Dopamine D2-like receptors on human peripheral blood lymphocytes: a radioligand binding assay and immunocytochemical study

1 Peripheral blood lymphocytes express dopamine D₁-like and D₂-like receptors which were investigated using radioligand binding assay and molecular biology techniques. Analysis of dopamine D₂-like receptors expressed by human peripheral blood lymphocytes with radioligand binding assay may offer a rapid technique for assessing receptor changes in disorders characterized by involvement of the dopaminergic system. However, the suitability of radioligand binding assay techniques to measure dopamine D₂-like receptors is questioned. 2 In view of the discrepancy between data of dopamine D₂-like receptor determination with molecular biology and radioligand binding assay techniques, we have assayed dopamine D₂-like receptors expressed by human peripheral blood lymphocytes using as radioligands the dopamine receptor agonist 7-[³H]-hydroxy-N,N-di-n-propyl-2-aminotetraline ([³H]-7-OH-DPAT) and two antagonists ([³H]-spiperone and [³H]-nemonapride). 3 Analysis of saturation curves revealed a concentration-dependent binding of all compounds to human peripheral blood lymphocytes. Dissociation constant (K_d) values averaged between 0.15 and 0.40 n m for different radioligands. The maximum density of binding sites (B_max) was low, ranging from 4.15 ± 0.05 fmol/10⁶ cells with [³H]-spiperone and 8.66 ± 0.04 fmol/10⁶ cells with [³H]-7-OH-DPAT. 4 Displacement curves of [³H]-7-OH-DPAT, [³H]-spiperone and [³H]-nemonapride binding to human peripheral blood lymphocytes revealed, using radioligand concentrations giving the highest specific:non-specific binding ratio, a pharmacological profile consistent with the labelling of dopamine D₂-like receptors. The use of higher radioligand concentrations resulted in a poorly displaceable and characterizable binding. 5 Detection of dopamine D₂, D₃ and D₄ receptor immunoreactivity in cytospin centrifuged peripheral blood lymphocytes revealed dopamine D₃ and D₄ but not D₂ receptor immunostaining. 6 The above findings indicate in agreement with molecular biology studies, that dopamine D₂-like receptors expressed by human peripheral blood lymphocytes belong to the D₃ and D₄ receptor subtypes. These receptors are detectable using either dopamine D₂-like receptor agonists and antagonists as radioligands if controlled experimental conditions are followed. The standardisation of immunocytochemical techniques for detecting human peripheral blood lymphocyte dopamine receptors may contribute to clarify their role in lymphocyte function or as a peripheral marker of the status of the dopaminergic system.     

Amphetamine Decreases α2C-Adrenoceptor Binding of [11C]ORM-13070: A PET Study in the Primate Brain

Background:

The neurotransmitter norepinephrine has been implicated in psychiatric and neurodegenerative disorders. Examination of synaptic norepinephrine concentrations in the living brain may be possible with positron emission tomography (PET), but has been hampered by the lack of suitable radioligands.

Methods:

We explored the use of the novel α_2C-adrenoceptor antagonist PET tracer [¹¹C]ORM-13070 for measurement of amphetamine-induced changes in synaptic norepinephrine. The effect of amphetamine on [¹¹C]ORM-13070 binding was evaluated ex vivo in rat brain sections and in vivo with PET imaging in monkeys.

Results:

Microdialysis experiments confirmed amphetamine-induced elevations in rat striatal norepinephrine and dopamine concentrations. Regional [¹¹C]ORM-13070 receptor binding was high in the striatum and low in the cerebellum. After injection of [¹¹C]ORM-13070 in rats, mean striatal specific binding ratios, determined using cerebellum as a reference region, were 1.4±0.3 after vehicle pretreatment and 1.2±0.2 after amphetamine administration (0.3mg/kg, subcutaneous). Injection of [¹¹C]ORM-13070 in non-human primates resulted in mean striatal binding potential (BP _ND) estimates of 0.65±0.12 at baseline. Intravenous administration of amphetamine (0.5 and 1.0mg/kg, i.v.) reduced BP _ND values by 31–50%. Amphetamine (0.3mg/kg, subcutaneous) increased extracellular norepinephrine (by 400%) and dopamine (by 270%) in rat striata.

Conclusions:

Together, these results indicate that [¹¹C]ORM-13070 may be a useful tool for evaluation of synaptic norepinephrine concentrations in vivo. Future studies are required to further understand a potential contribution of dopamine to the amphetamine-induced effect.     

Long-term effects of postnatal amphetamine treatment on striatal protein kinase A activity, dopamine D1-like and D2-like binding sites, and dopamine content

The purpose of the present study was to determine whether exposure to amphetamine during the preweanling period would alter dopaminergic functioning in the dorsal striatum of adult rats. In three experiments, we assessed the effects of repeated amphetamine treatment on striatal protein kinase A (PKA) activity, dopamine (DA) D₁-like and D₂-like binding sites, and DA content. Rats were pretreated with saline or amphetamine (2.5 mg/kg, ip) for 7 consecutive days starting on postnatal day (PD) 11. At PD 90, rats were killed and their dorsal striata (i.e., caudate–putamen) were removed and frozen until time of assay. Amphetamine pretreatment produced long-term reductions in both striatal PKA activity and DA content. Early amphetamine exposure also resulted in an upregulation of D₂-like binding sites, while leaving D₁-like binding sites unaffected. It is likely that the upregulation of D₂-like binding sites was stimulated by the persistent decline in striatal DA levels. Although speculative, it is possible that excess striatal D₂-like receptors were responsible for inhibiting PKA activity through actions on the cAMP signal transduction pathway. The behavioral relevance of these amphetamine-induced neurochemical changes has not yet be determined.     

HIGH-LEVEL EXPRESSION OF RAT D1A DOPAMINE RECEPTOR cDNA IN MOUSE FIBROBLAST LTK- CELLS BY n-BUTYRATE

1. In order to develop a simple, efficient system for the high-level expression of dopamine receptors in eukaryotic cells, we have studied the effects of n-butyrate on the expression of rat D_1A dopamine receptor cDNA in mouse fibroblast LTK^- cells as compared with those of n-butyrate on endogenous D₁ receptor levels in opossum kidney cells. 2. In the transfected LTK^- cell membranes with pRc/CMV-D_1A receptor cDNA, a selective D₁ dopamine antagonist, [³H]-SCH 23390, exhibited a K₄ of 0.9 ± 0.1 nmol/L and a B_max of 0.35 ± 0.05 pmol/mg protein (n= 5). 3. Addition of n-butyrate (2–10 mmol/L) to the culture medium for 48 h dose-dependently increased the D_1A receptor level up to 1.5 ± 0.3 pmol/mg protein (n= 7), although the K₄ values were not affected. The increase in receptor level was accompanied by an elevation of selective D₁ agonist-induced adenylyl cyclase activity. 4. In contrast, n-butyrate treatment (2–10 mmol/L) did not affect either endogenous D₁ receptor levels or fendoldopam-induced adenylyl cyclase activity in opossum kidney cells. 5. These results suggest n-butyrate is a useful tool for obtaining high-level expression of D_1A dopamine receptor cDNA in mouse fibroblast LTK^- cells.     

A PET Study on the Acute Effect of Ethanol on Striatal D2 Dopamine Receptors with [11C]Raclopride in Healthy Males

The effect of acute oral ethanol intake (1·0 g/kg) on cerebral D2-receptors ([¹¹C]raclopride binding) was studied in seven healthy volunteers, using water and alcohol in two separate 59-min PET sessions. In the alcohol experiments, the blood ethanol concentration at the beginning of the imaging was 26·4±3·8 mmol/l and remained stable during the PET session. Ethanol was not found to influence binding of [¹¹C]raclopride to the dopamine D2 receptors in the human striatum, as indicated by the unchanged ratio B_max/K_d of the whole (left and right) striatum. The T_max values of the control and ethanol experiments did not differ in the whole striatum, but the right to left difference of striatal T_max was turned from negative to positive by ethanol (P=0·02). However, the difference between hemispheres in B_max/K_d was not significantly altered by ethanol intake. There was considerable interindividual variation in the response of all the above parameters to acute ethanol exposure. According to the present results, the acute effects of peroral ethanol exposure on striatal D2 receptor binding potential are of relatively small magnitude in man. However, the changes in T_max suggest that ethanol may influence the right–left difference of [¹¹C]raclopride binding. © 1997 by John Wiley & Sons, Ltd.     

Chronic phencyclidine,like amphetamine,produces a decrease in [3H]spiroperidol binding in rat striatum

Rats were treated with d-amphetamine sulfate (5 and 10 mg/kg i.p.) and phencyclidine (PCP) (5 mg/kg i.p.) twice per day. After 21 days, [³H]spiroperidol binding in striatum was reduced by all treatments; receptor number (B_max) and not affinity (K_D) was affected. These results suggest that the psychotic effect of PCP may, like those of amphetamine, involve changes in dopamine receptors.     

Striatal D2 dopamine receptor binding characteristics in vivo in patients with alcohol dependence

Striatal D₂ dopamine receptor characteristics of nine male patients with alcohol dependence abstinent for 1–68 weeks and eight healthy male volunteers were studied in vivo with positron emission tomography. The selective D₂ receptor ligand [¹¹C]raclopride and equilibrium model was used for D₂ receptor density (B_max) and affinity (K_d) measurements. A trend for a decreased striatal D₂ receptor density and for reduced D₂ receptor affinity was observed in patients with alcohol dependence. These parameters were not statistically significantly different between alcoholics and controls, but the ratio between D₂ receptor density and affinity (B_max/K_d or the striatum/cerebellum ratio from the high specific activity scan) was highly significantly lower in alcoholics than that of controls. In conclusion, the low D₂ dopamine receptor B_max/K_d ratio (striatum/cerebellum ratio) indicates that specific aspects of striatal [¹¹C]raclopride binding in vivo are deviant in alcoholics compared to controls. The result is compatible with a reduced avidity of striatal dopamine D₂ receptors in alcoholics, which is in line with the idea that D₂ dopaminergic mechanisms are involved in the biology of alcohol dependence in man.     

Pharmacological characterization and autoradiographic localization of a putative dopamine D4 receptor in the heart

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Long-Term Exposure to Oral Methylphenidate or dl-Amphetamine Mixture in Peri-Adolescent Rhesus Monkeys: Effects on Physiology,Behavior, and Dopamine System Development

The stimulants methylphenidate and amphetamine are used to treat children with attention deficit/hyperactivity disorder over important developmental periods, prompting concerns regarding possible long-term health impact. This study assessed the effects of such a regimen in male, peri-adolescent rhesus monkeys on a variety of cognitive/behavioral, physiological, and in vivo neurochemical imaging parameters. Twice daily (0900 and 1200 hours), for a total of 18 months, juvenile male monkeys (8 per group) consumed either an unadulterated orange-flavored solution, a methylphenidate solution, or a dl-amphetamine mixture. Doses were titrated to reach blood/plasma levels comparable to therapeutic levels in children. [¹¹C]MPH and [¹¹C]raclopride dynamic PET scans were performed to image dopamine transporter and D₂-like receptors, respectively. Binding potential (BP_ND), an index of tracer-specific binding, and amphetamine-induced changes in BP_ND of [¹¹C]raclopride were estimated by kinetic modeling. There were no consistent differences among groups on the vast majority of measures, including cognitive (psychomotor speed, timing, inhibitory control, cognitive flexibility), general activity, physiological (body weight, head circumference, crown-to-rump length), and neurochemical (ie, developmental changes in dopamine transporter, dopamine D₂ receptor density, and amphetamine-stimulated dopamine release were as expected). Cytogenetic studies indicated that neither drug was a clastogen in rhesus monkeys. Thus, methylphenidate and amphetamine at therapeutic blood/plasma levels during peri-adolescence in non-human primates have little effect on physiological or behavioral/cognitive development.     

Dopamine D2 receptors selectively labeled by a benzamide neuroleptic: [3H]-YM-09151-2

Summary In order to label dopamine D₂ receptors selectively we tritiated the potent benzamide neuroleptic, YM-09151-2 (26.7 Ci/mmol). The binding of [³H]-YM-09151-2 to canine striatal membranes was saturable and specific with a K _D of 57 pmol/l and B _max of 36 pmol/g tissue as determined by Scatchard analysis. The K _D, but not the B _max, of [³H]-YM-09151-2 increased 6-fold in the absence of sodium chloride. [³H]-YM-09151-2 labeled 40% more sites than [³H]-spiperone in the same tissue homogenate. [³H]-YM-09151-2 binding was inhibited by dopaminergic drugs in a concentration and stereoselective manner with the appropriate dopamine D₂ receptor profile. Thus, dopamine agonists inhibited [³H]-YM-09151-2 binding to canine striatal membranes with the following rank order of potency: (–)-N-n-propylnorapomorphine > apomorphine > (±)-6,7-dihydroxy-2-aminotetralin > (+)-N-n-propylnorapomorphine > dopamine > (–)-noradrenaline > serotonin > (–)-isoprenaline. Dopaminergic antagonists competed for [³H]-YM-09151-2 binding with the following order of potency: spiperone > (+)-butaclamol > haloperidol > clebopride > (–)-sulpiride > SCH-23390 > (–)-butaclamol. Furthermore, dopamine agonists recognized 2 states of the receptor labeled by [³H]-YM-09151-2, D ₂ ^high and D ₂ ^low . The D ₂ ^high state of the receptor could be converted to D ₂ ^low by guanine nucleotides and sodium ions as is the case for [³H]-spiperone binding to D₂ receptors. [³H]-YM-09151-2 appears to be a more selective ligand for dopamine D₂ receptors than [³H]-spiperone, since YM-09151-2 displays approximately 9-fold lower affinity than spiperone for cortical serotonergic (S₂) receptors. [³H]-YM-09151-2 may become a useful tool for the selective characterization of dopamine D₂ receptors.Abbreviations used (±)ADTN (±)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene - NPA N-n-propylnorapomorphine - Gpp(NH)p 5-guanylylimidodiphosphate     

Imaging Nicotine- and Amphetamine-Induced Dopamine Release in Rhesus Monkeys with [11C]PHNO vs [11C]raclopride PET

The radiotracer [¹¹C]PHNO may have advantages over other dopamine (DA) D₂/D₃ receptor ligands because, as an agonist, it measures high-affinity, functionally active D₂/D₃ receptors, whereas the traditionally used radiotracer [¹¹C]raclopride measures both high- and low-affinity receptors. Our aim was to take advantage of the strength of [¹¹C]PHNO for measuring the small DA signal induced by nicotine, which has been difficult to measure in preclinical and clinical neuroimaging studies. Nicotine- and amphetamine-induced DA release in non-human primates was measured with [¹¹C]PHNO and [¹¹C]raclopride positron emission tomography (PET) imaging. Seven adult rhesus monkeys were imaged on a FOCUS 220 PET scanner after injection of a bolus of [¹¹C]PHNO or [¹¹C]raclopride in three conditions: baseline; preinjection of nicotine (0.1 mg/kg bolus+0.08 mg/kg infusion over 30 min); preinjection of amphetamine (0.4 mg/kg, 5 min before radiotracer injection). DA release was measured as change in binding potential (BP_ND). Nicotine significantly decreased BP_ND in the caudate (7±8%), the nucleus accumbens (10±7%), and in the globus pallidus (13±15%) measured with [¹¹C]PHNO, but did not significantly decrease BP_ND in the putamen or the substantia nigra or in any region when measured with [¹¹C]raclopride. Amphetamine significantly reduced BP_ND in all regions with both radiotracers. In the striatum, larger amphetamine-induced changes were detected with [¹¹C]PHNO compared with [¹¹C]raclopride (52–64% vs 33–35%, respectively). We confirmed that [¹¹C]PHNO is more sensitive than [¹¹C]raclopride to nicotine- and amphetamine-induced DA release. [¹¹C]PHNO PET may be more sensitive to measuring tobacco smoking-induced DA release in human tobacco smokers.     

Dopaminergic mechanisms mediating the long-term expression of locomotor sensitization following pre-exposure to morphine or amphetamine

The role of dopaminergic mechanisms in opiate- and psychostimulant-induced long-term locomotor sensitization was investigated. To that aim, rats were behaviourally sensitized with morphine or amphetamine and 3 weeks after cessation of treatment challenged with various direct and indirect dopamine agonists. Both morphine- and amphetamine-pretreated rats displayed sensitization of the locomotor effects of amphetamine, cocaine, and the selective dopamine reuptake inhibitor GBR-12909. Sensitization of the locomotor stimulant effects of the dopamine D₂/D₃ receptor agonist quinpirole was observed in amphetamine- but not morphine-pretreated rats. In contrast, morphine-, but not amphetamine-pretreated rats appeared hyposensitive to the locomotor inhibitory effects of a low, presumably D₂-autoreceptor selective, dose of quinpirole. Neither pretreatment induced sensitization to the dopamine D₁/D₂ agonist apomorphine or the dopamine D₁ agonist SKF-82958. In fact, the locomotor stimulant effects of SKF-82958 appeared to be decreased in animals pre-exposed to amphetamine. These results suggest that functional changes in presynaptic dopamine release mechanisms represent common neuroadaptations involved in the long-term expression of morphine- and amphetamine-induced locomotor sensitization. Presynaptic dopamine D₂ and postsynaptic D₂ and/or D₃ receptors are differentially involved in the expression of morphine- and amphetamine-induced locomotor sensitization. In a parallel study, we report that all of the drugs that elicited sensitized locomotor responses in morphine- or amphetamine-pretreated rats caused reinstatement of previously extinguished heroin- or cocaine-seeking behaviour, respectively. Taken together, these data suggest a marked relationship between drug-seeking behaviour and drug sensitization. Received: 22 May 1998/Final version: 12 October 1998     

Kinetic properties of the in vivo accumulation of 3H-(−)-N-n-propylnorapomorphine in mouse brain

Summary (1) The influence of various dopamine (DA) receptor agonists and antagonists on the kinetic properties of the specific binding of ³H(–)-N-n-propylnorapomorphine (NPA) in the mouse striatum in vivo was studied. The specific binding of ³H-NPA, defined as the difference between the radioactivity in the striatum and cerebellum, was completely antagonized by the selective D-2 receptor antagonist raclopride but not by the selective D-1 antagonist SCH 23390, showing that the binding occurs exclusively to the D2 receptors. (2) The selective D-2 receptor agonists pergolide and quinpirole inhibited the ³H-NPA binding biphasically at low doses, indicating that these DA receptor agonists have high affinities for a subfraction (10 to 30%) of the NPA binding sites. (3) Increasing the synaptic DA concentration by DA release [(+)-amphetamine] or uptake blockade (amfonelic acid and methylphenidate) inhibited the ³HNPA binding in a competitive manner (unchanged B _max, increased K _D). Depletion of the DA in the synapses by -butyrolactone or reserpine decreased the apparent K _D value. (4) The possibility of estimating changes in the synaptic DA concentration from changes in the apparent K _D is discussed. According to the results obtained, the normal concentration of DA in the synaptic cleft in mouse striatum in vivo is about 40 nmol/l and this concentration is increased 2 to 3 times by (+)-amphetamine and amfonelic acid in doses which evoke hyperactivity and stereotypic behaviour.Send offprint requests to S. B. Ross at the above address     

Kinetic properties of the accumulation of 3H-raclopride in the mouse brain in vivo

Summary The kinetic properties of the accumulation of ³H-raclopride, a selective dopamine (DA) D-2 receptor antagonist, in mouse striatum in vivo was examined under various experimental conditions. The accumulation in striatum was saturable in contrast to that in cerebellum, which linearily increased with the dose. The specific binding of ³-Hraclopride in the striatum, defined as the difference in the accumulation in striatum and cerebellum 30 min after the injection was completely inhibited by the D-2 receptor antagonists spiperone and (-I-)-butaclamol [but not (–)butaclamol] and the DA receptor agonist N-n-propylnorapomorphine. The mean B _max value of the specific binding was 40.7 ± 2.8 pmol/g tissue and the mean apparent K _D value, based on the dose injected, was 87.8 ± 11.5 nmol/kg i. v. (18 different experiments). Pretreatment of the mice with a single injection of reserpine 4 h or 3 days beforehand reduced the apparent K _D value which in part seemed to be due to the decreased concentration of synaptic DA. Similarly, -butyrolactone injected immediately before raclopride reduced the apparent KD value, whereas amfonelic acid and (–)-amphetamine increased the observed K _D values. These findings indicate competition between endogenous DA and raclopride for the D-2 receptors. Both reserpine and -butyrolactone increased the apparent B _max value by about 50% which indicates a receptor pool of DA for which raclopride does not compete.Send offprint requests to S. B. Ross at the above address     

Actions of roxindole at recombinant human dopamine D2, D3 and D4 and serotonin 5-HT1A, 5-HT1B and 5-HT1D receptors

Roxindole is a potential antidepressant agent. The present study determined its affinity and agonist efficacy at recombinant human (h) dopamine hD₂, hD₃ and hD₄ and serotonin (5-HT) h5-HT_1A, h5-HT_1B and h5-HT_1D receptors. Roxindole exhibited high affinity at hD₃ as well as at hD₂ (short isoform) and hD₄ (4-repeat isoform) receptors (pK _i values 8.93, 8.55 and 8.23, respectively). Further, it displayed high affinity at h5-HT_1A receptors (pK _i = 9.42) but modest affinity at 5-HT_1B and 5-HT_1D receptors (pK _i values 6.00 and 7.05, respectively). In [³⁵S]GTPγS binding experiments, roxindole was >20-fold more potent in stimulating [³⁵S]GTPγS binding at hD₃ than at hD₂ or hD₄ receptors (pEC₅₀ = 9.23 vs. 7.88 and 7.69). However, whereas roxindole exhibited partial agonist activity at hD₃ and hD₄ sites (E _max = 30.0% and 35.1%, respectively, relative to dopamine = 100%), it only weakly activated hD₂ receptors (E _max = 10.5%). Roxindole potently blocked dopamine-stimulated [³⁵S]GTPγS binding at hD₂ receptors (pK _B = 9.05). In comparison, the dopamine receptor agonist, (-)quinpirole, acted as a partial agonist at hD₃ and hD₄ sites (E _max = 67.4% and 66.3%, respectively) but surpassed the efficacy of dopamine at hD₂ receptors (E _max = 132%). At h5-HT_1A receptors, roxindole behaved as a high affinity (pK _i = 9.42) partial agonist (E _max = 59.6%, relative to 5-HT = 100%), whereas (-)quinpirole had negligible activity. The selective 5-HT_1A antagonist, WAY 100,635, blocked roxindole (100 nM)-stimulated [³⁵S]GTPγS binding at h5-HT_1A receptors in a concentration-dependent manner (pK _B = 9.28). Roxindole only weakly stimulated [³⁵S]GTPγS binding at 5-HT_1B and 5-HT_1D receptors (E _max = 27.1% and 13.7%). The present data suggest that roxindole activates mainly D₃ vs. D₂ or D₄ receptors and 5-HT_1A vs. 5-HT_1B or 5-HT_1D receptors. Activation of D₃ and/or 5-HT_1A receptors may thus contribute to its potential antidepressant properties. Received: 4 February 1999 / Accepted: 29 March 1999     

Interaction of two sulfhydryl reagents with a cation recognition site on the neuronal dopamine carrier evidences small differences between [3H]GBR 12783 and [3H]cocaine binding sites

We have compared the effect of treating rat striatal cell membranes with ionic hydrophilic sulfhydryl reagents on the specific bindings of [³H]cocaine and of [³H]GBR 12783 (1-[2-(diphenylmethoxy)ethyl]4-(3-phenyl-2-[1-³H]propenyl)-piperazine) to the neuronal transporter of dopamine. Treatment with 1 mmol/1 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) resulted in similar time-and concentration-dependent reductions of the specific binding of both radioligands. None of the uptake blockers tested afforded any protection against 1 mmol/1 DTNB. Addition of (sub)millimolar concentrations of CaCl₂ or MgCl₂, or 250 mmol/1 KCl to a treatment medium containing 10 mmol/l Na + significantly increased the DTNB-induced reduction of the specific binding of both radioligands. Cations were likely to be responsible for this effect since ions in combination with DTNB induced similar reductions in binding when either 1 mmol/l CaCl₂ or 50–250 mmol/l NaCl were added. Effects of cations on the DTNB-induced inhibition of binding were generally more marked on [³H]GBR 12783 than on [³H]cocaine binding. When added to a medium containing 10 mmol/1 Na⁺ 1 mmol/1 DTNB induced a reduction in the B_max of the specific binding of both radioligands. Addition of 1 mmol/l Ca²⁺ maintained or increased this B_max reduction and elicited a decrease in affinity which was significant for [³H]GBR 12783 binding.Treatment of membranes with the sodium salt of p-hydroxymercurybenzenesulfonate (pHMBS) induced time-and concentration-dependent decreases in [³H]GBR 12783 binding which were significantly greater than decreases in [³H]cocaine binding. However, 50mol/lpHMBS produced a similar decrease in the B_max of the specific binding of both radioligands. The pHMBS-induced reduction of [³H]GBR 12783 binding was not reversed by drugs whose action is purely that of uptake inhibition or by substrates of the dopamine carrier. Some of these drugs (100 mol/l dopamine, 1 mol/l mazindol or 100 mol/l cocaine) protected the specific binding of [³H]cocaine against the effects of pHMBS, whereas 1 mmol/1 p-tyramine, 10 mol/l nomifensine and 10 nmol/l GBR 12783 were ineffective. Addition of 120 mmol/l Na⁺, 1 mmol/l Ca²⁺ or 10 mmol/l Mg²⁺ to a treatment medium containing 10 mmol/l Na⁺ significantly reduced the effects of pHMBS on the specific binding of both radioligands. When striatal cell membranes were treated in a medium containing 130 mmol/1 Na⁺, there was a general decrease in the effects of ions on the reductions of specific binding produced by DTNB or pHMBS. Cation concentrations which interfered with the actions of DTNB and pHMBS were approximately those which blocked the specific binding of [³H]GBR 12783 when they were present during association of the radioligand (K⁺, Ca ²⁺, Mg²⁺), or, in the case of Na⁺, which are effective in reducing this blockade (Bonnet et al. 1988).The present data are consistent with the existence of mutually exclusive binding sites for [³H]GBR and [³H]cocaine on the neuronal dopamine transporter. The hypothesis of a cation recognition site which could gate admission of uptake inhibitors or carrier substrates to their binding domain on the transporter is discussed.     

Evidence for the sensitivity of operant timing behaviour to stimulation of D1 dopamine receptors

Rationale Temporal differentiation of operant behaviour is sensitive to dopaminergic manipulations. Previous studies using the fixed-interval peak procedure implicated D₂-like dopamine receptors in these effects. However, recent findings suggest that d-amphetamine alters timing performance on the free-operant psychophysical procedure via D₁-like receptors. It is not known whether this effect of d-amphetamine is mimicked by direct D₁-like receptor stimulation. Objective The effects of a D₁-like receptor agonist 6-chloro-2,3,4,5-tetrahydro-1-phenyl-1H-3-benzazepine (SKF-81297) on performance on the free-operant psychophysical procedure and the interaction between SKF-81297 and a D₁-like receptor antagonist 8-bromo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol (SKF-83566) and a D₂-like receptor antagonist haloperidol, were examined. Materials and methods Rats were trained to respond on two levers (A and B) under a free-operant psychophysical schedule, in which sucrose reinforcement was provided intermittently for responding on A during the first half and on B during the second half of 50-s trials. Logistic psychometric functions were fitted to the relative response rate data (percent responding on B [%B] vs time from trial onset [t]) under each treatment condition, and quantitative indices of timing (T₅₀ [value of t corresponding to %B = 50] and the Weber fraction [(T₇₅-T₂₅)/2T₅₀; T₂₅ and T₇₅ are values of t corresponding to %B = 25 and %B = 75] were compared among treatments. Results SKF-81297 (0.8 mg kg⁻¹) reduced T₅₀; this effect was antagonized by SKF-83566 (0.03 mg kg⁻¹) but not by haloperidol (0.05, 0.1 mg kg⁻¹). Conclusions Stimulation of D₁-like dopamine receptors affects performance in the free-operant psychophysical procedure.     

D1 dopamine receptor binding in mood disorders measured by positron emission tomography

D₁ dopamine receptor binding in mood disorders was studied by positron emission tomography (PET) using¹¹C-SCH23390. Ten patients with bipolar mood disorders and 21 normal controls were studied in the drug-free state. The patients were in euthymic (N=6), depressed (N=3) and manic (N=1) states. Regional radioactivity in the brain was followed for 40 min by PET. A two-compartment model was used to obtain the binding potential (k₃/k₄) for the striatum and frontal cortex. The binding potentials for the frontal cortex for the patients were significantly lower than those for normal controls, whereas those for striatum were not significantly different. These findings suggest that D₁ dopamine receptors in the frontal cortex may be in a different state in patients with bipolar mood disorders.Preliminary results of this study were presented at the 17th Congress of Collegium Internationale Neuro-Psychopharmacologicum, Kyoto International Conference Hall, Kyoto, Japan, September 10–14, 1990     

The partial dopamine D2-like receptor agonist terguride functions as an agonist in preweanling rats after a 5-day reserpine regimen

Rationale Treating children and adolescents with partial D₂-like agonists is becoming increasingly common, although few developmental animal studies have assessed the psychopharmacology of this class of drug. Contrary to results from adult rat studies, it has been reported that partial D₂-like agonists may not induce agonist-like behavioral effects in preweanling rats during states of low dopaminergic tone. Objective The purpose of the present study was to determine whether a partial D₂-like agonist would act as an agonist in preweanling rats after a 5-day regimen of the dopamine-depleting agent reserpine or the tyrosine hydroxylase inhibitor α-methyl-dl-p-tyrosine (AMPT). Methods: Sprague–Dawley rats were pretreated with reserpine (1 mg kg^-1 per day) or AMPT (3×200 mg kg^-1 per day) on postnatal day (PD) 16–PD 20. Either 2 h (AMPT) or 5 h (reserpine) after the last pretreatment injection, rats were treated with saline, the partial D₂-like agonist terguride, or the full D₂-like agonist R(−)-propylnorapomorphine (NPA). Distance traveled and repetitive motor movements were measured for 60 min. Results After repeated reserpine treatment, both terguride and NPA increased the distance-traveled scores of preweanling rats; however, only NPA, but not terguride, increased distance-traveled scores after a 5-day regimen of AMPT or an acute injection of reserpine. Conclusions It is now apparent that partial D₂-like agonists are capable of inducing agonist-like behavioral effects in preweanling rats during a state of low dopaminergic tone. For agonistic actions to be observed, the pretreatment regimen must result in substantial and prolonged dopamine depletion.     

The effect of ‘two hit’ neonatal and young-adult stress on dopaminergic modulation of prepulse inhibition and dopamine receptor density

Background and purpose:

A combination of early neurodevelopmental insult(s) and young-adult stress exposure may be involved in the development of schizophrenia. We studied prepulse inhibition (PPI) regulation in rats after an early stress, maternal deprivation, combined with a later stress, simulated by chronic corticosterone treatment, and also determined whether changes in brain dopamine receptor density were involved.

Experimental approach:

Rats were subjected to either 24 h maternal deprivation on postnatal day 9, corticosterone treatment from 8 to 10 weeks of age, or both. At 12 weeks of age, the rats were injected with 0.1, 0.3 or 1.0 mg·kg⁻¹ of apomorphine or 0.5 or 2.5 mg·kg⁻¹ of amphetamine and PPI was determined using automated startle boxes. Dopamine D₁ and D₂ receptor levels were assessed in the nucleus accumbens and caudate nucleus using receptor autoradiography.

Key results:

Young-adult treatment with corticosterone resulted in attenuated disruption of PPI by apomorphine and amphetamine. In some rats, maternal deprivation resulted in reduced baseline PPI which added to the effect of corticosterone treatment. There was no down-regulation of dopamine D₁ or D₂ receptors.

Conclusions and implications:

These results confirm and extend our finding of an inhibitory interaction of developmental stress on dopaminergic regulation of PPI. No corresponding changes in dopamine receptor density were observed in brain regions with a major involvement in PPI regulation, suggesting long-lasting desensitization of dopamine receptor signalling or indirect changes in PPI regulation.