Separation of cocaine stereoisomers by capillary electrophoresis using sulfated cyclodextrins.

A capillary electrophoretic method for the separation of cocaine and its stereoisomers was developed. In this study, the effect of organic modifier was also investigated. The separation was achieved using 1% sulfated cyclodextrin, 10 mmol L(-1) phosphate buffer, 10% methanol at pH 3. The method provides good reproducibility and easy application.

Satiety threshold regulates maintained self-administration: comment on Lynch and Carroll (2001).

In this commentary the authors argue that the satiety threshold is the only mechanism that is sufficient and necessary to explain the regulation of maintained self-administration. The other mechanisms have been proposed mainly because of 2 sources of confusion surrounding the self-administration paradigm: the failure to distinguish between separate phases of a self-administration session and the assumption that positive reinforcement underlies drug self-administration. The authors of this commentary emphasize that the direct effects and aversion mechanisms have been proven to be untenable for the reasons reviewed by W. J. Lynch and M. E. Carroll (2001) and that the "ascending limb" of the dose-response curve is an experimental artifact. These ideas have persisted only to salvage a role for positive reinforcement in drug self-administration. The authors conclude that reinforcement is not relevant to the regulation of maintained self-administration.

Satiety threshold during maintained cocaine self-administration in outbred mice.

Male Swiss Webster mice maintained cocaine self-administration in a regular and dose-dependent manner. These characteristics made it possible to apply the satiety threshold model of drug self-administration developed recently for cocaine self-administration in rats. Non-linear regression analysis revealed that cocaine satiety threshold was 1.3 +/- 0.6 mg/kg and the functional half-life of the cocaine was 8.1 +/- 2.2 min. Whether the self-administration of cocaine was maintained by lever presses or nose pokes did not influence the inter-injection intervals. The results are consistent with the pharmacological model of maintained cocaine self-administration. The ability to determine addiction-relevant phenotypes (the satiety threshold and functional half-life of cocaine) in inbred strains of mice may help to identify the genetic determinants of cocaine self-administration behavior.

Mechanisms of cocaine hydrolysis and metabolism in vitro and in vivo: a clarification.

There is confusion in the literature concerning the mechanisms by which the cocaine hydrolysis product, benzoylecgonine (BE), is formed in vitro and in vivo. Some authors assume that all BE is formed nonenzymatically. This review summarizes evidence that both enzymatic and nonenzymatic mechanisms exist. In vitro BE is produced exclusively by hydrolysis at alkaline pH, as esterases present in blood or serum do not catalyze formation of BE. In vivo BE is formed both nonenzymatically as well as through the action of esterases found in a number of tissues including hepatocytes. The enzymatic mechanism is the predominant one operating in vivo.

Satiety threshold: a quantitative model of maintained cocaine self-administration.

The intervals between self-injections of cocaine by rats are defined by an equation that contains only three parameters: the dose of cocaine administered, the elimination half-life of cocaine, and an amount of cocaine in the body, which we have termed the cocaine satiety threshold. This latter parameter is defined as the maximal level of cocaine at which the probability of self-administration approximates one and above which the probability of self-administration is low. The mathematical model generated mean values for the satiety threshold and the functional elimination half-life of cocaine of approximately 1.7 mg/kg (i.v.) and 8.2 min, respectively. Therefore, the simple equations presented here permit the measurement of the pharmacokinetics and pharmacodynamics of cocaine using self-administration behavior as a bioassay. Our satiety model predicts that when cocaine levels are maintained above the satiety threshold, rats would not self-administer cocaine. The elimination rate of cocaine at the satiety threshold was calculated to be approximately 2 microg kg(-1) s(-1). Therefore, an infusion of cocaine at this rate should maintain cocaine levels fractionally above the satiety threshold. A continuous infusion of cocaine at this rate prevented cocaine self-administration for the duration of the infusion, thereby confirming the validity of the satiety model. These equations provide a quantitative description of cocaine self-administration and contain no subjective terms, implying that concepts such as "craving", drug "wanting" and "liking" and "reinforcement", used in psychologically oriented models, are not necessary for descriptions of this behavior in rats.

Priming threshold: a novel quantitative measure of the reinstatement of cocaine self-administration.

The intravenous injection of cocaine has been reported to reliably reinstate (prime) the self-administration of cocaine in animals. We report herein that there is a cocaine priming threshold in rats trained to self-administer cocaine. The cocaine priming threshold is defined as the minimum level of cocaine in the body that will reinstate maintained cocaine self-administration. The mean cocaine priming threshold in rats was calculated to be approximately 186 to 212 microg kg(-1). Therefore, any injection, series of injections or continuous infusion that result in a level of cocaine equivalent to that produced by a single intravenous injection of this range of doses, will reinstate cocaine self-administration. The priming threshold was significantly increased by the D(1) dopamine receptor antagonist SCH23390 (10 microg kg(-1), i.v.), indicating a role for dopaminergic neurotransmission. The priming threshold, but not the inter-injection interval of maintained self-administration, was increased following withdrawal from a 7-day infusion of D-amphetamine. In addition, there was no correlation between the cocaine priming threshold and the inter-injection intervals of maintained cocaine self-administration. Therefore, the mechanisms underlying the reinstatement of cocaine self-administration are distinct from the mechanisms underlying the maintenance of cocaine self-administration and they are differentially regulated. It is possible that the priming threshold may represent a distinct target for medications development.

Olfactory receptor neurons express D2 dopamine receptors.

The role of the dopamine (DA) in the olfactory bulb (OB) was explored by determining which of the potential target cells express dopamine receptors (DARs). Previously, it was reported that D2-like DAR (D2, D3, and D4 subtypes) radioligand binding is restricted to the outer layers of the OB. The neuronal elements present only in these layers are the axons of the olfactory receptor neurons (ORNs) and the juxtaglomerular (JG) neurons of the glomerular layer. Based on this pattern of D2-like ligand binding, it was suggested that D2-like receptors might be located presynaptically on ORN terminals. The present study was undertaken to investigate this hypothesis. In the outer bulb layers of rats in which the ORNs were destroyed by nasal lavage with ZnSO(4), D2-like radioligand binding was reduced severely. The receptor subtype D2 mRNA, but not D3 mRNA, was detected in adult rat olfactory epithelial tissue. By using in situ hybridization, this D2 mRNA was located preferentially in epithelial layers that contain ORN perikarya. D2 mRNA was eliminated after bulbectomy, a manipulation known to cause retrograde degeneration of the mature ORNs. Taken together, the surgical manipulations indicate that mature ORNs express D2 DARs and are consistent with the hypothesis that functional receptors are translocated to their axons and terminals in the bulb. This suggests that dopamine released from JG interneurons could be capable of presynaptically influencing neurotransmission from the olfactory nerve terminals to OB target cells through the D2 receptor.

Low CSF concentration of a dopamine metabolite in tobacco smokers.

OBJECTIVE: To screen for dopaminergic abnormalities in tobacco smokers and patients with posttraumatic stress disorder (PTSD), the authors determined serial CSF and plasma concentrations of the dopamine metabolite homovanillic acid (HVA). METHOD: Continuous subarachnoid sampling was used to obtain 37 serial CSF samples over 6 hours in 13 normal volunteers and 11 patients with combat-related PTSD; 10 smoked and 14 had never smoked. The smokers were abstinent from tobacco for 1 1 to 17 hours. RESULTS: The smokers had markedly lower CSF, but not plasma, HVA levels. Their CSF HVA concentrations averaged only 54% of the concentrations of the nonsmokers, independent of diagnosis. CONCLUSIONS: Smokers' low CSF concentrations of HVA may be associated either with chronic inhalation of nicotine or other constituents of tobacco smoke or with acute abstinence. Any possible basal dopaminergic abnormalities in patients with PTSD are small relative to the abnormalities associated with smoking.

Transient amelioration of the sensitization of cocaine-induced behaviors in rats by the induction of tolerance.

Intermittent administration of cocaine produced a progressive increase in the stereotypy response of rats to a challenge dose of cocaine (7.5 mg/kg, i.p.). Continuous infusion of cocaine (80 mg/kg per day) via osmotic pumps for 7 days into the sensitized rats produced tolerance to the behavioral responses to the challenge dose of cocaine 1 day after the removal of the pumps. Therefore, tolerance can mask the expression of behavioral sensitization in rats. However, by 10 days after the removal of the pumps, the behavioral tolerance was reversed and the rats again displayed a sensitized response to cocaine. Therefore, the tolerance to cocaine was temporary while the underlying sensitization persisted. The development of tolerance did not alter the underlying sensitization demonstrating that these represent independent phenomena. The relationship between sensitization and tolerance observed in these studies may provide a model relevant to the progress in humans of addiction to psychomotor stimulants.

D1-dopamine receptor binding and tyrosine hydroxylase-immunoreactivity in the fetal and neonatal hamster suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) of the anterior hypothalamus is the site of an endogenous biological clock that regulates mammalian circadian rhythms. Circadian rhythms, although endogenously driven, are synchronized or entrained to daily environmental cues. Developmentally, the SCN begins to oscillate before birth and is entrained to the maternal circadian rhythm by a mechanism that is still unclear. Recent evidence in rats and hamsters suggests that a fetal dopaminergic system and D1-dopamine receptors may be involved in the process of entraining the fetal clock. The present study using [3H]SCH 23390 autoradiography and tyrosine hydroxylase (TH) immunocytochemistry determined the developmental time courses of the appearance of D1 receptor in, and catecholaminergic input to, the hamster SCN. [3H]SCH 23390 binding to D1-dopamine receptors was first detected in the fetal SCN on embryonic day (E) 15, the day before birth in this species, and persisted through adulthood. The TH immunoreactive fibers were first observed on day E15 coursing just ventral to the fetal SCN but TH-immunoreactive cells and fibers were not seen within the SCN until postnatal day (P) 5. The presence of D1-dopamine receptor binding in the fetal hamster SCN is consistent with the role of these receptors in entrainment of the fetal circadian pacemaker to maternal cues. However, a receptor-transmitter mismatch exists between D1-dopamine receptors and TH-immunoreactive fibers in the fetal SCN suggesting that the role of dopamine in maternal-fetal entrainment may be as a paracrine or humoral signal.

Cerebrospinal fluid neuroendocrinology of alcohol misusers.

Cerebrospinal fluid (CSF) neurohumors reflect central nervous system physiology in a way that peripheral indices can not. We reviewed clinical studies of CSF biogenic amines and neurohormones in alcohol misusers during various stages of withdrawal or abstinence and found them difficult to compare because of highly variable experimental methods, reliance on single time collections (lumbar punctures) that fail to control for potential stress-induced effects of the procedure, lack of control for tobacco use, and a paucity of non-alcoholmisusing controls. However, taken together, the data thus far show that a variety of neuroactive substances are reduced in concentration in the CSF of some alcohol misusers. Low CSF levels of corticotropinreleasing hormone, beta-endorphin, norepinephrine, diazepam-binding inhibitor, 5-hydroxyindoleacetic acid and somatostatin have all been reported. Whether the decreased CSF levels of these neurohormones and neurotransmitters are a cause or consequence of alcoholism has not been determined. In fact, further studies using serial or continuous CSF sampling techniques with homogeneous, better-characterized patients and normal volunteers are still needed to establish the precise CSF neurochemical abnormalities in alcohol misusers.

Permeability of the blood-brain barrier within rat intrastriatal transplants assessed by simultaneous systemic injection of horseradish peroxidase and Evans blue dye.

Both horseradish peroxidase (HRP) and Evans blue dye (EBD) have been used previously to characterize the status of the blood-brain barrier (BBB) within brain tissue transplants and host brain tissue. We investigated the possibility that a differential permeability of the vasculature to these two markers can account for discrepancies in the literature concerning the presence of an intact BBB within the grafted tissues. Intravascular injection of both HRP and EBD was used to evaluate the status of the BBB within intracerebral tissue transplants. Simultaneous injection of HRP and EBD in rats with adrenal medulla transplants or C-6 glioma tumors demonstrated a lack of a BBB within these grafts. Both markers produced consistent results within each tissue type, although HRP was generally a more sensitive marker. In contrast to the lack of a BBB in the C-6 gliomas or adrenal medulla transplants, 1-week-old fetal striatal transplants had a BBB essentially intact to both HRP and EBD. Any reported discrepancies in the characterization of the BBB are not likely due to differences between the properties of HRP or EBD. Fetal striatal transplants appear to have an intact BBB at 1 week following transplantation.

Autoradiographic measurement of tyrosine hydroxylase immunoreactivity in rat brain using enhanced chemiluminescence: a general method for combined quantitative and cytochemical detection.

Autoradiographic measurements of peroxidase labelled tyrosine hydroxylase immunoreactivity in brain sections from rats with unilateral 6-hydroxydopamine lesions were obtained using enhanced chemiluminescence. The autoradiograms required only 5-60 s exposure and displayed a good signal-to-noise ratio. The autoradiograms were quantitated using densitometry and by comparison with a standard curve. Following acquisition of the autoradiograms the sections were stained with the chromogen diaminobenzidine (DAB). Distribution of the DAB closely followed the grain densities observed in the autoradiograms on both the lesioned and unlesioned side of the brain. This method represents a simple, rapid and inexpensive technique for performing both quantitative autoradiography and cytochemical studies in individual tissue sections of any peroxidase labelled immunoreactivity.

Substance P containing polymer implants protect against striatal excitotoxicity.

The present study examined whether substance P (Sub P) could protect against quinolinic acid (QA)-induced lesions of the striatum, as measured by a loss of striatal D1 dopamine receptors. Sub P was extruded into Evac polymer rods for slow release. One 4 mm rod segment was implanted unilaterally into the striatum of each rat. One week later, animals received a striatal injection of QA (50, 75 or 100 nmol/microliters) medial to the implanted rod. Controls received QA alone. Three weeks later, there was a dose-dependent loss of D1 receptors following QA. Sub P rods protected the striatum from QA-induced D1 receptor loss at this time. These results support the neuroprotection role of Sub P on excitotoxicity.

Nicotine potentiation of haloperidol-induced catalepsy: striatal mechanisms.

Nicotine potentiated the catalepsy produced by haloperidol. The excitotoxin quinolinic acid (QA) selectively destroys striatal neurons when injected directly into the striatum. Bilateral QA lesions of the rat striatum (150 nmol) significantly reduced the catalepsy produced by haloperidol as well as the ability of nicotine to potentiate haloperidol-induced catalepsy. A second experiment examined whether the ability of nicotine to potentiate haloperidol-induced catalepsy was associated with a potentiation of dopamine turnover following haloperidol. Nicotine alone produced a mild increase in dopamine turnover relative to saline treated controls while haloperidol produced a marked increase in dopamine turnover relative to saline- and nicotine-treated controls. However, the combined administration of haloperidol and nicotine did not further elevate dopamine turnover over that observed following haloperidol alone. The results indicated that: 1) nicotine could not potentiate haloperidol-induced catalepsy without an intact striatum and 2) the behavioral effect of nicotine and haloperidol cotreatment was not due to any change in dopamine turnover.

Effect of fetal striatal and astrocyte transplants into unilateral excitotoxin-lesioned striatum.

Studies have suggested that neurotrophic mechanisms may underlie transplant-induced functional recovery. Astrocytes have been reported to be a source of neurotrophic factors. The present study examined the possible role of cultured astrocytes in promoting recovery of apomorphine-induced rotation behavior in rats with unilateral kainic acid (KA) lesions of the striatum. Five weeks after the lesions, one group of rats received fetal striatal tissue (E17) transplants, another group received transplants of cultured astrocyte suspension, and the remaining rats received sham transplants and served as controls. Apomorphine-induced rotation behavior was tested 4 weeks after the KA lesions, and 5 and 10 weeks following the transplantation. The KA-induced rotation behavior was reduced by the striatal transplants but not by the cultured astrocyte transplants 5 and 10 weeks following the transplantation. Histochemical analysis indicated that the striatal transplants had survived and grown and contained neurons and glia with similar morphology to those in the host brain. Immunocytochemical analysis of the astrocyte transplant sites revealed heavy glial fibrillary acidic protein and OX-42 staining in the transplant areas, suggesting that the transplanted astrocytes may have survived in the host brain. Although fetal striatal transplants can ameliorate apomorphine-induced rotation behavior, transplants of astrocytes alone may not be sufficient to reverse the functional deficits produced by KA lesions.

Long-term sensitization of apomorphine-induced rotation behavior in rats with dopamine deafferentation or excitotoxin lesions of the striatum.

Following unilateral 6-hydroxydopamine (6-OHDA)-induced deafferentation or unilateral kainic acid (KA) lesions of the striatum, rats displayed rotation behavior in response to apomorphine (0.25 or 1 mg/kg, SC, for 6-OHDA- and KA-lesioned rats, respectively). Three days following the initial apomorphine injection, rats were challenged under identical conditions with the same dose of apomorphine received previously. A third trial with apomorphine was again repeated after 3 days. Two more sets of behavioral data, each consisting of three trials, were collected under the same conditions as the first. Each set was separated by a period of 5-6 weeks. Following the second trial of the first set, rats showed a significant increase in the maximal number of rotations, demonstrating behavioral sensitization. Following the two 5-week intervals, rats were still sensitized to apomorphine, showing behavioral responses similar to the sensitized. Following the two 5-week intervals, rats were still sensitized to apomorphine, showing behavioral responses similar to the sensitized responses observed after the initial trials. Thus, the postsynaptically mediated sensitization of apomorphine-induced rotation behavior in 6-OHDA- or KA-lesioned rats is a long-lasting phenomenon. That lesions producing postsynaptic dopaminergic hypersensitivity and hyposensitivity can both show long-lasting sensitization may indicate multiple mechanisms underlying the sensitization.