The restructuring of dopamine receptor subtype gene transcripts in c-fos KO mice.

Although c-Fos protein is one of the principal molecules in intracellular signaling, c-fos gene disruption is associated with alterations in neuronal functions that do not correspond to its importance in function. The aim of the study was to evaluate the changes of dopaminergic system together with acetylcholinesterase (AChE) in c-fos disruption (KO). KO male mice showed an increase in D1-like receptor (279% of WT) and D2-like receptor (345% of WT) binding sites in the cortex. On the gene expression level (assessed by real-time PCR), lower quantities of D1R-mRNA (0.64) and D5R-mRNA (0.6) were found in females when compared to males in the frontal cortex, higher D2R-mRNA in the parietal (1.43) and temporal (2.64) cortex and lower AChE-mRNA (0.67). On the contrary, female striatum contained higher level of D2R-mRNA (1.62) and AChE-mRNA (1.57) but lower level of D3R-mRNA (0.73). Hypothalamic D1R-mRNA, D2R-mRNA and D4R-mRNA were higher in females (1.38, 1.63, and 1.68, respectively). Disruption of c-fos increased selectively D5R-mRNA (1.31) in male parietal cortex, D2R-mRNA (1.72) in male temporal cortex, and cerebellar D2R-mRNA in both males (1.43) and females (1.42), respectively. In females, we found rather decrease in DR-mRNA. Multiple correlations in mRNA quantities (in WT mice) were found, which changed considerably upon c-fos KO. Main interactions in WT were inter-regional, CNS of KO underwent an extensive restructuring comprising intraregional interactions in the frontal cortex, hypothalamus, and cerebellum. These changes in DR (between others) could be considered as one of the adaptive mechanisms in c-fos KO mice.

Transient and selective overexpression of dopamine D2 receptors in the striatum causes persistent abnormalities in prefrontal cortex functioning.

Increased activity of D2 receptors (D2Rs) in the striatum has been linked to the pathophysiology of schizophrenia. To determine directly the behavioral and physiological consequences of increased D2R function in the striatum, we generated mice with reversibly increased levels of D2Rs restricted to the striatum. D2 transgenic mice exhibit selective cognitive impairments in working memory tasks and behavioral flexibility without more general cognitive deficits. The deficit in the working memory task persists even after the transgene has been switched off, indicating that it results not from continued overexpression of D2Rs but from excess expression during development. To determine the effects that may mediate the observed cognitive deficits, we analyzed the prefrontal cortex, the brain structure mainly associated with working memory. We found that D2R overexpression in the striatum impacts dopamine levels, rates of dopamine turnover, and activation of D1 receptors in the prefrontal cortex, measures that are critical for working memory.

Investigation of the alternatively spliced insert region of the D2L dopamine receptor by epitope substitution.

Alternatively spliced variants of the D2 dopamine receptor have distinct neuronal function and localization. The long isoform (D2L) of this heptahelical transmembrane receptor differs from the short form only by the presence of a 29-amino acid insert in the third intracellular loop-a region known to be important for G protein coupling. Short and long isoforms have been shown to have distinct Galphai/o protein coupling specificities. However, the exact role of the alternatively spliced insert region in D2 dopamine receptor function needs a more comprehensive examination. One way to address this is to substitute the entire insert region with an equivalent length, yet nonhomologous protein sequence. This report demonstrates the feasibility of replacing the 29-amino acid insert with a hemagglutinin double epitope tag with no recognizable functional consequences. The D2L mutant is indistinguishable from the wild type D2L receptor in terms of its ligand binding characteristics, as well as two effector responses: the agonist-mediated inhibition of forskolin-stimulated cAMP production, and agonist-stimulated MAPK phosphorylation. These data demonstrate that the epitope substitution generates a functional receptor, and that the alternatively spliced insert region, itself, does not appear to play a direct role in signal transduction. The epitope substitution permits dissection of sequence-mediated effects from structural effects due to the presence of the alternatively spliced insert region. Thus, this new construct could be a valuable tool for the study of D2 receptor function.

Dopamine, oxytocin, and vasopressin receptor binding in the medial prefrontal cortex of monogamous and promiscuous voles.

Comparisons between monogamous and promiscuous vole species have proven useful in examining neurobiological mechanisms underlying social attachment. Reward processing is important for social attachment, and the medial prefrontal cortex (mPFC) exerts a direct influence on reward pathways. Dopamine (DA), oxytocin (OT), and arginine vasopressin (AVP) all have been implicated in the regulation of social attachment in monogamous voles. Therefore, we used radiolabeled ligands to examine dopamine D(1)- and D(2)-like, OT, and AVP V(1a) receptor binding densities in the mPFC of monogamous and promiscuous voles. Species differences were found; monogamous voles had higher densities of D(2)-like and OT receptor binding and lower densities of D(1)-like and V(1a) receptor binding than did promiscuous voles. Sex differences also were found; females had higher densities of OT receptor binding but lower densities of V(1a) receptor binding than did males in both species. Further, the laminar distribution of receptor binding indicates the possibility of an interaction between DA and OT systems in the mPFC in the regulation of social attachment. Differences in D(1)- and D(2)-like receptor binding between species are discussed in terms of how they might modulate cortical activity and subsequent DA release in the nucleus accumbens (NAcc).

Behavioral and neurochemical effects on rat offspring after prenatal exposure to ethanol.

The work studied behavioral and neurochemical alterations in 21-day-old pups, from both sexes (26 g on average) born from female Wistar rats administered daily with ethanol (0.5 or 4.0 g/kg, p.o.), for 30 days before mating, and throughout their gestational period. Ethanol administration continued from delivery up to weaning. The open field, elevated plus maze and forced swimming tests were used to evaluate effects of ethanol on locomotion, anxiety and depression, respectively. Binding assays were used to identify dopaminergic (D1- and D2-like) and muscarinic (M1 plus M2) receptors. Results of the plus maze test indicated significant and dose-dependent increases in the number of entrances in the open arms and in the time of permanence in the open arms, in the prenatally ethanol-exposed offspring, as compared to controls, indicating an anxiolytic effect. In the open field test, this group presented decreases in spontaneous locomotor activity as well as in the occurrences of rearing and grooming. Offspring also showed dose-dependent increases in their immobility time in the forced swimming test, characterizing despair behavior. Decreases in the hippocampal (D2: 32%; D1: 25%) and striatal (D2: 30%; D1: 52%) dopaminergic binding were detected in ethanol-exposed offspring. On the other hand, significant increases were observed in muscarinic binding in the hippocampus (40%) as well as in the striatum (42%). This study shows evidence that in utero ethanol exposure produces a long-lasting effect on development and pharmacological characteristics of brain systems that may have important implications in behavioral and neurochemical responsiveness occurring in adulthood.

2-[4-(3,4-Dimethylphenyl)piperazin-1-ylmethyl]-1H benzoimidazole (A-381393), a selective dopamine D4 receptor antagonist.

2-[4-(3,4-Dimethylphenlyl)piperazin-1-ylmethyl]-1H benzoimidazole (A-381393) was identified as a potent dopamine D4 receptor antagonist with excellent receptor selectivity. [3H]-spiperone competition binding assays showed that A-381393 potently bound to membrane from cells expressing recombinant human dopamine D4.4 receptor (Ki=1.5 nM), which was 20-fold higher than that of clozapine (Ki=30.4 nM). A-381393 exhibited highly selective binding for the dopamine D4.4 receptor (>2700-fold) when compared to D1, D2, D3 and D5 dopamine receptors. Furthermore, in comparison to clozapine and L-745870, A-381393 exhibits better receptor selectivity, showing no affinity up to 10 microM for a panel of more than 70 receptors and channels, with the exception of moderate affinity for 5-HT2A (Ki=370 nM). A-381393 potently inhibited the functional activity of agonist-induced GTP-gamma-S binding assay and 1 microM dopamine induced-Ca2+ flux in human dopamine D4.4 receptor expressing cells, but not in human dopamine D2L or D3 receptor cells. In contrast to L-745870, A-381393 did not exhibit any significant intrinsic activity in a D4.4 receptor. In vivo, A-381393 has good brain penetration after subcutaneous administration. A-381393 inhibited penile erection induced by the selective D4 agonist PD168077 in conscious rats. Thus, A-381393 is a novel selective D4 antagonist that will enhance the ability to study dopamine D4 receptors both in vitro and in vivo.

Effects of long-term biogenic amine transporter blockade on receptor/G-protein coupling in rat brain.

This study examines the effect of long-term elevation of brain monoamine levels on receptor/G-protein coupling by chronic administration of a highly potent tropane analog, WF-23 (2beta-propanoyl-3beta-(2-naphthyl) tropane). WF-23 blocks dopamine, serotonin and norepinephrine transporters with high affinity in vitro, and blocks transporters for at least two days following a single in vivo administration. Rats were chronically treated for 15 days with 1mg/kg WF-23, injected i.p. every two days. Receptor activation of G-proteins was determined by [35S]GTPgammaS autoradiography in brain sections for D2, 5-HT1A and alpha2-adrenergic receptors, as well as mu opioid receptors as a non-monoamine receptor control. Chronic treatment with WF-23 produced significant reductions in D2, 5-HT1A, and alpha2-adrenergic receptor-stimulated [35S]GTPgammaS binding in caudate/putamen, hippocampus and amygdala, respectively. There were no effects of WF-23 treatment on mu opioid-stimulated [35S]GTPgammaS binding. Additionally, there was no effect of WF-23 treatment on D2 receptor binding, as determined by [3H]spiperone autoradiography. These data show that chronic blockade of monoamine transporters produces specific uncoupling of receptors and G-proteins in specific brain regions in the absence of receptor downregulation.

Early administration of tiapride to young rats without long-lasting changes in the development of the dopaminergic system.

BACKGROUND: The benzamide tiapride, a selective dopamine D2/D3-receptor antagonist, can be used effectively in children to treat tic disorders and stuttering. Tiapride is a clinically safe substance (even during long-term treatment and when given to young children). Unfortunately, its probable effects on general brain development and the maturation of the dopaminergic system have not been investigated. Thus, important information for drug treatment in children is missing. Therefore, this study in rats describes tiapride's effects on several parameters of dopaminergic activity (dopamine transporter, D2 receptor, dopamine, DOPAC, and homovanillic acid in the striatum) seen after tiapride administration (30 mg/kg/day) to prepubertal (from day 25-39) and postpubertal (from day 50-64) rats. METHODS: Three groups of rats (n = 6) received tiapride within their drinking water for 14 days. Two groups were treated before puberty; one of those was killed at day 50, the other at day 90. The group treated after puberty was measured at day 90. A fourth group (n = 6) was treated from day 50 to day 53 and measured under tiapride at day 53. Changes were measured by ligand-binding assays (KD and Bmax values of dopamine transporter by [3H]-GBR binding and D2 receptor by [3H]- spiperone binding) and by HPLC (concentrations of dopamine, DOPAC, and homovanillic acid). RESULTS: The density of dopamine transporters and D2 receptors remained unaffected after early (day 25) and late (day 50) tiapride administration. Only during the treatment period could a significant reduction of D2-receptor binding (displacement of spiperone) and of dopamine and DOPAC levels be stated. CONCLUSIONS: These data suggest that tiapride treatment during postnatal brain development causes no long-lasting changes in the development of the central dopaminergic system and is in line with clinical experience in children.

Targeted mutation of CCK(2) receptor gene antagonises behavioural changes induced by social isolation in female, but not in male mice.

Neuropeptide cholecystokinin (CCK) regulates the adaptation of rodents in the novel environment. In the present study we analysed the behavioural changes induced by the individual housing in mice, lacking CCK(2) receptors. The wild-type (+/+) and homozygous (-/-) CCK(2) receptor deficient mice of both gender were used throughout the study. The weight gain during the 21-day isolation period and changes in the locomotor activity following the social separation were measured. The elevated plus-maze and resident/intruder tests were also performed to test alterations in the emotional behaviour. Social isolation induced locomotor hyperactivity, reduced weight gain and increased aggressiveness in the wild-type (+/+) and homozygous (-/-) male mice. In the wild-type (+/+) female mice the significant reduction of exploratory activity in the plus-maze was evident. By contrast, in female mice, lacking CCK(2) receptors, the exploration of the plus-maze was not significantly affected by the individual housing. This finding demonstrates that the social isolation does not cause anxiety-like state in the CCK(2) receptor deficient mice. Moreover, the targeted invalidation of CCK(2) receptors increased in male mice the affinity of dopamine D(2) receptors in the sub-cortical structures, whereas in female mice the increased affinity of 5-hydroxytryptamine(2) (5-HT(2)) receptors in the frontal cortex was established. The increased affinity of 5-HT(2) receptors is probably the compensatory change to the lack of CCK(2) receptors in female mice and probably reflects the reduced sensitivity of these animals to the anxiogenic manipulations. In conclusion, targeted mutation of CCK(2) receptors selectively antagonised the behavioural changes induced by the individual housing in females, but not in male mice.

MDMA-evoked changes in [11C]raclopride and [11C]NMSP binding in living pig brain.

Positron emission tomography (PET) studies with radiolabeled dopamine D2-like receptor ligands reveal d-amphetamine-evoked increases in the competition from endogenous dopamine. However, the corresponding effects of methylenedioxymethamphetamine (MDMA, "Ecstasy"), which releases catecholamines and also serotonin, are unknown. Using PET, we measured the binding potentials (pBs) of the benzamide [11C]raclopride and the butyrophenone N-[11C]methylspiperone ([11C]NMSP) in brain of living pigs first in a baseline condition and at 45 and 165 min after infusion of (+/-)-MDMA-HCl (1 mg/kg, i.v.). Concomitant studies of cerebral blood flow did not reveal significant perfusion changes in the cerebellum reference region or in striatum, supporting the present use of reference tissue methods for the mapping of MDMA-evoked pB changes. Relative to the baseline pB of [11C]raclopride for dopamine D(2/3) receptors in striatum (pB = 1.5-2.2), MDMA-treatment reduced pB by 35% in the first posttreatment scan and by 22% in the second posttreatment scan, comparable to changes typically evoked by d-amphetamine at a similar dose. In most previous studies, the in vivo binding of butyrophenones has been nearly insensitive to d-amphetamine-evoked dopamine release. However, we found the baseline pB of [11C]NMSP for dopamine D2-like receptors in striatum (pB = 4-5) was decreased by 30% in the first post-MDMA scan and by 50% in the second post-MDMA scan, irrespective of assumptions about the extent of equilibrium binding attained during the 90-min-long PET recordings. Distinct properties of MDMA such as simultaneous release of dopamine and serotonin in brain may account for the present finding of progressive decline in the availability of [11C]NMSP binding sites in striatum.

Postnatal haloperidol eliminates the deficit in circling behavior produced by prenatal exposure to the same drug.

Up to 35% of pregnant women take psychotropic drugs at least once during gestation [Austin and Mitchell, 1998]. From concurrent animal and human evidence, it has been proposed that exposure to several psychoactive medications in utero or during lactation increases the risk for permanent brain disorders. Present preventive or therapy practices applied on humans for this type of long-lasting behavioral alterations are mainly based on empirical results. Here, we test an experimental approach designed to counteract a circling performance deficit that appears in Sprague-Dawley rats at puberty on exposure to the dopaminergic blocker haloperidol (HAL) during gestation [J.L. Brusés, J.M. Azcurra, The circling training: A behavioral paradigm for functional teratology testing, in: P.M. Conn (Ed.), Paradigms for the study of behavior, Acad. Press, New York, 1993, pp. 166-179. Method Neurosci. 14]. Gestational exposure to HAL (GD 5-18, 2.5 mg/kg/day ip) induced the expected circling activity decrease in the offspring at the fifth week of life. When prenatal exposure to HAL was continued through lactation (PD5-21, 1.5 mg/kg/day ip), rats otherwise showed a control-like circling performance. No difference was yet found between lactation-only, HAL-exposed pups and saline (SAL)-treated controls (n=8 each group). We further performed saturating (3H)-spiroperidol (SPI) binding assays on striatal P2 membrane fractions 2 months later. The dopamine-type D2-specific binding results suggested that above circling behavior findings could be partially explained by enduring HAL-induced neurochemical changes. The role of critical periods of sensitivity as transient windows for opportunistic therapies for behavioral teratology is discussed.

Dopamine displaces [3H]domperidone from high-affinity sites of the dopamine D2 receptor, but not [3H]raclopride or [3H]spiperone in isotonic medium: Implications for human positron emission tomography.

Because the high-affinity state of the dopamine D2 receptor, D2High, is the functional state of the receptor, has a role in demarcating typical from atypical antipsychotics, and is markedly elevated in amphetamine-sensitized rats, it is important to have a method for the convenient detection of this state by a ligand. The present data show that, in contrast to [(3)H]spiperone or [(3)H]raclopride, [(3)H]domperidone labels D2High sites in the presence of isotonic NaCl in either striatum or cloned D2Long receptors, yielding a dopamine dissociation constant (1.75 nM) in agreement with that found with [(3)H]dopamine. Increased labeling of D2High sites occurred with [(3)H]domperidone after severe disruption of the cells, suggesting that [(3)H]domperidone has better access to the D2 receptor from the cytoplasmic aspect of the cell membrane. The density of the [(3)H]domperidone-labeled D2 receptors was the same as that of the [(3)H]raclopride-labeled D2 receptors, but twice the density of [(3)H]spiperone sites for human cloned D2Long receptors, compatible with the monomer-dimer concept of the D2 receptor. [(3)H]domperidone readily labels the D2High sites in postmortem human brain homogenates. Although [(3)H]spiperone or [(3)H]raclopride can occupy D2High sites, the inability of 1-10 nM dopamine to displace these ligands under isotonic conditions suggests that these ligands may not be suitable for monitoring the physiological high-affinity state of the dopamine D2 receptor by means of [(11)C]methylspiperone or [(11)C]raclopride in humans.

Effect of sabcomeline on muscarinic and dopamine receptor binding in intact mouse brain.

Sabcomeline [(R-(Z)-(+)-alpha-(methoxyiamino)-1-azabicyclo[2.2.2]octane-3-acetonitrile)] is a potent and functionally selective muscarinic M1 receptor partial agonist. However, little is known of the binding properties of sabcomeline under in vivo conditions. In this study, muscarinic receptor occupancy by sabcomeline in mouse brain regions and heart was estimated using [3H]quinuclidinyl benzilate (QNB) and [3H]N-methylpiperidyl benzilate (NMPB) as radioligands. In the cerebral cortex, hippocampus, and striatum, the estimated IC50 value of sabcomeline for [3H]NMPB binding was almost 0.2 mg/kg. Sabcomeline was not a selective ligand to M1 receptors as compared with biperiden in vivo. In the cerebral cortex, maximum receptor occupancy was observed about 1 hr after intravenous injection of sabcomeline (0.3 mg/kg), and the binding availability of mACh receptors had almost returned to the control level by 3-4 hr. These findings indicated that the binding kinetics of sabcomeline is rather rapid in mouse brain. Examination of dopamine D2 receptor binding revealed that sabcomeline affected the kinetics of both [3H]raclopride and [3H]N-methylspiperone (NMSP) binding in the striatum. It significantly decreased the k3 and k4 of [3H]raclopride binding resulting in an increase in binding potential (BP = k3/k4 = Bmax/Kd) in sabcomeline-treated mice, and an approximately 15% decrease in k3 of [3H]NMSP binding was also observed. Although the mechanism is still unclear, sabcomeline altered dopamine D2 receptor affinity or availability by modulations via neural networks.

GABA(A) alpha 1 subunit knock-out mice do not show a hyperlocomotor response following amphetamine or cocaine treatment.

The GABA(A) receptor system provides the major inhibitory control in the CNS, with the alpha 1 beta 2 gamma 2 subunit combination being the most abundant and widely distributed form of the receptor. The alpha1 subunit knock-out (alpha1 KO) mice had a surprisingly mild overt phenotype, despite having lost approximately 60% of all GABA(A) receptors. The alpha1 KO mice had normal spontaneous locomotor activity, but were more sensitive to the sedating/ataxic effects of diazepam than wildtype (WT) mice. Pharmacological modulation of dopamine and N-methyl-D-aspartate (NMDA) receptors also produced altered responses in alpha1 KO mice compared with WT mice. As expected, the NMDA receptor antagonist MK801, amphetamine and cocaine increased locomotor activity in WT mice. Although MK801 increased locomotor activity in alpha1 KO mice, amphetamine and cocaine induced stereotypy not hyperlocomotion. Binding studies showed no gross changes in the total number of D1, D2 or NMDA receptors. Furthermore, pre-pulse inhibition of acoustic startle and the effects of cocaine in conditioned place preference were similar in both alpha1 KO and WT mice, indicating selective rather that global changes in response to dopaminergic agents. These data demonstrate subtle changes in behaviours mediated by neurotransmitters other than GABA in alpha1 KO mice and suggest that compensation may have occurred beyond the GABAergic system.

Design and synthesis of a piperazinylalkylisoxazole library for subtype selective dopamine receptor ligands.

A piperazinylbutylisoxazole libary was designed, synthesized and screened for the binding affinities to dopamine D2, D3, and D4 receptors. Several ligands were identified to possess high binding affinity and selectivity for the D3 and D4 receptors over the D2 receptor. Compounds 6s and 6t showed K(i) values of 2.6 nM and 3.9 nM for the D3 receptor with 46- and 50-fold selectivity over the D2 receptor, respectively.

Positron emission tomography and ex vivo and in vitro autoradiography studies on dopamine D2-like receptor degeneration in the quinolinic acid-lesioned rat striatum: comparison of [11C]raclopride, [11C]nemonapride and [11C]N-methylspiperone.

With [11C]raclopride,[11C]nemonapride and [11C]N-methylspiperone, degeneration of dopamine D2-like receptors in the unilaterally quinolinic acid-lesioned rats was evaluated by positron emission tomography (PET) and ex vivo and in vitro autoradiography. PET showed a decreased uptake of [11C]raclopride in the lesioned striatum, but an increased uptake of [11C]nemonapride and [11C]N-methylspiperone despite a decreased binding in vitro. Ex vivo autoradiography showed an increased accumulation of the three ligands in the cortical region overlying the injured striatum, probably enlarging PET signals. PET has the limited potential for evaluating the receptor degeneration in the present animal model.

Pharmacological characterization of the D1- and D2-like dopamine receptors from the brain of the leopard frog, Rana pipiens.

The pharmacological profiles of D1- and D2-like dopamine receptors were investigated for native brain receptors in the leopard frog, Rana pipiens, using direct binding assays, which characterize functional receptors rather than assess total receptor protein. We used homogenate assays of R. pipiens fore- and midbrains to determine, via saturation isotherms, that the dissociation constant, Kd, for (3)H-SCH-23390 binding to the D1-like receptors was 0.29 nM, and the maximal receptor density, Bmax, was 40 fmoles/mg protein. This compares with the more than 10-fold higher density of D1 sites in rat striatum. Specific binding for the D2-like receptors was measurable using these methods with (3)H-spiperone as the ligand. However, saturation of binding was not achieved. This contrasts with the > 400 fmoles/mg protein Bmax in rat striatum. Pharmacological profiles (rank order of potency of displacing drugs) for each receptor type were determined. We used non-radioactive SCH-23390, SKF-38393, sulpiride, and spiperone to displace (3)H-SCH-23390 and (3)H-spiperone at D1 and D2 receptors, respectively. Parallel displacement assays were performed with rat striatal controls. Results indicated that the relative rank order displacements in anuran dopamine receptors were characteristic of D1- and D2-like receptors. However, the rank orders were not identical to those in mammals. The rank order for affinity at D1-like receptors in both rats and frogs was SCH-23390 > SKF-38393 > spiperone > sulpiride. The rank order for affinity at D2-like receptors was spiperone > SCH-23390 > sulpiride > SKF-38393 in frogs, and spiperone > sulpiride > SCH-23390 > SKF-38393 in rats. SKF-38393 and spiperone had similar affinities for the 'D1' receptors in both species. SCH-23390 had a slightly lower affinity for the D1-like receptors in Rana, whereas sulpiride had a significantly lower affinity for Rana D1-like receptors compared to rat D1 receptors. In Rana D2-like receptors, spiperone and sulpiride were significantly less potent compared to rat. However, SCH-23390 and SKF-38393 were equally potent for the D2-like receptors in both species. The results indicate that amphibian brain dopamine receptors fall into two classes similar to the mammalian D1 and D2 subfamilies, but with binding characteristics slightly different from those typically described in mammals. This work represents the first pharmacological characterization of native brain dopaminergic receptors in an anuran amphibian. Because direct binding assays measure the initial aspect of the functional interaction between transmitter and receptor, these data provide an important complement to studies using cell expression systems.

Effects of lithium on dopamine D2 receptor expression in the rat brain striatum.

Effects of lithium on the dopamine D2 receptor expression in the rat brain striatum were studied. Feeding the chow containing 0.2% LiCO3 for 6 days increased the level of the dopamine D2 receptor mRNA, and the transcription rate of the dopamine D2 receptor gene, indicating the stimulatory effects of lithium on the transcription of the dopamine D2 receptor gene. [3H] Spiperone binding to the striatal membranes increased in the rats treated with lithium, while the Western blotting analysis showed no change of the amount of the dopamine D2 receptors. These results suggested that lithium might induce the conformational changes of the dopamine D2 receptors. The methamphetamine-induced locomotor activity was enhanced by the pretreatment with lithium, whereas simultaneous increase in the methamphetamine concentration in the striatum was also observed. These observations suggested that the stimulation of methamphetamine-induced locomotor activity by lithium might be, at least partly, due to either increased sensitivity of the dopamine receptors, or increased concentration of methamphetamine in brain, or combination of both.

Agonist regulation of D(2) dopamine receptor/G protein interaction. Evidence for agonist selection of G protein subtype.

The D(2) dopamine receptor has been expressed in Sf21 insect cells together with the G proteins G(o) and G(i2), using the baculovirus system. Expression levels of receptor and G protein (alpha, beta, and gamma subunits) in the two preparations were similar as shown by binding of [(3)H]spiperone and quantitative Western blot, respectively. For several agonists, binding data were fitted best by a two-binding site model in either preparation, showing interaction of expressed receptor and G protein. For some agonists, binding to the higher affinity site was of higher affinity in D(2)/G(o) than in the D(2)/G(i2) preparation. Some agonists exhibited binding data that were best fitted by a two-binding site model in D(2)/G(o) and a one-binding site model in D(2)/G(i2). Therefore, receptor/G protein interaction seemed to be stronger in the D(2)/G(o) preparation. Agonist stimulation of [(35)S]GTP gamma S (guanosine 5'-3-O-(thio)triphosphate) binding in the two preparations also gave evidence for higher affinity D(2)/G(o) interaction. In the D(2)/G(o) preparation, agonist stimulation of [(35)S]GTP gamma S binding occurred at higher potency for several agonists, and a higher stimulation (relative to dopamine) was achieved in D(2)/G(o) compared with D(2)/G(i2). Some agonists were able to stimulate [(35)S]GTP gamma S binding in the D(2)/G(o) preparation but not in D(2)/G(i2). The extent of D(2) receptor selectivity for G(o) over G(i2) is therefore dependent on the agonist used, and thus agonists may stabilize different conformations of the receptor with different abilities to couple to and activate G proteins.

Behavioral responses to repeated cocaine exposure in mice selectively bred for differential sensitivity to pentobarbital.

Mice from the 20th generation of three lines divergently selected for response to pentobarbital-induced sedation times [long-sedation time (LST), short sedation time (SST), and randomly bred control (RBC)] were used to study cocaine-induced behavioral sensitization. These lines showed variable degrees of locomotor activities in response to cocaine. At a low cocaine dose and long withdrawal period (10 mg/kg, twice a day for 5 days followed by a 14-day withdrawal), the LST mice showed tolerance development. In response to cocaine, the locomotor activities of the SST were not significantly different from the RBC group. At a higher dose and a shorter withdrawal period (20 mg/kg, daily for 7 days followed by a 3-day withdrawal), the SST mice showed behavioral sensitization similar to the RBC mice, but the LST mice did not develop sensitization. The different responses in locomotor activity induced by cocaine suggest that genetic factors may play a role in determining the magnitude of response to this drug. Dopamine (DA) levels did not differ significantly in either striatum (STR) or nucleus accumbens (NAC) for the cocaine-treated animals to their corresponding saline-treated controls. The affinity (Kd) of D2 in the NAC decreased significantly, without changes in density (Bmax), in the cocaine-treated SST and RBC mice. On the other hand, the density of D2 binding sites in the SST and the RBC mice in the STR was significantly increased in cocaine-treated groups without change in Kd. The LST mice did not show any changes in the Kd and Bmax in either the STR or the NAC. Taken together, these findings suggest that the changes in the Kd of D2 in the NAC and the Bmax of D2 in the STR may contribute to the differences in locomotor responses to cocaine exposure in these mouse lines.