Tritiated Dopamine Research Articles

Pathophysiology of schizophrenia: dopamine hypothesis.

T he following artide is the second in our series to highlight the role of psychopharmacology in contributing to our theories of the etiologic basis of mental disorders. In parallel to the catecholamine hypothesis of depression (presented in the fall 1995 issue of the Journal, pp. 523533), the dopamine hypothesis for schizophrenia developed and became influential in the 1960s and 1970s. The origins of this hypothesis can be traced back to 1938, when Shube1 first reported on amphetamine-induced psychosis. This phenomenon was not widely accepted until 1958, when Connell1 published a monograph on a study of 42 cases. Carlsson and Lindqvist in 19632 were the first to suggest that the antipsychotic properties of neuroleptic medication were related to monoaminergic receptor blockade in the brain. The notion that dopamine had a specific role in the pathogenesis of psychosis gained support when it was shown that normal subjects and nonschizophrenic psychiatric patients could become psychotic when administered high-dose amphetamine and L-dopa.6 In 1975, Seeman and Lee7 reported that the dosages of 25 neuroleptic drugs required to inhibit electrically stimulated release of tritiated dopamine correlated closely with the average clinical dose required to control psychosis. They suggested that neuroleptics acted at presynaptic dopamine terminals. The following year, Creese, Burt, and Snyder reported in the article reprinted below that the ability of a wide array of antipsychotic medications to compete for tritiated haloperidol binding (at what is now referred to as the D2 receptor) predicted the dosage required to inhibit dopamine-mediated behavior in animals and to control psychosis in humans. Seeman et al. independently reported similar findings in a report that was published in Nature two months later.8 In a telephone interview, Dr. Creese described the historical context in which he conducted his study. Dr. Creese completed his Ph.D. at Cambridge University with Susan Iversen, with whom he had conducted behavior studies in 6-hydroxydopamine-lesioned rats. This crystallized his interest in the molecular mechanisms of dopamine-mediated behaviors. Dr. Creese took a postdoctoral position at Johns Hopkins with Professor Solomon Snyder, who, along with Candace Pert and Gavril Pasternak, had just identified opiate receptors. Dr. Creese described feeling exhilarated and privileged to work at Johns Hopkins, where many exciting discoveries were made in opiate, dopamine, GABA, glycine, muscarinic, adrenergic, and serotonin receptor identification. Dr. Creese used techniques for studying receptor pharmacology pioneered by Snyder to demonstrate definitively that the antipsychotic properties of multiple neuroleptic medications were related to their ability to bind to 1)2 dopamine receptors. Dr. Creese commented that current research in dopamine receptors continues to implicate dopamine in psychotic disorders, but the nature of that interaction now appears more complex. Postmortem studies of schizophrenic brains have found increased D2 receptors; however, the role of neuroleptic exposure has not been eliminated as a possible confounding factor. PET studies have variably found increased D2 binding in drug-naive patients; findings vary depending on the ligand and the mathematical model used for the analysis. A current hypothesis is that schizophrenic subjects may have an increase in the newly discovered D4 receptor, which has a structure and pharmacology similar to those of the 1)2 receptor. Dr. Creese reported that every year there are new discoveries about the complex nature of dopamine receptors, all of which may contribute to more specific and safer treatment of psychosis.

Acute stimulatory effect of estradiol on striatal dopamine synthesis.

The acute effect of physiological doses of estradiol (E2) on the dopaminergic activity in the striatum was studied. In a first series of experiments, ovariectomized rats were injected with 17 alpha or 17 beta E2 (125, 250, or 500 ng/kg of body weight, s.c.), and in situ tyrosine hydroxylase (TH) activity (determined by DOPA accumulation in the striatum after intraperitoneal administration of NSD 1015) was quantified. A dose-dependent increase in striatal TH activity was observed within minutes after 17 beta (but not 17 alpha) E2 treatment. To examine whether E2 acts directly on the striatum, in a second series of experiments, anesthetized rats were implanted in the striatum with a push-pull cannula supplied with an artificial CSF containing [3H]tyrosine. The extracellular concentrations of total and tritiated dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were measured at 20-min intervals. Addition of 10(-9) M 17 beta (but not 17 alpha) E2 to the superfusing fluid immediately evoked an approximately 50% increase in [3H]DA and [3H]DOPAC extracellular concentrations, but total DA and DOPAC concentrations remained constant. This selective increase in the newly synthesized DA and DOPAC release suggested that E2 affects DA synthesis rather than DA release. Finally, to determine whether this rapid E2-induced stimulation of DA synthesis was a consequence of an increase in TH level of phosphorylation, the enzyme constant of inhibition by DA (Ki(DA)) was calculated. Incubation of striatal slices in the presence of 10(-9) M 17 beta (but not 17 alpha) E2 indeed evoked an approximate twofold increase in the Ki(DA) of one form of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and binding properties of [ 3H]NNC 12-0781, a new radioligand for the dopamine reuptake system

The tritiated dopamine reuptake inhibitor [ 3H]NNC 12-0781 ([1-[2-(bis(4-fluorophenyl)-methoxy)-ethyl]-4-(3-(2-furanyl)-2,3-[ 3H]-propyl) -piperazine) was radiolabelled in one step starting from 1-[2-(bis(4-fluorophenyl)-methoxy)-ethyl]-4-(3-(2-furanyl)-2-propenyl)-piperazine, using tritium gas and PdO as catalyst. The radiochemical purity of [ 3H]NNC 12-0781 was higher than 99% after HPLC purification with a specific radioactivity of 21 Ci/mmol. [ 3H]NNC 12-0781 bound specifically to rat striatum in vitro at +4 °C with a K d of 1.76 nM and B max of 587 fmol/mg tissue. The nonspecific binding was about 10% at K d. At +37 °C no acceptable binding was observed. The association of [ 3H]NNC 12-0781 thus has the characteristics of a radioligand for the dopamine transporter in vitro at +4 °C.

Differential effects of nitric oxide gas and nitric oxide donors on depolarization-induced release of [ 3H]norepinephrine from rat hippocampal slices

The NO-generating compounds sodium nitroprusside (NP), nitroglycerin (NTG), and isosorbide dinitrate (ISDN) all significantly inhibited N- methyl- d-aspartate (NMDA)-stimulated release of tritiated norepinephrine ([ 3H]NA) from preloaded hippocampal slices of adult male Sprague-Dawley rats with IC 50 's of 114 μM, 1.2 mM, and 1.7 mM respectively. NTG and ISDN also inhibited KCl-stimulated release, while NP had no significant effect on KCl-stimulated release. Although these results suggest that the inhibitory effects of these compounds were mediated by release of NO, NTG and ISDN did not generate detectable levels of NO, and iron-cyanide complexes similar in structure to NP but lacking NO also inhibited release. In contrast, both S- nitroso-N- acetyl- d , l-penicillamine (SNAP) and authentic NO gas significantly enhanced NMDA-stimulated release of [ 3H]NA (EC 50's: 331 and 3.4 μ M respectively). This enhancement was not selective for NMDA-stimulated release, since both SNAP and NO potentiated KCl-stimulated release as well. In addition, NO gas significantly enhanced NMDA-stimulated release of tritiated dopamine ([ 3H]DA) from striatal slices and [ 3H]NA from cortical and cerebellar slices. Analogs of cyclic guanosine monophosphate (cGMP) had no significant effect on NMDA-stimulated transmitter release, suggesting that the observed increase in release is via a cGMP-independent mechanism. While exogenous NO enhanced both NMDA- and KCl-stimulated neurotransmitter release, it appears that endogenous NO does not play a role in this depolarization-induced release since NO synthase inhibitors did not significantly reduce NMDA-stimulated [ 3H]NA release. The possibility remains that endogenous NO could modulate neurotransmitter release in other circumstances.

Potent cocaine analogs inhibit [ 3H]dopamine uptake in rat mesencephalic cells in primary cultures: pharmacological selectivity of embryonic cocaine sites

The cellular localization of the cocaine binding sites in primary cultures of embryonic rat mesencephalic cells was previously reported to differ from that observed in adult rat brain. In order to know whether this different localization was associated with a different pharmacological selectivity, we tested the effect of new cocaine analogs on tritiated dopamine ([ 3H]DA) uptake in primary cultures of rat embryonic mesencephalic cells. In these cultures, [ 3H]DA was taken up by a nomifensine-sensitive, but desipramine and fluoxetine-insensitive process, reflecting selective uptake by the dopaminergic transporter. 3β-(4-Chlorophenyl)tropan-2β-carboxylic acid methyl ester (RTI-COC-31) was by far the most potent inhibitor of the [ 3H]DA uptake, presenting an IC 50 of 3.8 nM, while the corresponding analog with an unsubstituted phenyl ring (WIN 35,065-2) was 38 times less potent. The enantiomer of WIN 35,065-2, namely WIN 35,065-3, was 30 times less potent than the former. A similar pattern was found for the relative ability of these compounds to inhibit binding of the radiolabeled cocaine derivative [ 125I]RTI-55 to membranes prepared from mesencephalic cultures. The order of potencies found for the three cocaine analogs on mesencephalic cultures was similar to that previously obtained in [ 3H] WIN 35,428 binding experiments and [ 3H]DA uptake inhibition in adult rat striatum, suggesting that the pharmacological selectivity of cocaine sites functionally related to the DA transporter in cultured embryonic neurons does not differ from that obtained in adult rat brain.

Regulatory interactions among axon terminals affecting the release of different transmitters from rat striatal slices under hypoxic and hypoglycemic conditions.

An in vitro model of ischemia was utilized to study the effects of both oxygen and glucose depletion on transmitter release from rat striatal slices. The spontaneous and stimulation-evoked releases of tritiated dopamine, gamma-aminobutyric acid, glutamate, and acetylcholine were measured. Hypoxia increased the evoked release of glutamate and dopamine without effect on the resting release. In contrast, hypoglycemia itself increased the resting release of dopamine. Hypoxia in combination with hypoglycemia provoked a massive release of glutamate, dopamine, and gamma-aminobutyric acid. The effect on acetylcholine release was less pronounced. Ca2+ withdrawal partly reduced the effect of hypoxia combined with hypoglycemia on dopamine release and application of tetrodotoxin (1 microM) abolished it. MK-801 (3 microM), an N-methyl-D-aspartate receptor antagonist, attenuated the effect of hypoxia and hypoglycemia on [3H]dopamine release. omega-Conotoxin (0.1 microM) had a similar effect on stimulation-evoked release under a hypoxic condition. The D2 receptor antagonist sulpiride (100 microM) failed to enhance the release of [3H]acetylcholine in hypoxia combined with hypoglycemia. It was suggested that in response to hypoxia combined with hypoglycemia there is a massive release of glutamate due to the increased firing rate which in turn releases dopamine from the axon terminals through stimulation of presynaptic N-methyl-D-aspartate receptors. Dopaminergic inhibitory control on ACh release seems not to be operative under conditions of hypoxia combined with hypoglycemia.

Mesencephalic dopaminergic neurons in primary cultures express functional neurotensin receptors

The cellular distribution and functional aspects of neurotensin (NT) binding sites in rat mesencephalic cells in primary culture were investigated by an original approach combining anatomical and biochemical studies. Using a double-labeling protocol combining 125I-NT receptor radioautography and tyrosine hydroxylase (TH) immunocytochemistry, we obtained the first direct visualization of NT binding sites on TH-immunoreactive neurons. Eighty percent of the TH neurons were endowed with NT binding sites, which can be observed on both cell bodies and processes. TH-immunoreactive neurons were characterized as dopaminergic neurons by their ability to take up dopamine in a benztropine- and nomifensine-sensitive manner. In the mesencephalic cultures, NT increased potassium-evoked release of tritiated dopamine, and the relative potencies of various NT-related peptides to increase dopamine release were in good agreement with their abilities to bind to NT sites. These results show for the first time that cultured rat mesencephalic dopaminergic cells express functional NT receptors. Finally, the specificity and distribution of NT receptors on dopaminergic neurons in primary culture are quite similar to what was observed in the adult rat brain using pharmacological and radioautographic approaches. These data indicate that NT can influence the activity of dopaminergic neurons at very early stages of the rat brain development.

Autoradiographic identification of D 1 dopamine receptors labelled with [ 3H]dopamine: Distribution, regulation and relationship to coupling

On the basis of experiments made on striatal membranes, Leff and Creese [ Molec. Pharmac. (1985) 27, 184–192] have proposed that tritiated dopamine binds to a high-affinity agonist state of D 1 dopamine receptors (D 1h) which adopt this conformation when they are associated with the GTP-binding protein involved in the transduction process. Quantitative autoradiography was thus used to look for the distribution of these D 1h sites in the rat brain and to compare it with that of D 1 receptors labelled with [ 3H]7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine ([ 3H]SCH23390), a D 1 antagonist. The effects of unilateral 6-hydroxydopamine lesion of the ascending dopamine pathways on the density of [ 3H]dopamine D 1h and [ 3H]SCH23390 binding sites in the striatum and the nucleus accumbens were also analysed. In the striatum, when D 2 receptors were blocked by spiroperidol (20 nM), [ 3H]dopamine was found to bind specifically to dopamine receptors of the D 1 type. Complementary experiments made with dopamine uptake blockers indicated that high-affinity dopamine uptake sites were not labelled by [ 3H]dopamine under our experimental conditions. The anatomical distribution of [ 3H]dopamine D 1h binding sites was found to be markedly different from that of [ 3H]SCH23390 binding sites. This was particularly the case in the substantia nigra, some amygdaloid nuclei and the prefrontal cortex—structures in which the ratios between [ 3H]SCH23390 and [ 3H]dopamine binding sites were more than seven-fold higher than that observed in the striatum. [ 3H]SCH23390 binding was not significantly affected in either the striatum or the nucleus accumbens six weeks after a complete unilateral destruction of ascending dopamine pathways. In contrast, a marked decrease in [ 3H]dopamine D 1h binding sites was found in both structures, but this effect was lower in the medioventral (−60%) than in the laterodorsal (− 81%) part of the striatum, even though dopamine denervation was uniform throughout the structure. Preincubation of the sections with dopamine (0.5 μ M) led to a partial recovery (+ 126%) in the lesioned striatum and an increase of [ 3H]dopamine labelling in the control striatum (+68%). This suggests that the presence of dopamine stabilizes the D 1h state of D 1 receptors. The absence or low amounts of dopamine, either due to dopamine denervation or naturally occurring (prefrontal cortex), would then impair the [ 3H]dopamine D 1h binding. In addition, a lower coupling of D 1 receptors with adenylate cyclase was observed in the substantia nigra when compared to that in the striatum: this may explain the relatively weak [ 3H]dopamine binding in the substantia nigra. Similarly, the lower loss of [ 3H]dopamine binding sites seen specifically in the medioventral part of the striatum in the 6-hydroxydopamine-lesioned rats could be related to a supersensitive dopamine-stimulated adenylate cyclase activity found in this striatal area [Herve´ et al. (1989) J. Neurosci. 9, 3699–3707], In conclusion, the D 1h state of the D 1 receptors is dependent on the endogenous dopamine levels and on the intensity of the D 1 receptor coupling. In order to explain both influences, it is proposed that the D 1h binding sites observed in brain sections are generated by the dissociation of a ternary complex in which dopamine, D 1 receptor and GTP-binding protein are associated.

The putative polyamine antagonists ifenprodil and SL 82.0715 enhance dopamine efflux from rat striatal slices independent of NMDA receptor activation

NMDA stimulated the release of endogenous or tritiated dopamine from rat striatal slices and tritiated norepinephrine from cortical and hippocampal slices. The putative polyamine antagonists ifenprodil and SL 82.0715 inhibited [ 3H]norepinephrine release from cortical and hippocampal slices but enhanced the basal efflux of endogenous and tritiated dopamine from striatal slices. Incubation of striatal slices in a calcium-free buffer did not ameliorate these effects suggesting that the increase in dopamine efflux was not due to a calcium-dependent release process. Superfusion of striatal slices with 10 μM of either ifenprodil, cocaine, or amphetamine resulted in a significant release of tritiated dopamine which was reversed when the slices were again superfused with non-drug-containing buffer. The release observed in the presence of 10 μM ifenprodil (7-fold increase over basal) was intermediate between that observed for cocaine (3-fold increase) and amphetamine (12-fold increase). Both ifenprodil and SL 82.0715 also blocked the uptake of [ 3H]dopamine into striatal synaptosomes with IC 50 values of approximately 1.5 μM. This was again intermediate between the values obtained for cocaine (0.43 μM) and amphetamine (4.2 μM). These results suggest that ifenprodil and its analog SL 82.0715 may act as indirect dopamine agonists by both blocking presynaptic dopamine uptake and by directly increasing the basal efflux of dopamine.

Long‐Term Survival of Intrastriatal Dopaminergic Grafts: Modulation of Acetylcholine Release by Graft‐Derived Dopamine

The nigrostriatal dopaminergic system of rats was unilaterally lesioned with 6-hydroxydopamine. Part of the animals was grafted 2 weeks later with fetal dopaminergic cells on the lesioned side; untreated rats of the same strain served as controls. Both 3 and 12-14 months after surgery the striatal dopamine (DA) content and the in vivo rotational response following injection of D-amphetamine showed significant changes in grafted as compared to lesioned animals. At 12-14 months after transplantation, the electrically evoked release of tritiated DA and acetylcholine (ACh) in slices (preincubated with [3H]DA or [3H]choline, respectively) of striata of intact, lesioned, or grafted animals was also investigated. Electrical field stimulation of striatal slices of the lesioned side did not evoke any significant [3H]DA overflow, whereas a marked [3H]DA release was observed in slices of grafted and control striata. Moreover, both DL-amphetamine (3 microM) and nomifensine (10 microM) strongly enhanced basal 3H outflow in these slices. Electrically evoked [3H]ACh release was significantly reduced in slices from all striatal tissues by 0.01 microM apomorphine. In slices from denervated striata a clearcut hypersensitivity for this action of apomorphine was present, indicating supersensitivity of DA receptors on cholinergic terminals; this hypersensitivity was significantly reduced in graft-bearing striata. Furthermore, because this hypersensitivity was unchanged in slices of lesioned striata under stimulation conditions (four pulses/100 Hz) avoiding inhibition by endogenously released DA, it is concluded that lesion-induced DA receptor supersensitivity is caused by an increase in receptor density or efficacy rather than by a decreased competition between endogenous and exogenous agonists. Both reuptake blockade of DA with nomifensine (10 microM) and release of endogenous DA by DL-amphetamine (3 microM) potently reduced [3H]ACh release only in control and grafted but not in lesioned tissue. In experiments using potassium-evoked [3H]ACh release, tetrodotoxin had no effect on the inhibitory activity of amphetamine and nomifensine, indicating that the DA receptors involved in their indirect inhibitory action are located directly on the cholinergic terminals.

Dopamine transporter: expression in Xenopus oocytes

Xenopus oocytes can express biologically relevant transport activity after injection of mRNAs encoding several carrier molecules. mRNA from PC12 cells, as well as transcripts from a rat ventral midbrain library, can be expressed in these oocytes and allow them to display pharmalogically specific dopamine uptake. mRNA-injected oocytes incubated with tritiated dopamine contain tritiated dopamine and metabolites; lower amounts of radiolabeled dopamine and more radiolabeled metabolites are found in oocytes co-incubated with cocaine or in water-injected oocytes. Tritiated dopamine uptake into mRNA-injected oocytes is time, sodium, and temperature dependent. It is blocked by cocaine and mazindol, but not by haloperidol. It is not found after injection of mRNA from other brain regions. A size-selected rat midbrain library constructed in the plasma vector pCDM8 yields mRNA transcripts whose injection into oocytes causes cocaine-blockable [ 3H]dopamine uptake. These findings provide an assay for purification of the dopamine transporter cDNA by sib selection techniques.

Distribution of intracerebrally injected dopamine as studied by a punch-scintillation modeling technique

Three-dimensional distribution gradients of intracerebrally injected tritiated dopamine were calculated on the basis of concentrations in multiple punch-samples from sequential sections of Macaca fascicularis brain tissue. The monkey was pretreated systemically with a monoamine oxidase inhibitor to retard elimination. Gradients were best fit by cubic exponential equations relating concentration to distance from the center of the site. The concentration at the center, total amount of label, and total extent of the site injected just before perfusion were consistent with initial distribution in the extracellular space, if the volume fraction of the latter is estimated at 20%. The extent of distribution was distinctly greater in the mediolateral and dorsoventral dimensions than in the anteroposterior dimension. The total amount of label near the site decreased rapidly in the first few minutes after injection, then much more slowly, reaching about 30% of the injected amount after 2 h. Its distribution within the site changed steadily, the outer boundary gradually expanding and the peak at the center gradually decreasing. This pattern was consistent with an initial rapid dispersion by injection pressure and an initial loss of tritiated dopamine due to disruption of the blood-brain barrier at the center of the site, followed by a steady expansion of the site driven by diffusion and bulk flow.

Estradiol-induced prolactinomas: differential effects on dopamine in posterior pituitary and median eminence

Prolactin release is inhibited by dopamine and stimulated by estradiol. Dopamine is released from nerve terminals in the median eminence and posterior pituitary. Estradiol may act directly on the anterior pituitary or by modulating the two dopaminergic systems. Estradiol treatment induces the formation of prolactinomas in Fischer 334 rats. Therefore, this strain was chosen as the experimental model. The first objective was to determine whether estradiol differentially regulates the two dopaminergic systems. The second objective was to explore whether the anterior pituitary in estradiol-treated rats acquires the capability for de novo synthesis of dopamine. Rats were ovariectomized and implanted with estradiol capsules (OVEX + E2). Controls were untreated ovariectomized rats (OVEX). Three weeks thereafter, rats were killed. Anterior and posterior pituitaries and medial basal hypothalami (MBH) were removed and individually incubated for 60 min in Hank's balanced salt solution containing 10 μCi [ 3H]-tyrosine. The median eminence was then dissected from the MBH. Tissues were homogenized in perchloric acid and the supernatant fluids were extracted with alumina. Both endogenous and tritiated dopamine were simultaneously quantitated by HPLC. Prolactinoma formation in OVEX + E2 rats was confirmed by dramatic rise (50-fold) in plasma prolactin levels and marked enlargement (3-fold) of the anterior pituitary. Estradiol treatment caused a significant 60% reduction in both dopamine content and synthesis in the median eminence. In contrast, estradiol treatment affected neither dopamine content nor synthesis in the posterior pituitary. There was no evidence for de novo synthesis of dopamine in anterior pituitaries from either OVEX or OVEX + E2 rats. We conclude that the two dopaminergic systems which regulate prolactin secretion, exhibit a differential response to estradiol.

Kinetics and Block of Dopamine Uptake in Synaptosomes from Rat Caudate Nucleus

The dopamine (DA) uptake system in mammalian nerve terminals was studied by measuring the unidirectional influx of tritiated DA into synaptosomes prepared from rat caudate nucleus. Two distinct time-dependent components of DA uptake were observed. The principal component was saturable with respect to DA concentration, required both external Na and Cl, and was competitively blocked by micromolar concentrations of the psychotropic agents cocaine, benztropine, nomifensine, amphetamine, and methamphetamine. This principal component of uptake has the properties expected for a carrier-mediated transport system. The second component, which accounted for about 10-30% of the DA uptake at 2 microM DA, was not saturable, and was independent of external Na, Cl, and blockers of the carrier-mediated system. The saturable, Na-dependent component had an apparent Km(DA) of about 0.5 microM. The dependence of DA uptake on external Na was sigmoid [Hill coefficient = 2; Ka(Na) = 45 mM] whereas the dependence on Cl was best described by a rectangular hyperbola [Ka(Cl) = 15 mM]. Depolarizing conditions (elevated external K) reduced the rate of DA influx. The data are consistent with a carrier-mediated DA transport mechanism in which each DA molecule entering the nerve terminal via the carrier is accompanied by two or more Na ions and one Cl ion in a rheogenic process carrying one or more net positive charges into the cell. Net, concentrative accumulation of DA inside nerve terminals may be accomplished by utilizing the Na electrochemical gradient to drive DA against its electrochemical gradient via this carrier system.

Release of [ 3H]dopamine from rat prefrontal cortex: Modulation through presynaptic cholinergic heteroreceptors

The release of tritiated dopamine ([ 3H]DA) from slices of the rat prefrontal cortex was studied using a superfusion technique. Release appeared to be voltage-dependent and also dependent on external Ca 2+, suggesting the presence of a specific neurotransmitter release mechanism. γ-Aminobutyric acid (GABA), aspartate, glutamate and serotonin had no effect on either basal DA release or K +-stimulated release. Carbachol, a cholinergic agonist, inhibited K +-stimulated DA release. The results demonstrate that cholinergic heteroreceptors on dopaminergic terminals of the prefrontal cortex modulate DA release.

Neurotensin effects on evoked release of dopamine in slices from striatum, nucleus accumbens and prefrontal cortex in rat.

The effects of neurotensin (NT) on the K+-evoked release of endogenous and tritiated dopamine in striatum and on 3H-dopamine in slices from nucleus accumbens and prefrontal cortex were investigated. In striatum, NT (1-1,000 nM) elicited a dose-dependent increase in endogenous and 3H-dopamine release. The dose-response curves were comparable with the two methods. Concerning the comparison of NT modulation of 3H-dopamine release in the three cerebral structures, the peptide induced a more marked effect in striatum with a maximal effect of 150% increase. In accumbens, NT (1-1,000 nM) potentiated the K+-evoked 3H-dopamine release, but in contrast with striatum, the plateau corresponded to a 50% increase. In prefrontal cortex, NT (1-1,000 nM) induced small but significant effects, with a maximal increase of 50% at 100 nM. Acetyl-NT (8-13) displayed an action similar to the natural peptide while NT (1-8) did not exhibit any effect, suggesting that the action of NT involved a receptor. The presence of tetrodotoxin did not alter the facilitating effects of NT in the three structures, indicating that interneurons were not involved in the action of NT. The comparison of the effects of NT showed that in terms of efficacy, NT induced an increase in dopamine release more marked in striatum than in nucleus accumbens and prefrontal cortex. These results are consistent with differences in NT receptors localization in these three dopaminergic structures.

Distribution of dopamine D1 receptors in rat cortical areas, neostriatum, olfactory bulb and hippocampus in relation to endogenous dopamine contents.

The tritiated dopamine D1 antagonist SCH23390 was employed to determine the densities of D1 receptors in seven discrete and functionally identified cortical areas (cingulate, frontal, parietal, primary somatosensory, primary visual, retrosplenial and entorhinal-piriform) as well as in the neostriatum, hippocampus and olfactory bulbs. In addition, the tissue levels of the catecholamines NA, AD, DA, the indoleamine 5-HT and their main metabolites (MHPG, DOPAC, HVA, 3-MT, 5-HTP and 5-HIAA) were measured in the different regions by HPLC with electrochemical detection. The Scatchard analysis of saturation curves revealed the highest density of [3H]SCH23390 binding sites for the neostriatum, while the densities were 10-20 times lower for total cerebral cortex and hippocampus respectively. For the olfactory bulb and other cortical areas, D1 receptor densities were determined by equilibrium binding at a fixed radioligand concentration approaching saturation. The distribution of D1 receptors was heterogeneous with the greatest densities in entorhinal-piriform and cingulate cortices. The endogenous DA levels measured for all regions correlated with their metabolite (DOPAC, HVA and 3-MT) contents (r = 0.999; P less than 0.001). There was also a very good correlation (r = 0.981; P less than 0.001) between tissue DA and D1 receptor densities. This quantitative information reflects particular features of the organization of the DA systems and is discussed in relation to turnover and recently established aspects of the DA innervation.

Biochemical and radioautographic evidence for dopaminergic afferents of the locus coeruleus originating in the ventral tegmental area

The dopamine (DA) content of the locus coeruleus (LC) and the uptake of tritiated DA in the presence of desmethylimipramine into fresh vibratome sections of the LC-area were determined in control rats and in rats whose ventral tegmental area (VTA) had been destroyed by local application of 6-hydroxydopamine (6-OHDA). Destruction of the VTA reduced the DA content and the number of dopaminergic fibers visualized by radioautography in the LC area. This indicates that the DA containing afferents of the LC originate, at least partly, in the VTA.

Regional and laminar density of the dopamine innervation in adult rat cerebral cortex

The topographie distribution and density of the dopamine innervation in adult rat cerebral cortex were investigated by means of a recently improved radioautographic procedure for the light microscopic visualization and counting of monoamine axonal varicosities. Dopamine terminals were specifically labeled by high-affinity uptake in whole cerebral hemisphere slices incubated for 15 min at 35°C with 10 −6M tritiated dopamine in the presence of 10 −4M pargyline and 5 × 10 −6M desipramine. The slices were subsequently fixed, embedded in Epon and processed for light microscope radioautography as large 4-μm-thick (whole hemisphere) or smaller, semi-thin sections (selected areas). In radioautographs of serial semi-thin sections exposed for various periods of time, the number of labeled axonal varicosities reached a plateau after 12–14 days of exposure. Counts on such sections of increasing thickness allowed to calculate a correcting factor to transform numbers obtained from 4-μm-thick sections into their equivalent for a tissue thickness of 0.5 μm from which all varicosities were detected. The number of labeled varicosities could then be expressed per mm 3 of tissue after measuring their mean caliper diameter in electron microscope radioautographs. As visualized at 3 transverse levels representing most of the major cytoarchitectonic divisions of cerebral cortex, two novel aspects were recognized in the topographic distribution of dopamine terminals: (1) the presence of a dopamine innervation in layer VIb of the frontal, parietal, temporal and occipital neocortex, and (2) a significant contingent of dopamine varicosities within the deep and not only upper layers of supragenual cingulate cortex. A fair number of dopamine varicosities were also detected in the upper layers of the dorsomedial frontal area, in the retrosplenial and adjacent occipital cortex as well as in the ventral subiculum and field CA1 of the hippocampus. As measured in 10 sectors representing different cortical regions, the highest density of dopamine innervation was found in the supragenual cingulate cortex (1.7 × 10 6) and particularly in its layers II and III (3.1 × 10 6). A slightly lower density was measured in the anteromedian “prefrontal” cortex (1.0 × 10 6). The rostrorhinal and the perirhinal cortex showed moderate dopamine innervation (3.0 and 5.5 × 10 5) with varicosities in every layer. The piriform and the posterior entorhinal cortex were also moderately and ubiquitously innervated (2.5 and 3.0 × 10 5). In the dorsofrontal, parietal and temporal neocortex, overall dopamine innervation was of low density (5.0−6.0 × 10 4) and concentrated in layer VI(2 × 10 5 varicosities/mm 3). The primary visual and the lateral occipital neocortex had the lowest density, with terminals also confined to layer VI(4.0 × 10 4). Together with recent immunocytochemical observations on developing or adult rat cortex, these results justify some revision of current views regarding the organizational features of the mesocortical dopamine system.

Involvement of intraterminal dopamine compartments in the amine release in the cat striatum

A push pull superfusion technique has been used in the anesthetized cat to study the simultaneous release of tritiated and total dopamine (DA) during continuous labelling with tritiated tyrosine. The concentration of tritiated and total DA (1.3 and 70 nM respectively) and dihydroxyphenylacetic acid (1 μM) have been measured in the extracellular space under our experimental conditions. The specific activity of spontaneously released DA was found to be 0.76 Ci/mmol. The release of tritiated and total amine following 3 h superfusion with [ 3H]tyrosine did not occur in parallel in response to the local application of either α-methyl- p-tyrosine (0.1 mM) or amphetamine (1 μM). Amphetamine induced an increase followed by a decrease in the specific activity of released DA which reflects an initial release of newly synthesized DA followed by the release of stored amine. The transfer between intraterminal pools of the amine is thus clearly evidenced. The results show that the simultaneous determination of tritiated and total DA release allows the relative contribution of the two intraterminal pools to the amine release to be monitored.