Characterization of the serotonin transporter knockout rat: a selective change in the functioning of the serotonergic system.

Serotonergic signaling is involved in many neurobiological processes and disturbed 5-HT homeostasis is implicated in a variety of psychiatric and addictive disorders. Here, we describe the functional characterization of the serotonin transporter (SERT) knockout rat model, that is generated by N-ethyl-N-nitrosurea (ENU)-driven target-selected mutagenesis. Biochemical characterization revealed that SERT mRNA and functional protein are completely absent in homozygous knockout (SERT-/-) rats, and that there is a gene dose-dependent reduction in the expression and function of the SERT in heterozygous knockout rats. As a result, 5-HT homeostasis was found to be severely affected in SERT-/- rats: 5-HT tissue levels and depolarization-induced 5-HT release were significantly reduced, and basal extracellular 5-HT levels in the hippocampus were ninefold increased. Interestingly, we found no compensatory changes in in vitro activity of tryptophan hydroxylase and monoamine oxidase, the primary enzymes involved in 5-HT synthesis and degradation, respectively. Similarly, no major adaptations in non-serotonergic systems were found, as determined by dopamine and noradrenaline transporter binding, monoamine tissue levels, and depolarization-induced release of dopamine, noradrenaline, glutamate and GABA. In conclusion, neurochemical changes in the SERT knockout rat are primarily limited to the serotonergic system, making this novel rat model potentially very useful for studying the behavioral and neurobiological consequences of disturbed 5-HT homeostasis.

Ultrasonic vocalizations in rat pups: effects of serotonergic ligands.

Ligands with varying intrinsic activity and selectivity for the various subtypes of the serotonin receptor were tested in the rat pup ultrasonic vocalization (USV) model, a putative animal model reflecting anxiety. USV were elicited by isolating rat pups from their mother and littermates by placing them on a warm (37 degrees C) or a cold (18 degrees C) plate. Concurrently, the negative geotaxic (NG) response and rectal temperature were determined to assess the potentially sedative and hypothermic effects of putative anxiolytics. USV were reduced at low doses and in both temperature conditions by the full 5-HT1A receptor agonists flesinoxan and 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino) tetralin.HBr) and the partial 5-HT1A receptor agonists buspirone, ipsapirone and BMY 7378 (2-[4-[4-[2-pyrimidinyl]-1,2-piperazinyl]butyl]-1,2-benzi-isoth iozol-3-(2H)one-1,1-dioxide. 2HCl). The 5-HT1A receptor antagonists NAN-190 (1-(2-methoxyphenyl)-4-[4-(2-phtalimido)-butyl]piperazide.2H Cl), (+/-)-WAY 100,135 (+/-)-(N-tert-butyl-3(4-(2-methoxy phenyl)piperazin-1 -yl)-2-phenyl propionamine.2HCl), and ((S)-UH-301 (S)-5-fluoro-8-hydroxy-2-(di-n-propyl-amino)tetralin.HBr) reduced USV at higher doses and only in one of both test conditions. The selective 5-HT1A receptor antagonist DU 125530 (2-[4-[4[(7-chloro-2,3dihydro-4-benzodioxin-5-yl)-1-piperazi nyl]butyl]-1,2-benzisothiazol-3(2H)-one-1,1, dioxide, monomesylate), did not influence USV at the cold plate up to high doses, although concomitantly the negative geotaxis was disturbed. The negative geotaxis was impaired after all 5-HT1A receptor ligands, except BMY 7378 and (+/-)-WAY 100,135. Hypothermia coincided with USV-suppression, except for NAN-190 and (S)-UH-301. The USV-suppressing action of flesinoxan (3 mg/kg) could be antagonized by DU 125530, but not its NG effect. However, the hypothermia induced by flesinoxan was antagonized by DU 125530. USV were also suppressed by the 5-HT uptake inhibitors fluvoxamine (both warm and cold plate) and clomipramine (only warm plate). The tricyclic antidepressant imipramine only decreased USV on the cold plate, however, in a U-shaped dose-response curve. At the highest dose tested, no decrease was present. The 5-HT uptake stimulant tianeptine reduced USV under both conditions. Fluvoxamine had no side effects, clomipramine induced hypothermia and tianeptine clearly had sedative properties. The 5-HT1B/2C receptor agonist TFMPP (trifluorometaphenylpiperazine) stimulated USV at a low dose at the cold plate and suppressed USV at a high dose under both conditions. The 5-HT2A/2C receptor antagonist ketanserine enhanced USV at low doses under both conditions and had no effect at a higher dose. Concurrently heavy sedation and hypothermia occurred. The 5-HT3 receptor agonist phenylbiguanide and the 5-HT3 receptor antagonist ondansetron had no effect in this paradigm. Clearly, subtypes of the 5-HT receptor affect rat pup USV differentially.

Mutational analysis of the potential phosphorylation sites for protein kinase C on the CCK(A) receptor.

1. Many G protein-coupled receptors contain potential phosphorylation sites for protein kinase C (PKC), the exact role of which is poorly understood. In the present study, a mutant cholecystokininA (CCK(A)) receptor was generated in which the four consensus sites for PKC action were changed in an alanine. Both the wild-type (CCK(A)WT) and mutant (CCK(A)MT) receptor were stably expressed in Chinese hamster ovary (CHO) cells. 2. Binding of [3H]-cholecystokinin-(26-33)-peptide amide (CCK-8) to membranes prepared from CHO-CCK(A)WT cells and CHO-CCK(A)MT cells revealed no difference in binding affinity (Kd values of 0.72 nM and 0.86 nM CCK-8, respectively). 3. The dose-response curves for CCK-8-induced cyclic AMP accumulation and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) formation were shifted to the left in CHO-CCK(A)MT cells. This leftward shift was mimicked by the potent inhibitor of protein kinase activity, staurosporine. However, the effect of staurosporine was restricted to CHO-CCK(A)WT cells. This demonstrates that attenuation of CCK-8-induced activation of adenylyl cyclase and phospholipase C-beta involves a staurosporine-sensitive kinase, which acts directly at the potential sites of PKC action on the CCK(A) receptor in CCK-8-stimulated CHO-CCK(A)WT cells. 4. The potent PKC activator, 12-O-tetradecanoylphorbol 13-acetate (TPA), evoked a rightward shift of the dose-response curve for CCK-8-induced cyclic AMP accumulation in CHO-CCK(A)WT cells but not CHO-CCK(A)MT cells. This is in agreement with the idea that PKC acts directly at the CCK(A) receptor to attenuate adenylyl cyclase activation. 5. In contrast, TPA evoked a rightward shift of the dose-response curve for CCK-8-induced Ins(1,4,5)P3 formation in both cell lines. This demonstrates that high-level PKC activation inhibits CCK-8-induced Ins(1,4,5)P3 formation also at a post-receptor site. 6. TPA inhibition of agonist-induced Ca2+ mobilization was only partly reversed in CHO-CCK(A)MT cells. TPA also inhibited Ca2+ mobilization in response to the G protein activator, Mas-7. These findings are in agreement with the idea that partial reversal of agonist-induced Ca2+ mobilization is due to the presence of an additional site of PKC inhibition downstream of the receptor and that the mutant receptor itself is not inhibited by the action of PKC. 7. The data presented demonstrate that the predicted sites for PKC action on the CCK(A) receptor are the only sites involved in TPA-induced uncoupling of the receptor from its G proteins. In addition, the present study unveils a post-receptor site of PKC action, the physiological relevance of which may be that it provides a means for the cell to inhibit phospholipase C-beta activation by receptors that are not phosphorylated by PKC.

Short and long term plasticity after lesioning of the cell body or terminal field area of the dopaminergic mesocorticolimbic system in the rat.

To investigate within one study regenerative capacities of dopaminergic axons and cell bodies, short and long term recovery of behavioral and biochemical impairments following a bilateral 6-hydroxydopamine (6-OHDA) lesion of the ventral tegmental area (VTA)-nucleus accumbens (NAc) pathway was investigated in rats. Novelty-induced motility, presynaptic functions and the levels of dopamine (DA) and its metabolites were reduced when cell bodies in the VTA or axons in the NAc were lesioned. Spontaneous recovery of the behavioral deficit was observed 4 weeks after a lesion of the NAc. Subsequently presynaptic functions recovered as shown by the reappearance of low dose apomorphine (50 mg/kg)-induced hypomotility, normalization of [(3)H]dopamine uptake, reinnervation of the NAc and normalization of levels of DA and its metabolites within 24 weeks. In contrast, after a VTA lesion no recovery was observed during 48 weeks, neither from hypomotility and loss of the low dose apomorphine response nor from decreased [(3)H]dopamine uptake and levels of DA in the NAc. Short term postsynaptic supersensitivity (hypermotility upon a higher dose of apomorphine (125 mg/kg)) was present 1 and 4 weeks after the lesion but not thereafter. A near total absence of dopaminergic neurons in the VTA and axons in the NAc were found 24 weeks postlesion. Treatment with the ACTH-(4-9) analog ORG 2766 (10 mg/kg s.c., 6 days once daily) facilitated recurrence of presynaptic functions after a lesion of axons but had no short or long term effect when cell bodies were lesioned. These findings substantiate the postulate that the peptide facilitates recovery processes.

Topography and characteristics of specific binding sites for non-opioid gamma-type endorphins in the rat brain as studied by autoradiography with [35S]Met-desenkephalin-gamma-endorphin.

An in vitro autoradiographic study was performed to characterize specific rat brain binding sites for non-opioid neuroleptic-like gamma-type endorphins, using [35S]Met-des-enkephalin-gamma-endorphin ([35S]Met-DE gamma E; [35]S-beta-endorphins(5-17)) with high specific activity as radioligand. The binding sites appeared to be confined to rat forebrain regions, e.g., orbital cortex, frontal cortex, cingulate cortex, piriform cortex, nucleus accumbens, amygdala, mediodorsal nucleus of the thalamus and arcuate and periventricular nuclei of the hypothalamus. These regions are part of the mesocorticolimbic feedback circuit. Densitometric analysis of the autoradiographs revealed that the density of the binding sites was highest in the mediodorsal nucleus of the thalamus and the amygdala. Concentration-dependent displacement of [35S]Met-DE gamma E (500 pM) with DE gamma E yielded an IC50 of 0.6 nM whereas DE alpha E (beta-endorphin(6-16)) had an IC50 of 210 nM. Various endorphins, sharing the gamma-endorphin C terminus, displaced [35S]Met-DE gamma E to the same extent as non-labelled DE gamma E (at 10(-6) M) whereas non-endorphin peptides did not show displacing capacity. Possible relationships of the binding sites with opioid receptors were investigated. DAMGO (mu) and DPDPE (delta) displaced [35S]Met-DE gamma E to some extent at 10(-6) M whereas U69,593 (kappa) was inactive, suggesting that the binding sites for gamma-type endorphins may resemble mu- and delta-opioid receptors in some aspects. Similarly, relationships with dopamine receptors were investigated. Haloperidol partially displaced [35S]Met-DE gamma E whereas sulpiride, SKF38,393 and 3-PPP at 10(-6) M did not induce significant displacement. Thus, binding sites are distinct from dopamine receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucocorticoid and mineralocorticoid receptors differentially modulate cultured dopaminergic neurons of rat ventral mesencephalon.

Activation of kappa-opioid receptors by U69,593 in a concentration of 1 microM maximally inhibited dopamine release. Withdrawal from chronic (4 days) treatment with U69,593 (1 microM) induced a long-lasting (at least 7 days) increase (30-50%) in [3H]dopamine release induced by 25 mM K+ from cultured rat mesencephalic neurons without causing functional kappa-opioid receptor desensitization. Incubation of these cultured neurons with the glucocorticoid agonist, dexamethasone (3 nM), had no effect on basal or 25 mM K(+)-induced dopamine release, whereas the steroid completely blocked the development of opiate-induced neuronal supersensitivity to depolarization. This blockade was found to be concentration-dependent, with an EC50 of about 0.3 nM and was not associated with changes in corticotropin-releasing factor (CRF) receptor, dopamine D2 autoreceptor or kappa-opioid receptor functioning. Therefore, opioid and glucocorticoid receptors appear to be co-localized in dopaminergic neurons of rat ventral mesencephalon and interact in a functionally antagonistic manner. Interestingly, the mineralocorticoid agonist, aldosterone (5 nM), itself increased K(+)-stimulated [3H]dopamine release by about 25%, consistent with the putative role of mineralocorticoid receptors in maintaining neuronal excitability. Moreover, whereas 1 nM corticosterone appeared to mimic the dopamine release-facilitating effect of aldosterone, 30 nM corticosterone displayed a modulatory effect similar to that of dexamethasone. Thus, whereas mineralocorticoid receptors that are already activated at low plasma corticosterone levels may uphold neuronal sensitivity to depolarizing stimuli, activation of glucocorticoid receptors by higher plasma corticosterone levels may prevent the development of neuronal supersensitivity, e.g. following chronic activation of kappa-opioid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat pup ultrasonic vocalization: effects of benzodiazepine receptor ligands.

The involvement of the GABA(A)-benzodiazepine receptor complex in rat pup ultrasonic vocalisations was studied by testing benzodiazepine receptor ligands with varying intrinsic activity and selectivity for benzodiazepine subtype receptors. Ultrasonic vocalisations were recorded under two temperature conditions (37 degrees C and 18 degrees C), presumably reflecting a low and high stress state. The latency to the negative geotaxis response, a measure of motor coordination and the rectal temperature were determined to assess putative side effects of drugs. The full, non-selective benzodiazepine receptor agonists diazepam, chlordiazepoxide, alprazolam and oxazepam suppressed ultrasonic vocalisations both at 37 degrees C and 18 degrees C conditions, although more efficaciously at 37 degrees C. The partial, non-selective benzodiazepine receptor agonist bretazenil and the partial benzodiazepine, selective receptor agonist alpidem significantly reduced ultrasonic vocalisations at 37 degrees C, but not at 18 degrees C. The full benzodiazepine, selective receptor agonist zolpidem behaved like other full, non-selective benzodiazepine receptor agonists by reducing ultrasonic vocalisations under both high and low temperature. The effects of zolpidem indicate that activation of benzodiazepine, receptors alone already suffices to suppress ultrasonic vocalisations. The non-selective, benzodiazepine receptor antagonist flumazenil and the partial, non-selective benzodiazepine receptor inverse agonist FG 7142 (N'-methyl-beta-carboline-3-carboxamide) and the full, non-selective benzodiazepine receptor inverse agonist DMCM (6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) had no significant effect on ultrasonic vocalisations under both temperature conditions. The involvement of benzodiazepine receptors in rat pup ultrasonic vocalisations (37 degrees C-condition) was confirmed by antagonism of the ultrasonic vocalisations reducing effects of chlordiazepoxide by flumazenil (1 or 3 mg/kg). Using the rat pup ultrasonic vocalisations paradigm under 18 degrees C and 37 degrees C conditions combined with measurements of negative geotaxis-latencies and rectal temperatures it is possible to (1) distinguish benzodiazepine receptor agonists from other anxiolytics because of dissimilar dose response curves at 37 degrees C and 18 degrees C, (2) differentiate partial from full receptor agonists by absence of effects at the 18 degrees C condition, (3) suggest a key role for benzodiazepine, receptors in the modulation of ultrasonic vocalisations. These data contribute to the predictive validity of pup vocalizations as an animal model of anxiety.

Opioid receptor-mediated inhibition of evoked catecholamine release from cultured neurons of rat ventral mesencephalon and locus coeruleus.

The effects of selective opioid agonists on the evoked release of [3H]dopamine and [3H]noradrenaline were studied in cultured dopaminergic neurons of the ventral mesencephalon (containing the substantia nigra and ventral tegmental area) and in cultured neurons of the noradrenergic locus coeruleus, respectively. The cultures were prepared from embryonic day 15 rat brains. After 9 days in culture, the calcium-dependent release of [3H]dopamine from dopaminergic substantia nigra/ventral tegmental area neurons induced by 23 mM k+ appeared to be inhibited exclusively by activation of kappa-opioid receptors, as [3H]dopamine release was inhibited selectively by the kappa agonists U69,593 and dynorphin-(1-13) (EC50 8 and 5 nM, respectively), and this inhibitory effect was antagonized by the kappa-selective antagonist nor-binaltorphimine (Ki 0.07 nM). In contrast, cultured noradrenergic locus coeruleus neurons appeared to contain release-inhibitory mu-opioid receptors only, as evoked [3H]noradrenaline release was inhibited selectively by the mu agonist [D-Ala2, MePhe4, Gly-ol5]enkephalin (EC50 45 nM), a response that was antagonized by the preferential mu antagonist naloxone (Ki = 0.7 nM). The delta-opioid receptor agonist [D-Ser2(O-butyl), Leu5]enkephalyl-Thr6 did not affect catecholamine release. Dopamine release from cultured ventral mesencephalic neurons, induced by 100 microM N-Methyl-D-Aspartate (NMDA), also appeared to be subject to kappa receptor-mediated inhibition, whereas NMDA-induced noradrenaline release from cultured locus coeruleus neurons was under the inhibitor control of mu receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Anxiolytic effects of flesinoxan in the stress-induced hyperthermia paradigm in singly-housed mice are 5-HT1A receptor mediated.

In the stress-induced hyperthermia paradigm in singly-housed male mice, two sequential rectal temperature measurements reveal the basal temperature (T1) and, 10 min later, an enhanced body temperature (T2), due to the stress of the first rectal measurement. The difference T2 - T1 (deltaT) is the stress-induced hyperthermia and putatively reflects a stress-induced anxiogenic response. The full 5-HT1A receptor agonist flesinoxan ((+)-enantiomer), its (-)-enantiomer and the racemic mixture reduced stress-induced hyperthermia effects, indicating putative anxiolytic properties. The ratio of their potencies to reduce stress-induced hyperthermia was similar to their potency in receptor binding affinities for 5-HT1A receptors, supporting that the anti-hyperthermia effects are mediated by the 5-HT1A receptor. This was further substantiated when the 5-HT1A receptor antagonists WAY 100635 ((N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclo-hexane carboxamine trihydrochloride) and DU 125530 (2-[4-[4-(7-chloro-2,3-dihydro-1,4-benzodioxin-5-yl)-1-piperazinyl ]butyl]-1,2-benzisothiazol-3(2H)-one-1,1-dioxide, monomesylate) both were able to antagonize the anti-stress-induced hyperthermia effects of flesinoxan. The stress-induced hyperthermia paradigm in singly-housed mice represents a simple and robust paradigm to measure putative anxiolytic effects of drugs.

Repeated and chronic morphine administration causes differential long-lasting changes in dopaminergic neurotransmission in rat striatum without changing its delta- and kappa-opioid receptor regulation.

Repeated, once daily morphine treatment (14 days) as well as chronic morphine administration (6 days) caused a rebound reduction in the electrically evoked release of [3H]dopamine from superfused rat striatal slices 1 day after the last subcutaneous injection. Interestingly, whereas [3H]dopamine release remained significantly reduced for at least 3 weeks following morphine withdrawal in chronically treated (tolerant/dependent) rats, neurotransmitter release from dopaminergic nerve terminals gradually increased above control values following cessation of repeated morphine administration. Postsynaptically, dopamine D1 receptor-stimulated adenylate cyclase appeared to be sensitized 1-3 days but was unchanged 3 weeks after chronic morphine treatment. In contrast, such an enhanced postsynaptic dopamine D1 receptor efficacy did not occur 1-3 days following repeated morphine administration, but appeared to develop slowly resulting in a profound increase of dopamine-sensitive adenylate cyclase 3 weeks after the last injection. The inhibitory effect of dynorphin A-(1-13) on [3H]dopamine release, as well as that of [Met5]enkephalin on dopamine D1 receptor-stimulated adenylate cyclase appeared to be unchanged subsequent to repeated or chronic morphine treatment. These data indicate that, long after cessation of drug treatment, chronic morphine treatment causes a reduction whereas repeated morphine administration gradually induces an enhancement of opioid receptor-regulated dopaminergic neurotransmission due to local adaptive changes within the rat striatum. Such distinct long-lasting alterations of dopaminergic neurotransmission induced by different temporal patterns of morphine administration in projection areas of mesencephalic dopaminergic neurons may be related to the enduring effects of drug abuse such as behavioural sensitization and drug craving.

Patterns of c-fos expression induced by fluvoxamine are different after acute vs. chronic oral administration.

Fluvoxamine is a selective serotonin (5-HT) reuptake inhibitor (SSRI) with a broad spectrum of behavioral and therapeutic effects, e.g. in depressive illness. We used the expression of c-fos, after both acute and chronic oral administration of fluvoxamine in the rat, to study its immediate and long-term effects, in relation to the distribution of Galanin (GAL) and Vasoactive Intestinal Polypeptide (VIP). After acute oral administration, most consistent increases were apparent in (parts of); the nucleus of the solitary tract, medial part; the lateral parabrachial nucleus, external part; the bed nucleus of the stria terminalis, dorsolateral part; and the central nucleus of the amygdala, lateral part. After chronic administration, distribution of Fos-IR was similar to acute administration, although numbers of Fos-IR neurons were no longer significantly different from control values. It is concluded that activation of 5-HT3-receptors in the caudal brainstem or gastro-intestinal afferents of the vagal nerve may play a role in the observed pattern of Fos-IR after fluvoxamine administration. The relationship with the antidepressant effects of fluvoxamine needs further investigations.

Chronic activation of mu- and kappa-opioid receptors in cultured catecholaminergic neurons from rat brain causes neuronal supersensitivity without receptor desensitization.

The effects of chronic activation of mu- and kappa-opioid receptors on the release of [3H]dopamine from cultured ventral mesencephalic neurons and that of [3H]noradrenaline from cultured locus ceruleus neurons were studied. It was found that a 4-day exposure of cultured mesencephalic neurons to the kappa-agonist U69,593 (5 alpha, 7 alpha, 8 beta)-(-)-N-methyl-N-[7(1-pyrrolidinyl)-1-oxaspiro(4,5)-dec-8-yl] benzeneacetamide; 1 microM] increased markedly 25 mM K(+)-induced dopamine release upon opioid withdrawal by about 70%, with no change in the release-inhibitory effects of U69,593 (EC50, 3-6 nM; maximal inhibition about 50%). Moreover, 1.2 mM Ca++ (in the continuous presence of K+) and 100 microM N-methyl-D-aspartic acid receptor-mediated dopamine release also was potentiated to a similar extent after chronic kappa receptor activation. Similarly, 4-day treatment of cultured locus ceruleus neurons with morphine (1 microM) caused an enhanced release of [3H]noradrenaline induced by K+, Ca++ or N-methyl-D-aspartic acid upon opioid withdrawal by 55 to 150%. In addition, the release-inhibitory effect of the mu-selective agonist [D-Ala2,MePhe4,Gly-ol5]-enkephalin on the K(+)-induced [3H]noradrenaline release remained unaffected (EC50, 10-12 nM; maximal effect, 80-90%). Interestingly, chronic activation of autoreceptors on cultured mesencephalic neurons by the dopamine D-2 receptor-selective agonist LY171,555 [(trans)-(-)-(4aR)-4, 4a,5,6,7,8,8a,9-octahydro-5-propyl-1H-pyrazolo[3,4g]quinoline; 1 microM] or those of cultured locus ceruleus neurons by the alpha-2 adrenoceptor-selective agonist clonidine (1 microM) did not affect K(+)-induced neurotransmitter release upon agonist withdrawal.(ABSTRACT TRUNCATED AT 250 WORDS)

Interacting presynaptic kappa-opioid and GABAA receptors modulate dopamine release from rat striatal synaptosomes.

The presynaptic regulation of stimulated dopamine release from superfused rat striatal synaptosomes by opioids and gamma-aminobutyric acid (GABA) was studied. It was found that in addition to dopamine D2 autoreceptors, calcium-dependent K(+)-stimulated [3H]dopamine release was inhibited through activation of a homogeneous population of kappa-opioid receptors in view of the potent inhibitory effect of the kappa-selective agonist U69,593 (EC50 0.2 nM) and its antagonism by norbinaltorphimine. Neither mu- nor delta-selective receptor agonists affected release of [3H]-dopamine. In addition, GABA potently inhibited the evoked [3H]dopamine release (EC50 0.4 nM) through activation of GABAA receptors in view of the GABA-mimicking effect of muscimol, the sensitivity of its inhibitory effect to picrotoxin and bicuculline, and the absence of an effect of the GABAB receptor agonist baclofen. In the presence of a maximally effective concentration of GABA, U69,593 did not induce an additional release-inhibitory effect, indicating that these receptors and the presynaptic D2 receptor are colocalized on the striatal dopaminergic nerve terminals. The excitatory amino acid agonists N-methyl-D-aspartate and kainate, as well as the cholinergic agonist carbachol, stimulated [3H]dopamine release, which was subject to kappa-opioid receptor-mediated inhibition. In conclusion, striatal dopamine release is under regulatory control of multiple excitatory and inhibitory neurotransmitter by activation of colocalized presynaptic receptors for excitatory amino acids, acetylcholine, dopamine, dynorphins, and GABA within the dopaminergic nerve terminals. Together, these receptors locally control ongoing dopamine neurotransmission.

Functional characteristics of a series of N4-substituted 1-(2,3-dihydro-1,4-benzodioxin-5-yl)piperazines as 5-HT1A receptor ligands. Structure-activity relationships.

The agonistic/antagonistic profile of a series of 10 N4-substituted 1-(2,3-dihydro-1,4-benzodioxin-5-yl)piperazines is evaluated in the in vitro adenylyl cyclase assay. The profile is severely affected by the characteristics of the N4-substituents ranging from full agonism (benzamidoethyl derivative 1), mixed agonism/antagonism (phthalimidobutyl derivative 7) to predominantly antagonism (saccharinbutyl derivate 9). A novel full antagonist 10, as potent as WAY 100635, is obtained by substitution of Cl at C-7 of the benzodioxinyl moiety in 9.

Dopamine displays an identical apparent affinity towards functional dopamine D1 and D2 receptors in rat striatal slices: possible implications for the regulatory role of D2 receptors.

In this study we examined the selectivity of dopamine (DA) for rat striatal DA D1 and D2 receptors. In a Krebs-HEPES buffer, the Ki values of DA for D1 binding sites (labelled with [3H]SCH23390) and D2 binding sites (labelled with [3H]spiroperidol) in striatal membranes amounted to about 30 and 0.3 microM, respectively. However, the EC50s of DA (3 microM) and the DA releasing drug amphetamine (1 microM) were identical considering D1 receptor-stimulated and D2 receptor-inhibited adenylate cyclase activity in superfused striatal slices. Moreover, these EC50 values were also obtained studying DA- and amphetamine-induced D2 receptor activation, resulting in inhibition of the electrically evoked release of [14C]acetylcholine from the slices. Therefore, with regard to the apparent affinity of exogenous and endogenous DA for D1 and D2 receptors in rat striatal slices, the ligand-receptor binding data appeared to be misleading. Thus, our data show that in rat striatal slices DA has an identical apparent affinity towards functional D1 and D2 receptors, which is particularly intriguing in view of the very high receptor selectivity of synthetic D1 and D2 receptor agonists for these functional receptors in superfused brain slices as predicted on the basis of binding assays. This may have important implications for our understanding of central DA neurotransmission. For instance, since the inhibitory effect of opioid and muscarinic receptor activation on adenylate cyclase activity has been shown to be inversely related to the degree of DA D2 receptor activation, the degree of activation of D1 and D2 receptors by released DA is suggested to act as a functional gate allowing distinct neurotransmitters to play a role in striatal neurotransmission.

Acute effects of oxidized low density lipoprotein on metabolic responses in macrophages.

The immediate effects of oxidized low density lipoprotein (OxLDL) on the metabolic activity of cultured macrophages (RAW 264.7) were studied using a microphysiometer. Administration of OxLDL acutely induced a concentration-dependent increase in metabolic activity, with an EC50 of 16 +/- 3 microg/ml OxLDL and a maximal effect of 35% +/- 4% (mean +/- SEM; n=5). A biphasic response was measured after administration of 75 or 100 microg/ml OxLDL consisting of an initial sharp increase, followed by the induction of a long-lasting hypoactivity of 80% of the control value. Incubation of cells with polyinosinic acid (polyI; 100 microg/ml) for 30 min prior to OxLDL administration could completely block the effect of 25 microg/ml OxLDL. In addition, polyI acted as a full antagonist on the decrease of the biphasic response of cells generated by 75 and 100 microg/ml OxLDL. Macrophages used in this study possessed a specific binding site for OxLDL, with a dissociation constant (KD) of 9 +/- 2 microg/ml and a maximal binding of 610 +/- 32 ng 125I-OxLDL/mg cell protein. Binding of 125I-OxLDL to macrophages could be completely competed for by unlabeled OxLDL, by polyI for 58%, and by AcLDL for 46%. In conclusion, OxLDL can acutely activate the metabolic state of macrophages by a receptor-mediated process in a concentration-dependent fashion, which could be antagonized by polyI. Metabolic responses to OxLDL may underlie the changes observed in macrophages in the early atherosclerotic plaque.

In vitro and in vivo characterization of newly developed iodinated 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]piperazine derivatives in rats: limited value as dopamine transporter SPECT ligands.

A series of 1-{2-[bis(4-fluorophenyl)methoxy]ethyl}piperazines (CYD1, 2, 3, 5) with a 4-substituent incorporating a 1-hydroxy-3-iodo-2-propenyl moiety, except CYD2 which lacks the hydroxy, was synthesized as potential in vivo imaging ligands for the dopamine transporter. For two of the piperazine derivatives (CYD3 and 5), possible stereoselectivity was considered as well (both E- and Z-form). Their in vitro potency for inhibition of [3H]dopamine uptake in rat striatal synaptosomes was 10-fold lower than that of GBR 12,909 used as a reference. The highest Ki values were 137 and 101 nM for CYD1E and CYD3E, respectively. Inhibition potency was higher for the E- than for the Z-isomers. In vivo distribution of radioactivity in rats injected with the 123I-labeled CYDs showed preferred striatal uptake for CYD1E and CYD3E as compared to the cerebellum and occipital cortex. Although the E-isomer of CYD3 showed the best in vitro and in vivo binding characteristics, its striatal uptake ratios (maximal value: 2.7 for striatum-to-cerebellum at 4 h p.i.) are too low to consider application in human Single Photon Emission Computed Tomography studies.