Development and validation of a bioanalytical assay for the measurement of total and unbound teicoplanin in human serum.

BACKGROUND: The glycopeptide teicoplanin is considered first-line treatment for severe infections caused by Gram-positive bacteria. Individualized treatment of teicoplanin is gaining interest. As only protein-unbound drug is pharmacologically active, a sensitive assay measuring unbound and total teicoplanin is indispensable for pharmacological research and dose optimization. OBJECTIVES: To develop and validate a UPLC-MS/MS method to quantify unbound and total teicoplanin in human serum. METHODS: The developed assay was validated according to the ICH guideline M10 on Bioanalytical Method Validation and study sample analysis. Unbound teicoplanin was obtained by ultrafiltration. The assay was cross-validated with a quantitative microsphere (QMS) immunoassay in a side-by-side comparison using 40 patient samples. RESULTS: With the developed and validated method, all main teicoplanin components (A2-1, A2-2/A2-3, A2-4/A2-5 and A3-1) can be quantified. Total run time was 5.5 min. Concentration range was 2.5-150 mg/L for total and 0.1-25 mg/L for unbound teicoplanin. Precision (coefficient of variation) and accuracy (bias) of total teicoplanin were 5.97% and 107%, respectively, and 7.17% and 108%, respectively, for unbound teicoplanin.Bland-Altman analysis showed total concentrations measured with the UPLC-MS/MS method were equivalent to the results of the QMS immunoassay. A total of 188 samples from 30 patients admitted to the ICU and haematology department were measured; total concentrations ranged between 2.92 and 98.5 mg/L, and unbound concentrations ranged between 0.37 and 30.7 mg/L. CONCLUSIONS: The developed method provided rapid, precise and accurate measurement of unbound and total teicoplanin. The developed method is now routinely applied in pharmacological research and clinical practice.

Behavioral arousal and increased dopamine efflux after blockade of NMDA-receptors in the prefrontal cortex are dependent on activation of glutamatergic neurotransmission.

Blockade of NMDA/glutamate receptors induces altered behavior in humans and experimental animals. At the same time a differential activation of dopaminergic (DA) systems has been reported. To study the involvement of the medial prefrontal cortex (mPFC) in these effects, we used bilateral perfusions of the rat mPFC with the competitive NMDA-antagonist D-AP-5 and simultaneous determination of spontaneous behavior and local DA efflux. D-AP-5 concentration-dependently induced arousal and motor activity and also increased DA efflux. These effects were shown to have a similar time-scale but no causal relationship: combined D1/D2 receptor blockade in the mPFC did not inhibit the behavioral activation. As bilateral perfusion of the nucleus accumbens with D-AP-5 resulted in similar behavioral effects, but no change in DA efflux, we conclude that DA is not involved in the behavioral activation induced by these local perfusions. However, local blockade of non-NMDA glutamate receptors or stimulation of GABA-B receptors completely blocked the effects on behavior and DA efflux, suggesting that the arousal and locomotor activity induced by NMDA receptor blockade in mPFC is primarily dependent on activation of glutamatergic mechanisms. The mPFC appears to be an important site of action for NMDA antagonists to induce behavioral alterations.

Dopamine and noradrenaline efflux in the prefrontal cortex in the light and dark period: effects of novelty and handling and comparison to the nucleus accumbens.

We used on-line microdialysis measurements of dopamine and noradrenaline extracellular concentrations in the medial prefrontal cortex of awake, freely moving rats during the dark and the light period of the day to study whether (i) basal efflux would be higher in the active, dark period than in the inactive, light period; (ii) the activation induced by environmental stimuli would be dependent on these conditions. When determined one day after cannula placement, noradrenaline and dopamine levels were higher during the dark. Maximal relative increases induced by novelty and handling were 150% and 175-200%, respectively, and were very similar in the light and the dark, but the net increases were higher in the dark. Separate groups were tested one week after cannula placement to ensure recovery of possibly disturbed circadian rhythms. While basal levels in the dark were now approximately twice those in the light, the maximal relative and net increases after both novelty and handling were very similar. Basal levels of dopamine in the nucleus accumbens (one day after cannula placement) were not different in the light or dark, but were increased by novelty and handling to about 130% only in the light period, not in the dark. Thus, in the prefrontal cortex, dopamine strongly resembles noradrenaline, in that basal efflux was state dependent, whereas activation by stimuli was not. In the nucleus accumbens, basal dopamine efflux was not state dependent, but activation by stimuli was. These results suggest that there are differential effects of circadian phase on basal activity and responsiveness of the mesolimbic vs the mesocortical dopamine system.

C-Fos expression in the rat brain after pharmacological stimulation of the rat "mediodorsal" thalamus by means of microdialysis.

In order to visualize target cells of thalamic projections in the rat brain we examined the induction of c-fos messenger RNA and Fos-like immunoreactivity following stimulation of the "mediodorsal" thalamus (midline, mediodorsal and intralaminar nuclei) in freely moving rats. The thalamic neurons were activated through disinhibition by perfusion of the GABAA antagonist bicuculline-methyl chloride via a microdialysis cannula placed in the mediodorsal nucleus of the thalamus. The rats were allowed a recovery period of at least 20 h after surgery before being coupled to the perfusion pump. Cannula implantation with or without 4 h of Ringer perfusion caused hardly any detectable c-fos expression in the brain, but 20 min of bicuculline (0.1 mM) perfusion induced high levels of c-fos messenger RNA and Fos protein expression in the area adjacent to the dialysis membrane, indicating activated thalamic neurons. In situ hybridization as well as immunohistochemical analysis of the frontal cortical areas and limbic structures showed a rapid, specific and transient c-fos expression in the medial and lateral prefrontal cortex, nucleus accumbens, mediodorsal striatum, claustrum, nucleus reticularis of the thalamus and amygdala. The overall spatial distribution of the c-fos response was comparable to the innervation patterns of thalamic efferents known from anatomical tracing experiments. The rats were perfused with Ringer while asleep, but they woke up during treatment with bicuculline and displayed an increase in general behavioural activity, which could be correlated to the amount of bicuculline measurable in the dialysate. Pathological behaviours, such as epilepsy, were not noticeable during bicuculline treatment. These results show that it is possible to selectively activate defined anatomical pathways by pharmacological application of drugs using microdialysis in unanesthetized unrestrained animals and to visualize the transsynaptically activated target neurons of these projections. We conclude that this novel experimental approach is indeed suitable for studying functional anatomical pathways.

Changes in adrenoceptors and monoamine metabolism in neonatal and adult rat brain after postnatal exposure to the antihypertensive labetalol.

1. The purpose of the present study was to investigate the acute (single injection), direct (chronic treatment) and the long-lasting effects after exposure to the alpha 1/beta-adrenoceptor antagonist labetalol during rat brain development on adrenoceptors and monoamine metabolism. 2. In 10-day-old rat pups, subcutaneously administered labetalol (10 mg kg-1) passed the blood-brain barrier, reaching a level of 2.1 micrograms g-1 tissue in the brain 90 min after injection. 3. Chronic labetalol treatment (10 mg kg-1, s.c., twice daily) during the first 10 days of life significantly increased alpha 1-adrenoceptor binding in the hypothalamus (+39%), but not in the occipital cortex. 4. This chronic postnatal labetalol treatment did not result in long-lasting changes in alpha 1- and beta-receptors measured on day 60. 5. A single labetalol injection (10 mg kg-1, s.c.) on postnatal day 10 significantly increased noradrenaline (NA) metabolism in all brain regions tested (+25 to 105%), but had no effects on 5-hydroxytryptamine (5-HT) or dopamine metabolism. 6. Chronic labetalol treatment between postnatal (PN) days 1 and 10 also increased NA metabolism on PN 10 (3-methoxy-4-hydroxyphenylglycol (MHPG)/NA, +20 to 100%), suggesting that tolerance to the acute effect of labetalol did not occur. A slight increase in 5-HT metabolism (20%) was induced by the chronic labetalol treatment in the hippocampus and meso-limbic system. 7. In general, long-lasting effects on NA metabolism could not be detected on day 60 more than one month after the treatment. However, 5-HT metabolism was significantly increased in all four brain regions measured (+20 to 70%). 8. We conclude that chronic labetalol exposure during early postnatal rat brain development does not cause long-lasting changes in beta-receptor number or NA metabolism, but appears to be critical for the rate of 5-HT metabolism in later life.

Monoclonal antibodies against haemocyte molecules of Penaeus monodon shrimp react with haemolymph components of other crustaceans and disparate taxa.

In a previous study, monoclonal antibodies (mAbs) against different haemolymph molecules of the marine shrimp Penaeus monodon were produced and characterised. It was suggested that these mAbs could be used in studying haemocyte differentiation, behaviour and function in P. monodon. In the present study, the reaction of these mAbs on P. monodon was compared with other crustaceans and disparate taxa. The mAbs also reacted with haemolymph components of three freshwater crustaceans, a terrestrial isopod crustacean and with coelomic fluid of an annelid. No reactions were observed with haemolymph of an insect and a mollusc, nor with blood cells of two vertebrates. This comparative study shows reactivity of the mAbs with a wide range of crustaceans and related animals and suggests that well conserved molecules are recognised, which may indicate functional importance. Well-described mAbs can be used in studies of the crustacean defence system and may finally result in a better insight into this system.

Developmental changes in rat brain monoamine metabolism and beta-adrenoceptor subtypes after chronic prenatal exposure to propranolol.

The aim of this study was to investigate whether a chronic prenatal beta-blockade can alter the maturation of the noradrenergic system in the rat brain. Pregnant female and adult male rats were treated for 10 days with the beta-antagonist propranolol dissolved in the drinking water (40-50 mg/kg/day). Direct and long-term effects on beta-adrenoceptors and monoamine metabolism in various rat brain regions were determined. After the prenatal treatment the propranolol level in the foetal brain was 0.9 micrograms/g, while in the adult brain 2.0 micrograms/g was present. The foetal beta 1-receptors were significantly up-regulated by propranolol (200%), whereas the beta 2-receptor number remained unaltered. On postnatal days 4 and 21 the number of both beta-subtypes was the same as that of controls. Noradrenaline, its metabolite 3-methoxy-4-hydroxyphenylglycol and their ratio were unaltered directly after the prenatal treatment. In the PN 21 offspring, however, the metabolite level had increased in the frontal cortex (+ 17%) and hippocampus (+ 32%), and the ratio in the hippocampus (37%) and medulla pons (+ 34%). Prenatal treatment also induced a significant increase of the 5-hydroxyindoleacetic acid.5-hydroxytryptamine ratio (+ 15%) in the medulla pons at GD 21. No direct or lasting effects were found on dopamine metabolism. Propranolol treatment of adult rats gave no direct changes in monoamine metabolism. We concluded that chronic prenatal propranolol exposure (a) reversibly up-regulates foetal beta 1-adrenoceptors, and (b) increases the NA activity in the brain in later life.

Rapid sampling of extracellular dopamine in the rat prefrontal cortex during food consumption, handling and exposure to novelty.

We report the effects of physiological stimuli on extracellular dopamine (DA) in the medial prefrontal cortex (PFC) of the rat determined on-line in dialysates obtained every 5.5 min. The detection limit for DA was 0.03-0.1 pg/5 microl injection using a conventional HPLC set-up. Basal levels in PFC were at the detection limit, therefore 3 microM nomifensine was included in the Ringer perfusion fluid, producing readily detectable DA levels of 0.9 pg/injection. Perfusion with 3 microM TTX for 30 min decreased DA within 11 min to 10% of control. The routine use of rapid sampling of extracellular DA was applied to study cortical DA release in relation to behaviour. Exposure to a novel environment for 5.5 min led to an increase to 135%. Presentation of a food pellet to food-deprived rats resulted in a rapid increase to 150% within 5.5 min, which lasted 30-40 min, which is 10-20 min more than the time spent eating. Handling the rat for 5.5 min increased DA in PFC within 5.5 min to 160% and in 11 min to 190% of control followed by a 25-min period of a 50% increase, probably reflecting increased arousal. The results suggest that emotional arousal is a common denominator of increased cortical DA release and that responses are graded depending on the intensity of the stimulus.

Increased alpha 2-adrenoceptor mediated regulation of adult rat brain noradrenaline overflow after chronic neonatal exposure to propranolol; a microdialysis study.

Direct and persistent effects of chronic neonatal administration of the beta-adrenoceptor antagonist propranolol on brain noradrenergic activity were investigated by measuring tissue concentrations of noradrenaline and its metabolites and in vivo overflow of noradrenaline during adulthood. Rat pups were chronically treated with propranolol from postnatal day 1 to day 10. Determination of monoamine metabolism after the last injection showed an increase in noradrenaline metabolism in frontal cortex, limbic system and hippocampus of propranolol-exposed rats, but 47 days after this last injection it was apparent that these effects were not long-lasting. Moreover, basal noradrenaline overflow in vivo in the hippocampus of 40-55 day-old propranolol-exposed rats did not differ from that in controls. However, the regulation of noradrenaline release seemed to have been altered, since a pharmacological challenge with the alpha 2-adrenoceptor antagonist idazoxan induced an enhanced increase in the in vivo noradrenaline overflow in propranolol-exposed rats compared to controls. It is suggested that the neonatal beta-blockade induced a supersensitivity of the presynaptic alpha 2-adrenoceptor. The precise mechanism underlying this effect has to be elucidated.

Modulatory role of catecholamines in the transsynaptic expression of c-fos in the rat medial prefrontal cortex induced by disinhibition of the mediodorsal thalamus: a study employing microdialysis and immunohistochemistry.

We studied the interaction of catecholaminergic and thalamic afferents of the medial prefrontal cortex (PFC) by analyzing the effects of catecholamine depletion on thalamus-induced c-fos expression in the PFC of freely moving rats. Thalamic projections to the PFC were pharmacologically activated by perfusing the GABA-A receptor antagonist bicuculline (0.03 mM or 0.1 mM) through a dialysis probe implanted into the mediodorsal thalamic nucleus. Bicuculline perfusion induced Fos-like immunoreactivity in the thalamic projection areas, including the PFC, and in the thalamic nuclei surrounding the dialysis probe. 6-Hydroxydopamine lesions of the ventral tegmental area causing a 70-80% depletion of catecholamines in the PFC did not influence the increase in the number of Fos-like immunoreactive nuclei in the prefrontal cortex in response to thalamic stimulation. However, densitometric image analysis revealed that the intensity of Fos-like immunoreactivity in the PFC of lesioned rats perfused with 0.1 mM bicuculline was higher than in correspondingly treated controls. The behavioral activity to bicuculline perfusion, an increase of non-ambulatory activity (0.03 mM) followed by locomotion and rearing (0.1 mM), was not changed in 6-hydroxydopamine-lesioned rats. It is suggested that the thalamically induced c-fos response is directly mediated by excitatory, presumably glutamatergic, transmission and not indirectly by an activation of catecholaminergic afferents of the PFC. The increase in the intensity of Fos-like immunostaining in strongly stimulated, catecholamine-depleted rats suggests that catecholamines modulate the degree to which thalamic activity can activate the PFC of awake animals.

Early postnatal appearance of enhanced noradrenaline content in the brain of vasopressin-deficient Brattleboro rat; normal adrenoceptor densities and aberrant influences of vasopressin treatment.

The course of postnatal development of noradrenaline (NA) and its unconjugated free metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG), as well as the influence on early chronic vasopressin treatment, were investigated in various brain regions of the hereditary vasopressin-deficient (homozygous di/di) Brattleboro rat. In addition, the densities of the adrenergic receptor subtypes were measured in adult brain. Brain NA levels of di/di pups appeared enhanced already at 7 days of age when compared with data of heterozygous (+/di) controls. This was also seen in areas not known to receive a vasopressinergic input, e.g. the frontal cortex. Levels of MHPG also differed between genotypes, but changes were slight and either a decrease or increase, depending on age and region tested. Saturation analyses of alpha 1-, alpha 2-, and beta-adrenoceptor binding on crude membrane preparations of some brain regions revealed no differences in adulthood. Chronic treatment with vasopressin between 6 and 13 days of age reduced the enhanced NA brain levels throughout the brain of the di/di Brattleboro pups. The known vasopressin-mediated enhancement of NA turnover in adult brain was also measurable in +/di pups of this neonatal period (MHPG/NA ratios), indicating the early maturation of the interaction of vasopressinergic and NAergic systems. However, the dose-response in the di/di Brattleboro rat was biphasic with a decrease at a low dose of vasopressin. Since changes were found throughout the brain, it was concluded that vasopressin deficiency had altered the maturation of NA neurons of the locus coeruleus which may be due to the absence of a presumed inhibitory control of vasopressin on synthesis and storage mechanisms at the perikaryal level.

Postnatal exposure to the beta-adrenoceptor agonist clenbuterol has regionally selective direct and long-term effects on rat brain beta-adrenoceptors and monoamine metabolism.

The effects of chronic postnatal beta 2-adrenoceptor activation on the maturation of the rat brain noradrenergic system have been studied. For that purpose, rat pups have been treated twice daily during the first 10 days of life with the beta 2-agonist clenbuterol-HCl (2.5 mg/kg s.c.), and the effects on the beta-adrenoceptor number and monoamine metabolism have been determined directly after the treatment and in adulthood. On postnatal day 10, 90 min after the last clenbuterol injection 4.5 micrograms/g of the drug was present in the brain. At the end of the treatment the beta-receptor binding had decreased in the cerebellum (35%), but not in the frontal cortex or mesolimbic system. Clenbuterol significantly increased the steady-state brain levels of noradrenaline (NA) in the striatum 90 min after the last injection, whereas the levels in the frontal cortex, meso-limbic system, medulla pons and cerebellum were unaffected. The NA metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG), had significantly increased in the frontal cortex and striatum. The serotonergic (5-HT) and dopaminergic (DA) system were not altered. In general, no long-lasting effects on beta-adrenoceptor number and affinity or monoamine metabolism were measurable, except for the frontal cortex which showed a sustained increase of MHPG, a decrease of 5-HT and an increase of 5-HIAA/5-HT on PN 60. In conclusion, chronic postnatal activation of beta 2-adrenoceptors by clenbuterol treatment selectively causes changes in the setting of the neurochemical parameters investigated in the frontal cortex.

Novelty-induced increase in dopamine release in the rat prefrontal cortex in vivo: inhibition by diazepam.

The effects of graded stressful conditions on extracellular concentrations of dopamine (DA) in the medial prefrontal cortex of rats were measured in vivo using microdialysis. Picking up the rat twice with a 20-min interval increased extracellular DA to 120%, exposure to a novel environment by placement in a clean cage for 20 min to 150% and holding the rat in the hands for 20 min to over 200%. Diazepam (5 mg/kg) decreased DA to about 75% and attenuated the novelty- and handling-induced increases. Exposure to novelty or handling are easy and simple methods to obtain graded increases of in vivo cortical DA release.

Concentration-dependent dual action of locally applied N-methyl-D-aspartate on extracellular dopamine in the rat prefrontal cortex in vivo.

Using microdialysis, the glutamate agonist N-methyl-D-aspartate (NMDA) was perfused for 20 min through the medial prefrontal cortex of freely moving rats, and its effects on extracellular concentrations of dopamine (DA) were determined. NMDA (1 mM) increased DA to 170-1500%, depending on the intensity and duration of the clonic forelimb jerks and convulsions that were induced. NMDA (0.1 mM), however, decreased DA to 61%. Metabolites of DA were decreased after both concentrations of NMDA. The effects of both 0.1 mM and 1 mM NMDA were blocked by 0.5 mM of the competitive NMDA-antagonist D-AP-5. The NMDA-induced decrease in release and metabolism possibly results from an indirect action via an inhibitory local interneuron or polysynaptic circuit.

Dopamine and noradrenaline release in the prefrontal cortex of rats during classical aversive and appetitive conditioning to a contextual stimulus: interference by novelty effects.

Extracellular dopamine and noradrenaline concentrations in the medial prefrontal cortex were determined using online microdialysis in freely moving rats during classical appetitive or aversive conditioning to a contextual conditioned stimulus (CS). As CS we used placement in a Skinner box where either foot shocks or food pellets were delivered as unconditioned stimuli. In the aversive conditioning group we observed a conditioned increase in noradrenaline, but not in dopamine release, upon a final exposure to CS-alone. Other conditioned effects were possibly masked by the continued presence of novelty and handling effects of the transfer to the Skinner box.

Chronic neonatal NMDA receptor blockade with MK-801 alters monoamine metabolism in the adult rat.

Administration of non-competitive N-methyl-D-aspartate (NMDA) antagonists in rodents leads to a characteristic motor syndrome which has been related to changes in monoamine metabolism in a variety of brain regions. We examined the question whether chronic MK-801 treatment in neonatal rats from postnatal day 8 through 19, which has been shown previously to alter NMDA receptor function, would also affect monoamine metabolism in striatum and frontal cortex of adult rats. Monoamines and their metabolites were determined 5 months after the treatment using high-performance liquid chromatography with electrochemical detection. Dihydroxyphenylacetic acid (DOPAC) concentration was elevated (greater than 40%) in both regions tested, while 5-hydroxyindoleacetic acid (5-HIAA) concentration was significantly elevated only in the cortex (19%), and 3-methoxy-4-hydroxyphenylglycol (MHPG) only in the striatum (47%). These results demonstrate that the long-lasting effects of chronic neonatal MK-801 treatment are not restricted to glutamate transmission, but include monoamine transmission as well.

Decreased hypothalamic serotonin levels in adult rats treated neonatally with clomipramine.

Early postnatal treatment with the antidepressant drug clomipramine has repeatedly been shown to lead to behavioural and physiological changes in adult rats. To provide some neurochemical correlates to these studies we have measured a number of monoaminergic parameters in the brains of adult (one year old) rats that were treated twice daily with 15 mg/kg clomipramine from postnatal day 2-14. The most consistent finding was that the hypothalamic levels of serotonin (5-HT) were decreased and those of the dopamine metabolite dihydroxyphenylacetic acid (DOPAC) were increased in rats irrespectively whether they went through a range of behavioural and physiological tests or not. The numbers of beta-adrenoceptors in the frontal cortex and of alpha 2-adrenoceptors in the amygdala/piriform cortex were not changed. The decrease in hypothalamic 5-HT concentrations appears to be up to now the most consistent neurochemical alteration in adult rats that were neonatally treated with antidepressant drugs. It is, however, not clear what the relation is with the functional changes in these rats, that are proposed by some authors as an animal model for depression.

Preliminary study on haemocyte response to white spot syndrome virus infection in black tiger shrimp Penaeus monodon.

White spot syndrome virus (WSSV) has been a major cause of shrimp mortality in aquaculture in the past decade. In contrast to extensive studies on the morphology and genome structure of the virus, little work has been done on the defence reaction of the host after WSSV infection. Therefore, we examined the haemocyte response to experimental WSSV infection in the black tiger shrimp Penaeus monodon. Haemolymph sampling and histology showed a significant decline in free, circulating haemocytes after WSSV infection. A combination of in situ hybridisation with a specific DNA probe for WSSV and immuno-histochemistry with a specific antibody against haemocyte granules in tissue sections indicated that haemocytes left the circulation and migrated to tissues where many virus-infected cells were present. However, no subsequent haemocyte response to the virus-infected cells was detected. The number of granular cells decreased in the haematopoietic tissue of infected shrimp. In addition, a fibrous-like immuno-reactive layer appears in the outer stromal matrix of tubule walls in the lymphoid organ of infected shrimp. The role of haemocytes in shrimp defence after viral infection is discussed.

Determination of propranolol, labetalol and clenbuterol in rat brain by high-performance liquid chromatography.

A rapid and simple HPLC method for the measurement of adrenergic drugs (propranolol, labetalol and clenbuterol) in rat brain is described. This method was applied to establish if these drugs can pass the blood-brain barrier in prenatal or early post-natal life. The chromatography was performed using a C18 column and a phosphate buffer (pH 3)-acetonitrile (65:35, v/v) mixture. After homogenization of the brain tissue in perchloric acid, the supernatant was buffered at pH 9 and extracted with diethyl ether, followed by back-extraction in sulphuric acid. Recoveries of between 80 and 100% were achieved. The method was found to be accurate (100%) and precise (coefficient of variation around 10%). All three drugs were readily detected in the brain of neonatal rats after peripheral administration. In addition, we demonstrated the presence of propranolol in the fetal brain after maternal administration.

Local activation of metabotropic glutamate receptors inhibits the handling-induced increased release of dopamine in the nucleus accumbens but not that of dopamine or noradrenaline in the prefrontal cortex: comparison with inhibition of ionotropic receptors.

On-line in vivo microdialysis was used to determine the effects of a 16-min handling period on release of dopamine (DA) in the nucleus accumbens and of DA and noradrenaline (NA) in the medial prefrontal cortex of awake, freely moving rats. DA and NA were determined in one HPLC run. Handling resulted in an immediate and strong increase of both catecholamines in the prefrontal cortex. Maximal values for DA were 295%, and for NA 225%, of controls. DA in the nucleus accumbens was also increased (to 135% of controls) but only after a short delay. Local inhibition of ionotropic glutamate receptors by continuous reversed dialysis of the drugs 6-cyano-7-nitroquinoxaline, D-2-amino-5-phosphonopentanoic acid, or dizocilpine did not significantly affect handling-induced increases in cortical DA and NA release. Neither did the agonist of metabotropic glutamate receptors, trans-(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), or the GABA-B agonist baclofen. Reversed dialysis of dizocilpine in the nucleus accumbens was equally ineffective, but ACPD inhibited the increase in DA release in this area. Stimulation of metabotropic glutamate receptors in the nucleus accumbens was previously reported to inhibit activation of DA release in that area after stimulation of glutamatergic or dopaminergic afferents. It is concluded that metabotropic receptors in the nucleus accumbens are important for the control of activation of DA release in the accumbens by physiological stimuli but that a similar mechanism is lacking in the prefrontal cortex.