Biochemical characterization of the intramembrane interaction between neurotensin and dopamine D2 receptors in the rat brain

In order to better understand the neuroleptic-like effects of neurotensin in vivo, the effects of neurotensin in vitro on dopamine D₂ and D₁ agonist and antagonist binding sites were characterized in membranes from the neostriatum and the subcortical limbic area. Neurotensin increased theK_D bu not theB_max value ofS(−)[N-propyl-³H(N)]propylnorapomorphine ([³H]NPA) binding sites with a maximal increase of 20–40% at 3–10 nM of neurotensin in both areas. TheK_D increase was preferentially due to an increase in the dissociation rate. The maximal reduction of [³H]NPA binding (35%) was obtained within 5 min from the addition of neurotensin. Neurotensin increased theK_H of dopamine vs [³H]raclopride binding and, in the presence of GTP, alsoK_L. Neurotensin did not affect the percentage of binding sites in the high vs low affinity states or the binding characteristics of [³H]spiperone, [³H]SKF 38393, and [³H]SCH 23390. Serotonin (10 nM), neuropeptide Y (10 nM), Substance P (10 nM), dynorphin A (10 nM), morphine (10 nM), nicotine (100 nM),γ-amino-n-butyric acid (1 μM), orN-methyl-d-aspartate (1 μM) did not affect [³H]NPA binding. These results indicate that neurotensin in vitro selectively reduces D₂ agonist affinity by an enhancement of the dissociation rate. This antagonistic intramembrane interaction may underlie the neuroleptic-like effects of neurotensin at low concentrations in vivo on D₂ agonist binding, dopamine release, and on D₂-mediated behaviours.     

Kinetic and pharmacologic characterization of dopamine binding in the mouse cerebellum and the effects of the reeler mutation

The purpose of this study was to characterize the dopaminergic system in the mouse cerebellum and to determine whether the dyskinesia of the reeler mutant is accompanied by alterations in cerebellar and/or striatal dopamine binding. From the analysis of (3H) dopamine ((3H)DA) and (3H)spiperone ((3H)Sp) binding, the study of the effects of several drugs on this binding, and the comparison of these parameters between the cerebellum and striatum, we conclude that a dopaminergic system exists in the cerebellum with properties common to the striatal system but also with some differences. That is, 1) with (3H)DA as ligand, we find two binding sites in cerebellum with similar Kd to those of striatum but of lower density, 2) with (3H)Sp as ligand we observe two binding sites in cerebellum and one in striatum, and 3) the competition of (3H)DA binding by various drugs shows that among the cerebellar sites, relative to striatum, there is a higher proportion that corresponds to high affinity D3 and D4 (D2 high) binding sites. In cerebellum and striatum of reeler mice, (3H)DA binding increases 125-174% and 14%, respectively.     

Characterisation of dopamine receptors in insect (Apis mellifera) brain

In vitro binding experiments using the vertebrate D₁ dopamine receptor ligand [³H]SCH23390 and the vertebrate D₂ dopamine receptor ligand [³H]spiperone were conducted on membrane preparations of honey bee (Apis mellifera) brain. Specific binding of [³H]SCH23390 was saturable and reversible. Analysis of saturation data gave an apparent K_d of 6.3 ± 1.0 nM and B_max of 1.9 ± 0.2 pmol/mg protein for a single class of binding sites. The specificity of high affinity [³H]SCH23390 binding was confirmed in displacement experiments using a range of dopaminergic antagonists and agonists. The rank order of potency for antagonists was: R(+)-SCH23390 > cis-(Z)-flupentixol ≥ chlorpromazine > fluphenazine> S(+)-butaclamol > spiperone. R(±)-SKF38393 and dopamine were the most effective agonists tested. [³H]SCH23390 labels a site in bee brain that is similar, but not identical to the vertebrate D₁ dopamine receptor subtype. [³H]Spiperone also bound with high affinity to bee brain homogenates. Scatchard analysis of [³H]spiperone saturation data revealed a curvilinear plot suggesting binding site heterogeneity. The high affinity site had a apparent K_d of 0.11 ± 0.02 nM and B_max of 9.2 ± 0.5 fmol/mg protein. The calculated values for the low affinity site were a K_d of 19.9 nM and B_max of 862 fmol/mg protein. Kinetic analyses also indicated that [³H]spiperone recognises a heterogeneous population of sites in bee brain. Furthermore, agonist competition studies revealed a phenolaminergic as well as a dopaminergic component to [³H]spiperone binding in bee brain. The rank order of potency of dopaminergic antagonists in competing for [³H]spiperone binding was: spiperone > fluphenazine> S(+)-butaclamol > domperidone> R(+)-SCH23390 > S(−)-sulpiride.     

[H]Spiperone binds selectively to rat striatal D2 dopamine receptors in vivo: a kinetic and pharmacological analysis

We have determined the kinetic, equilibrium saturation, and pharmacological characteristics of [³H]spiperone ([³H]SPIP) binding to rat brain regional particulate fractions following i.v. injections of [³H]SPIP and compared these parameters to those determined in vitro with traditional ligand-homogenate binding assays. [³H]SPIP binding to rat striatum in vivo and in vitro occurs to a single class of non-interacting binding sites which possess the pharmacological properties of a D₂ dopamine (DA) receptor. The potencies of neuroleptic drugs in inhibiting DA receptor-mediated behaviors correlate with their potencies at displacing striatal [³H]SPIP binding in vivo. While striatum possesses a similar density of [³H]SPIP binding sites in vivo (34 pmol/g) and in vitro (31 pmol/g), binding affinity in vivo is about 200 times lower than in vitro. This difference in binding affinities appears to arise from alterations of [³H]SPIP association and dissociation rate constants brought about by tissue homogenization. The implications of our findings for external imaging of DA receptors and studies of DA receptor function in human brain homogenates are discussed.     

The effects of intranigral GABA and dynorphin A injections on striatal dopamine and GABA release: Evidence that dopamine provides inhibitory regulation of striatal GABA neurons via D2 receptors

The effects of injections of γ-aminobutyric acid (GABA) and dynorphin A into the substantia nigra, pars reticulata on the levels of extracellular dopamine (DA) and GABA in the ipsilateral striatum of halothane-anaesthetized rats were studied using microdialysis. The effects of intranigral injections of substance P and neurokinin A were also studied. Intranigral GABA (300 nmol) or dynorphin A (0.5 nmol) injections produced a simultaneous decrease in DA and increase in GABA levels, while intranigral substance P (0.07 nmol) or neurokinin A (0.09 nmol) injections produced an increase in DA but had no effect on GABA levels. DA agnonists, apomorphine (D₁/D₂), SKF 38393 (D₁) and pergolide (D₂) were applied locally by perfusing them through the microdialysis probe, each at a concentration of 10⁻⁵ M. All 3 agonists decreased the levels of DA in the striatum. However, while apomorphine and SKF 38393 increased, pergolide decreased the levels of GABA in the striatum. The increase in striatal GABA produced by intranigral injections of GABA (300 nmol) was reversed by local perfusion with pergolide (10⁻⁵ M), but was not reversed by local perfusion with SKF 38393 (10⁻⁵ M). These findings suggest that D₁ and D₂ receptors differentially regulate striatal GABA release, and are stimulatory and inhibitory, respectively. Furthermore, it is suggested that nigrostriatal DA functions as an inhibitory modulator of striatal GABA neurons, acting via D₂ receptors.     

Neurochemical correlates of behavioral sensitization following repeated apomorphine treatment: Assessment of the role of D1 dopamine receptor stimulation

Previous research has revealed a role of repeated D₁ dopamine receptor stimulation in the development of behavioral sensitization to the D₁/D₂ agonist apomorphine. The present experiments assessed the role of repeated D₁ receptor stimulation in neurochemical changes accompanying locomotor sensitization to apomorphine. To assess direct effects of D₁ stimulation on dopamine synthesis, rats were injected with the D₁ agonist SKF 38393 (8 mg/kg), followed by an injection with the 3,4-dihydroxyphenylalanine (DOPA) decarboxylase inhibitor, NSD-1015. DOPA accumulation, assessed in striatal, nucleus accumbens-olfactory tubercle (NAOT), and ventral mesencephalon (VM) tissue samples, was not affected by acute SKF 38393. In the second experiment, rats were treated with 10 daily injections of vehicle, apomorphine (5 mg/kg) or the D₁ agonist SKF 38393 (8 or 16 mg/kg). Daily measures of locomotor activity demonstrated a progressive increase in the apomorphine-treated rats, but not the SKF 38393-treated rats, across the 10 days. On day 11, all rats were injected with NSD-1015 for measurement of DOPA accumulation. Dopamine synthesis was enhanced in the striatum after repeated apomorphine treatment. In contrast, repeated SKF 38393 treatment resulted in either a small decrease or no change in DOPA accumulation in the different brain regions (striatum, NAOT, VM). In the third experiment, tissue levels of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and [³H]SCH 23390 binding to D₁ receptors were measured in rats treated with 10 daily injections of vehicle, apomorphine (5 mg/kg), or SKF 38393 (16 mg/kg). In the striatum and NAOT, none of the repeated drug treatments had an effect on DOPAC or dopamine levels. In the VM, DOPAC levels were enhanced following repeated apomorphine, but not repeated SKF 38393, whereas dopamine levels were not affected by either drug treatment. D₁ binding was not altered by the repeated drug treatments. Since repeated D₁ stimulation by SKF 38393 did not produce the same alterations in dopamine synthesis and DOPAC levels as repeated apomorphine, the neurochemical effects accompanying locomotor sensitization to apomorphine probably are not mediated by D₁ receptors. © 1993 Wiley-Liss, Inc.     

Differential pharmacological profile of striatal and cerebellar dopamine receptors labeled by [3H]quinpirole: Identification of a discrete population of putative D3 receptors

We have previously identified [³H]quinpirole-labeled dopamine receptors in the molecular layer of cerebellar lobule 10 which have a D₂-like pharmacological profile, are guanine nucleotide-insensitive, and are juxtaposed to putative D₃ receptor mRNA. This study compares the pharmacological profiles of [³H]quinpirole-labeled dopamine receptors in striatum and cerebellar lobule 10 using quantitative autoradiography. Dopaminergic compounds inhibited the specific binding of [³H]quinpirole in the caudate/putamen with the following rank order of potencies: spiperone > haloperidol ≥ (+)butaclamol ≥ quinpirole ≥ 7-OH-DPAT ≥ bromocriptine > clozapine > (-)sulpiride. In cerebellar lobule 10, a somewhat different rank order of potencies was observed: 7-OH-DPAT > quinpirole ≥ bromocriptine > spiperone > (+)butaclamol > haloperidol > clozapine > (-)sulpiride. Quinpirole possessed equal affinity for [³H]quinpirole-labeled receptors in the caudate/putamen and cerebellum. 7-OH-DPAT exhibited 5-fold greater affinity for cerebellar receptors than those in the caudate/putamen. Spiperone, haloperidol, (+)butaclamol, and clozapine were more potent in competing for [³H]quinpirole binding at striatal dopamine receptors than cerebellar receptors by 83-, 59-, 11-, and 6-fold, respectively. The relative potencies of these compounds at striatal and cerebellar dopamine receptors are generally similar to the differential affinities reported at D₂ and D₃ dopamine receptors expressed in CHO cells, respectively. These data provide additional evidence that the dopamine receptors observed in cerebellar lobule 10 represent a discrete population of putative D₃ receptors. © Wiley-Liss, Inc.     

In vitro and in vivo comparison of [³H](+)-PHNO and [³H]raclopride binding to rat striatum and lobes 9 and 10 of the cerebellum: a method to distinguish dopamine D₃ from D₂ receptor sites

In vitro binding characteristics of the dopamine D₃/D₂ antagonist [³H]raclopride were compared to the D₃/D₂ agonist [³H](+)‐PHNO in membrane preparations from rat striatum, cerebellum Lobules 9 and 10 (CB L9,10), and other cerebellar regions. In striatum, both radioligands labeled a single binding site. [³H](+)‐PHNO showed higher affinity, though lower B_max, compared with [³H]raclopride and was sensitive to inhibition by Gpp(NH)p. [³H](+)‐PHNO showed significant specific binding to CB L9,10 membranes with higher affinity compared to striatal membranes. [³H](+)‐PHNO binds to a high‐ and a low‐affinity binding site in CB L9,10 membranes; the high‐affinity site was not Gpp(NH)p‐sensitive. [³H](+)‐PHNO did not significantly bind cerebellum left hemisphere membranes. Very low specific binding of [³H]raclopride was found in CB L9,10. The selective dopamine D₃ antagonist SB‐277011 did not displace the binding of either ligand to striatal membranes but potently inhibited the binding of [³H](+)‐PHNO in CB L9,10 membranes. The highly selective D₂ antagonist SV‐156 showed the opposite profile. In vivo experiments were consistent with and supported by in vitro results. In summary, [³H](+)‐PHNO and [³H]raclopride mainly label dopamine D₂ receptors in rat striatum, with [³H](+)‐PHNO labeling a D population. In vitro and in vivo, [³H](+)‐PHNO labels CB L9,10 dopamine D₃ receptors that are apparently in a high affinity state whereas [³H]raclopride gave only very low signal in this region. The present approaches appear useful for selectively labeling dopamine D₃ and D₂ receptors in different rat brain regions and offer the possibility to demonstrate D₃ versus D₂ receptor selectivity of compounds using native rat brain tissue. Synapse, 2011. © 2010 Wiley‐Liss, Inc.     

Kainic acid receptor sites in the cerebellum of nervous, Purkinje cell degeneration, reeler, staggerer and weaver mice mutant strains

[³H]Kainic acid binding to crude cerebellar membrane preparations from mice nervous, Purkinje cell degeneration, reeler, staggerer and weaver mutant strains and their corresponding wildtype strains was determined. Specific kainate binding was strongly reduced in staggerer mutants, less in reeler and weaver mutation and was little affected by the nervous and Purkinje cell degeneration mutation. It is concluded that in the cerebellum kainate receptor sites are associated with the parallel fiber-Purkinje cell contacts and that kainate binding sites are most likely localized on the presynaptic side.     

D1 dopamine receptor stimulation inhibits firing activity of midbrain dopamine neurons in reserpine treated rats: An effect eliminated after hemitransection of diencephalon

It has been reported that systemic administration of the D₁ dopamine (DA) receptor agonist SKF 38393 inhibits the firing rate of substantia nigra pars compacta (SNC, A9) DA neurons after repeated reserpine treatment in locally anesthetized rats, although SKF 38393 induces little effect on the firing of midbrain DA neurons in normal rats. The present study found that local pressure microejection of SKF 38393 (10⁻² M, 20–100 nl) to SNC or substantia nigra pars reticulata (SNR) failed to influence the firing of SNC DA neurons in reserpinized rats (reserpine 1 mg/kg × 6 days, s.c.); subsequent intravenous (i.v.) injection of SKF 38393 (4 mg/kg), however, inhibited their firing and the inhibition was reversed by the D₁ receptor antagonist SCH 23390. Similarly, systemic administration of SKF 38393 (4 mg/kg, i.v.) inhibited the firing of ventral tegmental area (VTA, A10) DA cells in reserpinized rats, while local microejection of SKF 38393 (10⁻² M, 30–60 nl) did not affect their firing. Furthermore, the inhibitory effect of systemic SKF 38393 on firing rate of either SNC or VTA DA neurons in reserpinized rats was eliminated after hemitransection of diencephalon. These results suggest that repeated reserpine treatment renders midbrain DA neurons responsive to D₁ receptor stimulation and that D₁ receptor agonist-induced inhibition of midbrain DA cell firing in reserpinized rats may require the involvement of long-loop feedback pathways. © 1996 Wiley-Liss, Inc.     

Long-term treatment with haloperidol or clozapine does not affect dopamine D4 receptors in rat frontal cortex

Summary We examined the effects of long-term treatment with haloperidol and clozapine on dopamine D₄ receptors in rat frontal cortex. Dopamine D₄ receptor binding sites were indirectly determined from the displacement experiments of [³H]clozapine binding using nemonapride. Three-weeks administration of haloperidol (0.5mg/kg) or clozapine (10mg/kg) did not significantly affect the D₄ receptors in the frontal cortex. The density of D₂ receptors, determined by [³H]spiperone binding to striatum, was increased by long-term treatment with haloperidol, but it was not significantly changed by that with clozapine.     

Effect of reserpine-induced depletion of synaptic dopamine on [11C]Raclopride binding to D2-dopamine receptors in the monkey brain

Positron emission tomography was used to examine the in vivo binding of [¹¹C]raclopride to D₂-dopamine (DA) receptors in the striatum of two Cynomolgus monkeys after a single dose of reserpine (1 mg/kg, i.v.). A Scatchard procedure was repeated five times to follow D₂ receptor density and apparent affinity for 7 weeks after reserpine. Reserpine-induced depletion of DA lead to a marked increase in [¹¹C]raclopride binding, which was still detectable 20 days after treatment. Scatchard analyses indicated that the measured increase in [¹¹C]raclopride binding reflected an increase in receptor affinity but no evident change in receptor density (B_max). Thus, the increase in [¹¹C]raclopride binding after reserpine should correspond to a reduced competition with endogenous DA for binding to D₂ receptors. The results were used to estimate the DA-induced D₂ occupancy to be about 40% at physiological conditions. Synapse 25:321–325, 1997. © 1997 Wiley-Liss, Inc.     

5-HT2 receptor regulation of acetylcholine release induced by dopaminergic stimulation in rat striatal slices

The role of 5-hydroxytryptamine (5-HT) receptor subtypes in acetylcholine (ACh) release induced by dopamine or neurokinin receptor stimulation was studied in rat striatal slices. The dopamine D₁ receptor agonist SKF 38393 potentiated in a tetrodotoxin-sensitive manner the K⁺-evoked [³H]ACh release while SCH 23390, a dopamine D₁ receptor antagonist, had no effect. [³H]ACh release was decreased by the dopamine D₂ receptor agonist LY 171555 (quinpirole) and slightly potentiated by the dopamine D₂ receptor antagonist haloperidol. The selective neurokinin NK₁ receptor agonist [Sar⁹, met(O₂)¹¹]SP also potentiated K⁺-evoked release of [³H]ACh. GR 82334, a NK₁ receptor antagonist, blocked not only the effect of [Sar⁹, met(O₂)¹¹]SP but also the release of ACh induced by the D₁ receptor agonist SKF 38393. Among the 5-HT agents studied, only the 5-HT_2A receptor antagonists ketanserin and ritanserin were able to reduce the ACh release induced by dopamine D₁ receptor stimulation. Mesulergine, a more selective 5-HT_2C antagonist, showed an intrinsic releasing effect but did not affect K⁺-evoked ACh release induced by SKF 38393. Methysergide and methiothepin, mixed 5-HT_1/2 antagonists, as well as ondansetron, a 5-HT₃ receptor antagonist, showed an intrinsic effect on ACh release, their effects being additive to that of SKF 38393. 5-HT₂ receptor agonists were ineffective. However, the 5-HT₂ agonist DOI was able to prevent the antagonism by ketanserin of the increased [³H]ACh efflux elicited by SKF 38393, suggesting a permissive role of 5-HT_2A receptors. None of the above indicated 5-HT agents was able to reduce the ACh release induced by the selective NK₁ agonist. The results suggest that 5-HT₂ receptors, probably of the 5-HT_2A subtype, modulate the release of ACh observed in slices from the rat striatum after stimulation of dopamine D₁ receptors. It seems that this serotonergic control is exerted on the interposed collaterals of substance P-containing neurons which promote ACh efflux through activation of NK₁ receptors located on cholinergic interneurons.     

Localization and Characterization of Dopamine D1 Receptors in Sheep Hypothalamus and Striatum

Dopamine receptors are pharmacologically grouped as D₁ and D₂ receptors. Previous research in the ewe has shown that central D₁ receptors may have a role in facilitating prolactin release. The aims of this study were therefore to localize and characterize D₁ binding sites in the hypothalamus of sheep. For comparison, a known D₁ receptor-rich tissue (striatum) was also studied. The bioactivities of several D₁ analogues were also assessed for their efficacy in sheep tissue. In vitro autoradiography with [¹²⁵I]-SCH23982 was used to localize D₁ binding sites. The ventromedial hypothalamic nucleus (VMH) displayed moderate levels of specific binding, localized to the medial portion of the nucleus. Low levels of specific binding were seen in the preoptic area, supraoptic nucleus and anterior hypothalamic area. The suprachiasmatic nucleus, median eminence and arcuate nucleus did not show specific binding. As expected the striatum displayed high levels of specific binding. The VMH, preoptic area, median eminence, striatum and anterior pituitary were examined with radioligand binding studies to quantify and characterize D₁ binding sites. Scatchard analysis gave K_D 1.04 nM and B_max 127.4 fmol/mg protein for VMH and K_D 1.99 nM and B_max 454.6 fmol/mg protein for striatum. While specific binding occurred in the preoptic area and median eminence this binding did not show saturation characteristics. Specific binding was not observed in the anterior pituitary. Affinities determined by competitive binding studies showed that the binding sites in both VMH and striatum have the characteristics of a D₁ receptor, that is, high affinity for the D₁ agonists and antagonists, low affinity for dopamine and the serotonergic antagonist ketanserin and extremely low affinity for the D₂ agonists and noradrenaline. Adenylate cyclase studies showed that in the striatum dopamine and the D₁ agonists, fenoldopam and SKF38393, were able to cause significant dose-dependent increases in adenylate cyclase activity. In contrast the D₁ agonist, SKF82958, was inactive in this system. The D₁ antagonists SCH23390 and SCH39166, but not SKF83566, abolished the adenylate cyclase response to 50 μM dopamine. In the VMH the D₁ agonist SKF38393, but not dopamine, stimulated adenylate cyclase activity. In conclusion, these results demonstrate that D₁ binding sites exist within the hypothalamus in the VMH and that these binding sites have the characteristics of D₁ receptors. These receptors are a potential site of action for dopamine in facilitating prolactin release. In addition, the results show that at least for some dopamine analogues, receptor binding affinity does not always correlate with biological activity.     

[H]Nemonapride Binding in Human Caudate and Putamen

The binding of [³H]nemonapride to human postmortem caudate and putamen tissue was autoradiographically investigated using several antipsychotic drugs. Saturation experiments revealed a single population of binding sites (dissociation constant (K_D) 0.38 ± 0.01 nM, and total binding capacity (B_MAX) 55 fmol/TE). Prototypic dopamine (DA) receptors antagonists displaced [³H]nemonapride in a monophasic manner. The order of displacement potency was expected for DA D₂-like receptors: spiperone > (+)butaclamol ≥ chlorpromazine > (−)sulpiride > ketanserin. Displacement with serotonergic antagonists suggests that in human caudate and putamen tissue [³H]nemonapride may have a very low affinity serotonergic component. However, [³H]nemonapride displays a high affinity and selectivity for DA D₂-like receptors and should make it a preferred compound for tritium-based autoradiography.     

Regional adaptation of muscarinic receptors and choline uptake in brain following repeated administration of diisopropylfluorophosphate and atropine

The specific [³H]QNB binding and high-affinity uptake of [¹⁴C]choline in 8 brain regions (cerebral cortex, hippocampus, hypothalamus, thalamus, striatum, midbrain, cerebellum and brainstem) after repeated administration of DFP and atropine to guinea-pigs were simultaneously measured. Following repeated DFP administration, AChE was markedly depressed in each brain region. In these animals, there was a significant decrease in specific [³H]QNB binding in the cerebral cortex, hippocampus and striatum, whereas the [³H]QNB binding in the rest of brain regions was unchanged. Scatchard analysis revealed a 36% decrease in the B_max value for the striatal [³H]QNB binding without a change in theK_d value, suggesting a change in the receptor density. In contrast, repeated atropine administration produced a significant enhancement in the [³H]QNB binding only in the hippocampus and striatum. The B_max value in the striatum increased by 21% without a change in theK_d value.In addition to the receptor alteration, high affinity uptake of [¹⁴C]choline in the hippocampus and striatum was significantly decreased by DFP treatment, while that in the striatum increased by atropine treatment. Thus the present study has demonstrated that a prolonged activation and blockade of central muscarinic receptors resulted in specific adaptation in both the receptor density and ACh availability at the synapses in the cerebral cortex, hippocampus and striatum.     

Evaluation of dopamine D₂/₃ specific binding in the cerebellum for the positron emission tomography radiotracer [¹¹C]FLB 457: implications for measuring cortical dopamine release

In a recent positron emission tomography (PET) study, we demonstrated the ability to measure amphetamine‐induced dopamine (DA) release in the human cortex with the DA D_2/3 radioligand [¹¹C]FLB 457. As previous studies in animals have shown that a relatively high fraction of the [¹¹C]FLB 457 signal in the cerebellum represents specific binding to D_2/3 receptors, there was concern that the use of the cerebellum as a measure of nonspecific binding (i.e., reference region) to derive [¹¹C]FLB 457 binding potential (BP) (BP_ND) would bias cortical DA release measurements. Thus, we evaluated the fractional contribution of specific binding to D_2/3 receptors in the human cerebellum for [¹¹C]FLB 457. Six healthy human subjects (5M/1F) were studied twice with [¹¹C]FLB 457, once at baseline and again following a single oral dose of 15 mg of aripiprazole, a D_2/3 partial agonist. [¹¹C]FLB 457 distribution volume (V_T) was estimated using kinetic analysis in the cortical regions of interest and potential reference regions. The change in [¹¹C]FLB 457 V_T following aripiprazole ranged from ?33 to ?42% in the cortical regions of interest (ROIs). The aripiprazole‐induced change in [¹¹C]FLB 457 V_T in three potential reference regions suggests significant specific binding the cerebellum (CER, –17 ± 12%), but not pons (PON, –10 ± 10%) and centrum semiovale (CESVL, –3 ± 12%). Nevertheless, a reanalysis of the published [¹¹C]FLB 457 test–retest and amphetamine studies suggests that the use of the PON V_T and CESVL V_T as an estimate of nonspecific binding to derive [¹¹C]FLB 457 BP_ND in DA release studies is unlikely to be successful because it leads to less reproducible outcome measures, which in turn diminishes the ability to measure DA release in the cortex. D_2/3 blocking studies with aripiprazole and [¹¹C]FLB 457 suggest specific binding to D_2/3 receptors in the cerebellum. These data also suggest that the contribution of specific binding to D_2/3 receptors in the cerebellum is lower than that in the cortical ROIs and that CER V_T is mostly representative of nonspecific binding. Nevertheless, caution is advised when using reference tissue methods that rely solely on the cerebellum signal as an input function to quantify [¹¹C]FLB 457 BP_ND. Synapse, 2011. © 2011 Wiley‐Liss, Inc.     

Desensitization of D1 dopamine receptors down-regulates the Gsα subunit of G protein in SK-N-MC neuroblastoma cells

The effect of long-term (72 h) treatment with dopamine D₁ receptor agonists, SKF 38393 and dopamine on D₁ dopamine receptor and G-protein (G_Sα) was investigated in SK-N-MC neuroblastoma cells. The prolonged treatment of cells with 10 μM SKF 38393 or 10 μM dopamine resulted in a decrease in dopamine D₁ receptor by 41 and 81%, respectively, as measured by specific antagonist [³H]SCH 23390 binding. Similary, the prolonged treatment of SK-N-MC cells with 10 μM SKF 38393 or 10μM dopamine resulted in a reduction of the level of G_Sα subunit of G-protein. The results indicate that agonist-induced down-regulation of D₁ dopamine receptor may also modulate G-proteins.     

Development of beta-adrenergic receptors in normal and mutant mouse cerebellum

[3H]-dihydroalprenolol was used to examine the development of beta-adrenoceptors in the cerebellum of weaver, reeler, staggerer, and jimpy neurologically mutant mice and their normal counterparts. In normal animals the greatest increase in [3H] binding occurred during the postnatal interval of 10-15 days, and maximum values were obtained at about 3 weeks. Binding was saturable with an apparent dissociation constant of 0.26 nM, and the affinity of [3]-dihydroalprenolol for its receptor did not change significantly during development. [3H]-dihydroalprenolol binding was significantly reduced in weaver, reeler, and staggerer (14-22% of control values) at 20 days, but not in the jimpy mutant. These results are discussed in relation to the ontogeny of beta-adrenoreceptors with the known noradrenergic innervation of the cerebellum, and to the paucity of both granule neurons and myelination which occurs in the neurological mutants.     

Kinetics of dopamine and noradrenaline transport in synaptosomes from cerebellum, striatum and frontal cortex of normal and reeler mice.

Recent evidence indicates that the cerebellum has a dopaminergic system. In order to elucidate further the dopaminergic system in the cerebellum, we investigated the transport of dopamine (DA) in synaptosomal preparations of normal and reeler mice. For comparative purposes we also studied DA transport in synaptosomal preparations from striatum and frontal cortex and compared DA transport to noradrenaline (NA) transport. [3H]-DA transport into cerebellar synaptosomes was found to be a Na(+)-dependent, two component system--a high affinity, low capacity and a low affinity, high capacity. In striatum [3H]-DA is transported by a similar high but different low affinity component. Maximal velocities of both transport components in the striatum were higher than the corresponding ones in the cerebellum. In the frontal cortex we also observed two [3H]-DA transport components with affinities significantly lower than those in cerebellum and striatum. [3H]-NA transport into synaptosomes, prepared from the three brain regions studied, showed two transport components with similar Kt and Vmax values, except for the high affinity component in striatum whose affinity is lower. In reeler mice [3H]-DA transport was different from normal only in the cerebellum where the maximal velocity for both transport components was significantly higher (2x). In contrast, no significant difference was observed in the transport of [3H]-NA. The accumulated [3H]-DA from cerebellar slices was found to be releasable by K+ stimulation, in a Ca(++)-dependent manner, and most of the released radioactivity was in the form of [3H]-DA. These results indicate that in the cerebellum there is a low-density dopaminergic system which is distinct from the corresponding noradrenergic system.