administration of these agonists does not attenuate the ability of AP to inhibit the release of the neurotransmitter induced by amphetamine. The regulatory functions (i.e., presynaptic receptor control) of release appears to remain intact, but the level of neuronal activity has been increased.

Speed of movement initiation performance predicts differences in [3H]spiroperidol receptor binding in normal rats.

Speed of movement initiation is altered in normal aging and in neurological disorders such as Parkinson's disease. This study was undertaken to extend our previous results, which suggested a relationship between nigrostriatal dopamine function and an animal model of movement initiation speed (reactive capacity). Fisher 344 rats exhibiting exceptionally fast or slow reactive capacity but otherwise normal were examined for differences in the striatal binding of the dopaminergic ligand, [3H]spiroperidol. Rats with fast reactive capacity (Fast rats) exhibited significantly higher binding than did rats with slow reactive capacity (Slow rats). Also, Fast rats responded nearly maximally on the reactive capacity task regardless of the duration of time provided in which to respond, whereas Slow rats reacted more slowly when more response time was provided. The differences in [3H]spiroperidol binding and the differential influence of time provided to respond on the response latency of these two normal groups of rats was similar to that observed in old or model Parkinson's disease rats having nigrostriatal dopamine deficits. These results strengthen the relationship between an animal model of reactive capacity and nigrostriatal dopamine function.

Hypothermic effects of N-n-propylnorapomorphine in mice: antagonism by neurotransmitter receptor blockers.

Six receptor blockers were compared in mice for their ability to alter hypothermia induced by the dopamine agonist N-n-propylnorapomorphine (NPA). The dopamine antagonist haloperidol inhibited NPA-induced hypothermia whereas phentolamine, propranolol, sotalol, atropine, cyproheptadine, or naloxone (alpha-adrenergic, beta-adrenergic, muscarinic cholinergic, serotonergic, and opiate antagonists, respectively) failed to inhibit it. The degree of antagonism of the hypothermic effect of NPA induced by haloperidol pretreatment suggests that the hypothermic response may serve as a useful model of specific dopaminergic activity of aporphines. Hypothermia induced by NPA and other aporphines may supplement the measure of aporphine-induced stereotypic cage-climbing previously reported to be specifically dopaminergic.

Endurance training effects on striatal D2 dopamine receptor binding and striatal dopamine metabolites in presenescent older rats.

Endurance training is associated with higher binding of 3H-spiperone to striatal D2 dopamine receptors of rats sacrificed 48 h following the last exercise bout (Gilliam et al. 1984). In the present study we investigated the effects of endurance training in presenescent older rats on the relationship between steady-state levels of DA and its metabolites in striatum versus the affinity and density of striatal D2 DA receptors. Citrate synthase activity of the gastrocnemius-plantaris muscle was 29.06 +/- 2.27 mumole/g wet wt in 21-month-old trained rats versus 22.88 +/- 1.13 mumole/g wet wt in 21-month-old untrained animals. DOPAC levels and DOPAC/DA ratios were greater in the old controls. Endurance training was associated with lower DOPAC levels in the 21-month-old animals. Thus, endurance training may postpone selectively changes in DA metabolism over a portion of the lifespan. As expected, the number of D2 DA binding sites was reduced with age (6 months Bmax:429 +/- 21 fmoles/mg protein; 21 months:355 +/- 20) with no change in affinity. The Bmax of old runners was significantly higher (457 +/- 38 fmoles/mg protein) than that of old controls. Thus, endurance training appears to exert a protective effect on D2 dopamine receptors during the lifespan. Taken together, the present results suggest that there may be a possible reciprocal relationship between changes in DA metabolites and DA binding as a function of exercise in presenescent older rats, and that endurance training may decelerate the effects of age both on nigrostriatal dopamine neurons and on striatal D2 dopamine receptors during a portion of the lifespan.

Behavioral facilitation following chronic administration of N-n-propylnorapomorphine.

Chronic administration of drugs which interfere with normal neurotransmission within animal nervous tissue (e.g. neurotransmitter receptor antagonists) is known to result in the development of behavioral supersensitivity. During recent years, evidence has been presented which indicates that neurotransmitter receptor agonists also produce behavioral supersensitivity. This study shows that, using stereotypic cage-climbing behavior in mice, chronic administration of apomorphine, and N-n-propylnorapomorphine (two direct-acting dopamine agonists) and d-amphetamine (an indirect dopamine agonist) produced an enhanced behavioral response to a test dose of apomorphine 4, 8 and 12 days after cessation of chronic drug injections.

Dopamine receptor sensitivity after chronic dopamine agonists. Striatal 3H-spiroperidol binding in mice after chronic administration of high doses of apomorphine, N-n-propylnorapomorphine and dextroamphetamine.

Several previous reports have demonstrated that chronic administration of both directly and indirectly acting dopamine agonists produces a supersensitive behavioral response to challenge doses of dopamine agonists when compared to the responses induced by acute administration of these drugs. That is, a given dose of a dopamine agonist will produce a greater response after chronic dopamine agonist treatment than is observed upon acute administration of that dose. A similar behavioral phenomenon resulting from chronic administration of dopamine antagonists has been suggested to be due to an increase in the number of dopamine receptors present in relevant brain areas. The same hypothesis has been put forward for the hypersensitivity induced by chronic dopamine agonist administration. The present study was designed to investigate the effect of chronic administration of high doses of both direct and indirect dopamine agonists on the dopamine receptors labeled by 3H-spiroperidol. Groups of animals (CD-1 mice) were sacrificed 1, 3 and 5 days following the last chronic injection. Striatal tissue from these mice was incubated with 3H-spiroperidol and dopamine receptor binding evaluated. Affinity of the receptors for the ligand was unaltered by treatments. The receptors labeled by 3H-spiroperidol showed no significant differences in number following the chronic administration of high doses of apomorphine (30 mg/kg). The Bmax was significantly decreased at only one time period following chronic administration of dextroamphetamine (4 mg/kg); however, there was a dramatic 30% reduction in the Bmax in striatal tissue from those mice treated with N-n-propylnorapomorphine. These results suggest that the hypersensitive behavioral response in mice following chronic administration of direct and indirect acting dopamine agonists is not due to an increase in the number of dopamine receptors in the striatum which are labeled by 3H-spiroperidol.

Effects of apomorphine and piribedil on pentylenetetrazol-induced seizures in mice.

Based on previous work examining the effects of dextroamphetamine on pentylenetetrazol (PTZ)-induced clonic seizure threshold, the objective of the present study was to determine the effects of two other dopamine agonists, apomorphine (AP) and piribedil, on PTZ seizures. TD50 and LD50 values for CD-1 mice were determined initially for the two drugs. Subsequently, dose- and time-response analyses established that AP decreased PTZ seizure threshold 15 min after administraton, but increased the threshold at 60 min. Piribedil elevated the seizure threshold, but like AP, did not exhibit a clear dose-response relationship. Subsequent blocker studies with phentolamine, (-)sotalol, pimozide, and atropine suggested the possible neurotransmitter systems involved in the modulation of the PTZ-induced seizures by AP and piribedil. Pimozide blocked the changes in seizure threshold induced by both drugs. Atropine also decreased the AP-induced increase in threshold at 60 min. The pattern of inhibition of seizure threshold changes induced by the neurotransmitter blockers suggested that piribedil blocked seizures by means of indirect actions on several neurotransmitters.

Effects of ascorbate on a dopaminergic response: apomorphine-induced modification of pentylenetetrazol-induced seizures in mice.

The threshold for pentylenetetrazol (PTZ)-induced clonic seizures is below control levels 15 min after administration of apomorphine (APO) but above them 60 min after APO administration. Pretreatment with ascorbic acid (10 mg/kg) or the presence of ascorbic acid in the APO solution (1 mg/ml) inhibited the early (15 min) decrease in seizure threshold caused by 60 mg/kg APO and reversed the increase in seizure threshold 60 min after a 50 mg/kg APO challenge. Ascorbate co-administration concomitant with APO also counteracted the increase in seizure threshold 60 min after 70/kg APO. Results suggest a potential model for modulation of a seizure system through dietary supplementation.

Startle and sensorimotor correlates of ventral thalamic dopamine and GABA in rodents.

Recent studies have implicated the thalamus as a possible site for neuroanatomical and neurochemical changes in schizophrenia. In the present study, we investigated thalamic neurochemical correlates of behaviors potentially linked to schizophrenia. Whole thalamic DOPAC levels were elevated in rats that had poor extinction of the acoustic startle response. The dopamine agonist apomorphine microinjected into the ventromedial thalamus (VmT) disrupted prepulse inhibition of startle. Catalepsy was induced by VmT microinjections of the GABA-A agonist muscimol. A previous study revealed attentional disturbances and suppression of frontal cortical metabolic activity after muscimol microinjections into the mediodorsal thalamic nucleus. Together with recent findings of neuron cell loss and elevated DA levels in the thalamus of schizophrenics, these data suggest the involvement of disturbances of thalamic neurotransmission in schizophrenia.

Interaction of aerobic exercise training and clenbuterol: effects on insulin-resistant muscle.

The effects of aerobic exercise training, chronic administration of the selective beta 2-adrenergic agonist clenbuterol, and the combination of these two treatments on muscle insulin resistance were compared in female obese (fa/fa) Zucker rats. Rats were randomly assigned to trained, clenbuterol, clenbuterol-trained, or control groups. Training consisted of treadmill running for 2 h/day at 18 m/min up an 8% grade. Clenbuterol was administered by intubation (0.4-0.8 mg.kg body wt-1 x day-1) approximately 30 min before the rats ran each day. After 8 wk of treatment, muscle insulin resistance was assessed via hindlimb perfusion in the presence of 8 mM glucose and a submaximal (500 microU/ml) insulin concentration. Training increased citrate synthase activity (mumol.g wet wt-1 x min-1) by 32-74% and insulin-stimulated glucose uptake by 45%. Clenbuterol ingestion induced a 17-29% increase in muscle mass but decreased citrate synthase activity by 34-42% and had no effect on muscle glucose uptake. Administration of clenbuterol to rats that exercise trained prevented the training-induced improvement in insulin-stimulated glucose uptake and attenuated the increases in citrate synthase activity. In addition, both clenbuterol-treated groups displayed a 42% decrease in beta-adrenergic receptor density. The results indicate that clenbuterol administration, possibly through beta-adrenergic receptor downregulation, attenuated a cellular reaction essential for the exercise training-induced increase in citrate synthase activity and improvement in skeletal muscle insulin resistance of the obese Zucker rat.

Dose and time response analysis of apomorphine's effect on prepulse inhibition of acoustic startle.

Previous studies have suggested a dopaminergic modulation of the acoustic startle response. Using a 73 dB tone as a prestimulus to 100 dB white noise (PP trials), and 100 dB white noise alone (WN trials) during a 5-30 min or 5-90 min session, we found that IP injections of apomorphine (APO; 1.6 and 3.2 mg/kg) initially elevated PP trial means to levels statistically indistinguishable from WN trial means. These results suggest that APO can dose-dependently disrupt prepulse inhibition of the acoustic startle response. After the initial startle amplitude elevation 5-10 min post-injection in the 5-90 min protocol, startle response amplitude of the 3.2 mg/kg APO dose group decreased during successive testing periods, in contrast to the pattern of vehicle controls, which did not decrease during the session. The results of this extended time course study suggest complex time and dose effects of APO on reflex modification mechanisms. The finding that systemic APO administration produces a loss of prepulse inhibition in rodents underscores the pivotal role of DA neurotransmission in the mediation of startle behavior.

A quantitative estimate of the role of striatal D-2 receptor proliferation in dopaminergic behavioral supersensitivity: the contribution of mesolimbic dopamine to the magnitude of 6-OHDA lesion-induced agonist sensitivity in the rat.

Rats with unilateral depletions of neostriatal dopamine display increased sensitivity to dopamine agonists estimated to be 30 to 100 x in the 6-hydroxydopamine (6-OHDA) rotational model. Given that mild striatal dopamine D-2 receptor proliferation occurs (20-40%), it is difficult to explain the extent of behavioral supersensitivity by a simple increase in receptor density. This study was designed to investigate the quantitative aspects of the rotational behavior model utilizing constrained non-linear curve fitting routines. A dose-response curve for the rotational response arising from apomorphine stimulation of the normosensitive striatum was obtained in animals bearing unilateral lesions of striatal efferents (predominantly the striato-nigral pathway as previously described). After the control dose-response experiment, rats received a dopamine- (DA) depleting lesion in the contralateral hemisphere. In one group, 6-OHDA was infused into the medial forebrain bundle (MFB), a placement which is common in the literature and is known to deplete DA in both the striatum and nucleus accumbens. In a second group of rats, 6-OHDA was infused into the globus pallidus at a site which depletes caudate DA, but leaves n. accumbens DA relatively intact. The two experimental groups were tested in identical apomorphine-induced rotation dose-response experiments. The ED50's of the MFB- and caudate-lesioned rats were reduced by 36 and 5.8 fold, respectively, as compared to the control dose-response curve. The MFB and caudate lesions depleted striatal DA and produced a 30 and 36% increase in striatal D-2 binding sites, respectively. Modeling the behavioral and biochemical data with the null model for receptor occlusion indicated that increased striatal D-2 receptor density could account for the magnitude of behavioral supersensitivity in neither the MFB-lesioned group, nor even in the caudate-lesioned group. Thus simple up-regulation or D-2 receptors is unlikely to account for supersensitization as measured in the rotational model. Further, we suggest that quantitative modeling of such hypotheses is a valuable experimental technique for assessing relationships between biochemical and behavioral variables.

Modulation of G-protein linked cAMP accumulation in immortalized murine cortical astrocytes by retroviral infection.

The present study characterized beta-adrenergic receptors (beta-AR) in a clonal cell line (C1) immortalized from cerebral cortical astroglial cells of FVB/N mice. We also determined whether the wild type Moloney murine leukemia virus (wt-MoMuLV) and one of its neuropathogenic mutants, ts1-MoMuLV, modulated the beta-AR system in these cells. We observed that C1 cells possess a functional beta-AR system coupled to cAMP accumulation and capable of normal agonist-induced regulation (desensitization). Significant increases were observed in forskolin stimulated cAMP accumulation in C1 cells infected by wt MoMuLV and by ts1-MoMuLV. In contrast, the cAMP response to beta-AR stimulated by isoproterenol was relatively spared after viral exposure.

Reaction time and nigrostriatal dopamine function: the effects of age and practice.

Normal aged and Parkinsonian individuals lose the ability to initiate movements rapidly (increased reaction time) in parallel with changes in the nigrostriatal dopamine (DA) system. However, the ability of these individuals to improve their reaction time with practice has not been adequately assessed. We have developed a rodent model of human reaction time in which reaction time performance correlates highly with neurochemical measures of nigrostriatal DA integrity. In the present report, 15 young and 10 old male Sprague-Dawley rats were conditioned in a reaction time task to release a lever quickly in response to external stimuli in order to avoid a mild footshock. In order to examine the effects of practice on this reaction time task, the young animals were tested for 5 days at 3, 6 and 9 months of age and the old animals were tested for 5 days at 18, 21, and 24 months of age. From this well-practiced task, reaction time response latencies were measured and compared to measures of nigrostriatal DA function (steady-state levels of DA and its metabolites, D2DA receptor affinity and density). The old animals were slower in response latencies than the young animals. These age differences in response latencies, however, disappeared after several days of testing at each of the 3 test sessions, so that the old animals were not significantly slower than the young animals on days 4 and 5 of each session. As expected, the old animals showed reduced striatal D2DA receptor density with no age differences in DA receptor affinity.(ABSTRACT TRUNCATED AT 250 WORDS)