Localized alterations of dopamine receptor binding in rat brain by repeated electroconvulsive shock: an autoradiographic study

The effects of repeated electroconvulsive shock (ECS) on binding parameters of D1 and D2 type dopamine (DA) receptors were investigated in different brain regions of male rats using quantitative autoradiography. D1 binding was studied with [3H]SCH 23390 as the ligand and D2 binding with [3H]spiroperidol. The distribution patterns of both D1 and D2 receptor sites were in good agreement with previously published reports. Repeated ECS induced upregulation of D1 receptors in the olfactory tubercle, the endopiriform nucleus and the substantia nigra without appreciably affecting D1 binding sites in the striatum, n. accumbens or in other brain regions containing D1 binding sites. Upregulation of D2 binding sites, after ECS, was seen in the accumbens, the olfactory tubercle, the amygdaloid nuclei, the claustrum and the endopiriform nucleus, but not in the caudate-putamen or in other brain regions containing D2 binding sites. The present finding that repeated ECS can selectively upregulate DA receptor binding sites in discrete brain areas, including limbic structures, renders important support to a large number of previous studies that demonstrated effects of repeated ECS on DA receptor function in behavioral models.     

Comparative development of D1-dopamine and mu opiate receptors in normal and in 6-hydroxydopamine-lesioned neonatal rat striatum: dopaminergic fibers regulate mu but not D1 receptor distribution.

The postnatal development of D1 dopaminergic receptors (D1 receptors) was investigated in the rat striatum in relation to distribution of mu opiate receptor patches and islandic tyrosine hydroxylase (TH)-immunoreactive fibers. The possible influence of dopaminergic (DA) fibers originating from the substantia nigra on the postnatal distribution of striatal D1 and mu receptors was also examined by producing an early 6-hydroxydopamine (6-OHDA) lesion of DA fibers. D1 and mu receptors were labeled with selective ligands: [3H]SCH 23390 and [3H]DAGO, respectively. During the first postnatal week, control rats showed patches of dense D1 binding sites in the entire rostro-caudal extension of the striatum. The localization of D1 receptor patches corresponded to striosomes identified by TH-immunoreactive islands. The striatal distribution of mu receptors was relatively homogeneous at postnatal day 0 (P0) but was clearly patchy at P3-P4. During the second postnatal week the striosomal pattern of D1 binding sites disappeared along a dorso-ventral gradient whereas mu binding sites remained distributed in patches. Densitometric measurements showed that there was a parallel increase of D1 binding sites in both striosomes and the surrounding matrix from P0 to P4. The disappearance of D1 receptor patches observed in the dorsal striatum at P9 was due to a faster increase of D1 binding sites in the matrix than in striosomes between P4 and P9 whereas a significant difference was still observed between these two compartments in the ventral striatum of P9 rats. During the third postnatal week, the density of D1 binding sites still increased but became progressively uniform in the whole striatum. The intrastriatal injection of 6-OHDA in 2-day-old rats produced a local disappearance of TH-immunoreactive fibers in the striatum and a distal degeneration of TH-immunoreactive cell bodies in the substantia nigra. However an early lesion of striatal DA fibers did not modify the pattern of development or the density of D1 binding sites during the postnatal period examined (1 and 3 weeks after the lesion). The distribution of mu receptors was unchanged 1 week after the lesion but showed a clear disorganization 3 weeks after the lesion. We discuss the differential influence of DA fibers on the distribution of D1 and mu receptors in the rat striatum and the possible role of DA in the regulation of the expression of mu receptors.     

Quantitative autoradiography of ligands for dopamine receptors and transporters in brain of Göttingen minipig: comparison with results in vivo

The pig has been used as animal model for positron emission tomography (PET) studies of dopamine (DA) receptors and pharmacological perturbations of DA neurotransmission. However, the binding properties of DA receptors and transporters in pig brain have not been characterized in vitro. Therefore, the saturation binding parameters of [3H]SCH 23390 for DA D1 receptors and [3H]raclopride for DA D2/3 receptors were measured by quantitative autoradiography in cryostat sections from brain of groups of 8 week old and adult female G?ttingen minipigs. The magnitudes of Bmax and Kd for these ligands were similar in young and old pigs, and were close to those reported for rat and human brain. Furthermore, gradients in the concentrations of D1 and D2/3 sites in striatum measured in vitro agreed with earlier findings in PET studies. However, the dopamine transporter (DAT) ligand [3H]GBR12935 did not bind in pig brain cryostat sections. Whereas the tropane derivative [125I]RTI-55 labeled serotonin transporters (serotonin transporter (SERT)) in pig brain, use of the same ligand under conditions specific for DAT, revealed a pattern of binding similar to that observed for SERT conditions. Parallel studies revealed the presence of DAT in rat and ferret brain. The distribution volume (Vd) of the selective DAT ligand [11C]NS2214 ([11C]Brasofensine) was mapped in groups of normal and MPTP-lesioned G?ttingen miniature pigs. The in vivo pattern of Vd matched the distribution of SERT in vitro, and did not differ between the normal pigs and the lesioned animals with documented 60% DA depletions. However, the pattern of specific binding of the selective noradrenaline transporter ligand (S,S)-[11C]MeNER in a single Landrace pig showed that, of the three monoamine transporters, only DAT could not be detected in pig brain. We conclude that the pig is a suitable model for PET studies of DA D1 and D2/3 binding sites, which are fully developed on the eighth postnatal week. However, well-characterized piperazine and tropane radioligands failed to recognize DAT in pig brain; the two tropane radioligands lacked pharmacological specificity for DAT and SERT in pig brain in vitro and in vivo.     

Autoradiography of dopamine receptors and dopamine uptake sites in the spontaneously hypertensive rat

We examined the status of dopamine (DA) D1 and D2 receptors by using [3H]SCH 23390 and [3H]spiperone binding, respectively, and DA uptake sites by using [3H]mazindol binding in spontaneously hypertensive rats (SHR) and Sprague-Dawley (SD) rats. SHR showed significantly higher [3H]SCH 23390 and [3H]spiperone binding in the caudate-putamen (CPu), the nucleus accumbens (NAc) and the olfactory tubercle (OT) in comparison to the SD rats. There were no significant differences in [3H]mazindol-labeled DA uptake sites between the two strains. Unilateral 6-hydroxydopamine (6-OHDA) injection into the striatum resulted in more than 90% depletion of DA uptake sites in the CPu in both strains. 6-OHDA-induced DA depletion was associated with significant increases in striatal [3H]spiperone binding which were of similar magnitude in the SD rats (+64.1%) and SHR (+51.3%). There were only small decreases (-5.4%) in D1 receptor binding in the dorsolateral aspect of the CPu in the SHR, whereas there were no changes in striatal D1 receptors in the SD rats. These results indicate that, although the SHR have higher concentrations of both D1 and D2 receptors in the basal ganglia, these receptors are regulated in a fashion similar to DA receptors in SD rats after 6-OHDA-induced striatal DA depletion.     

Postnatal development of striatal dopamine function. II. Effects of neonatal 6-hydroxydopamine treatments on D1 and D2 receptors, adenylate cyclase activity and presynaptic dopamine function

To evaluate the influence of patch and matrix ingrowth of DA terminals upon striatal DA (dopamine) receptor function, we performed bilateral intrastriatal (i.s.) or single intracisternal (i.c.) injections of 6-hydroxydopamine (6-OHDA) into rat pups at various postnatal ages and determined D1 and D2 receptor binding, adenylate cyclase activities and markers for presynaptic DA terminal density and turnover as the animals matured. All injection schedules yielded: (a) variable and partial loss of DA, (b) increased DA turnover, (c) small (15-40%) increases in D1 receptor number but no change in affinity for antagonist ([3H]SCH 23390), (d) 2-3-fold increases in affinity of D1 receptors for agonist (SKF 38393) with preserved regulation of agonist affinity by guanine nucleotide, (e) no significant changes in DA-, guanine-nucleotide-, manganese- and forskolin-stimulated AC (adenylate cyclase) activity. D2 receptor binding was evaluated between 1 and 7 weeks of age in animals with i.s. treatment and 7 and 10 weeks of age in animals with i.c. treatment and was reduced by 40-50% with both treatment regimens. [3H]mazindol binding, a marker for presynaptic terminal DA transport sites, was reduced 30-40% by multiple i.s. or i.c. treatment regimens. In animals treated with one i.s. injection, [3H]mazindol binding was reduced 70% at 1 week of age, equal to control by 2 weeks and 14-46% greater than control between 3 and 7 weeks. We conclude that striatal D1 receptor sites maintain their density and second messenger function independently of postsynaptic DA terminal ingrowth, whereas the development of D2 receptor sites is sensitive to disruptions of DA terminal ingrowth.     

Neurotensin receptors and dopamine transporters: effects of MPTP lesioning and chronic dopaminergic treatments in monkeys.

The effect of denervation with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) of the dopamine (DA) nigrostriatal pathway on neurotensin (NT) receptor and DA transporter (DAT) in basal ganglia of monkeys (Macaca fascicularis) was investigated. The MPTP lesion induced a marked depletion of DA (90% or more vs. control) in the caudate nucleus and putamen. The densities of NT agonist binding sites labeled with [125I]NT and the NT antagonist binding sites labeled with [3H]SR142948A decreased by half in the caudate-putamen of MPTP-monkeys. In addition, the densities of [125I]NT and [3H]SR142948A binding sites markedly decreased (-77 and -63%, respectively) in the substantia nigra of MPTP-monkeys. Levocabastine did not compete with high affinity for [125I]NT binding in the monkey cingulate cortex, suggesting that only one class of NT receptors was labelled in the monkey brain. An extensive decrease of [3H]GBR12935 DAT binding sites (-92% vs. Control) was observed in the striatum of MPTP-monkeys and an important loss of DAT mRNA(-86% vs. Control) was observed in substantia nigra. Treatments for 1 month with either the D1 agonist SKF-82958 (3 mg/kg/day) or the D2 agonist cabergoline (0.25 mg/kg/day) had no effect on the lesion-induced decrease in NT and DAT binding sites or DAT mRNA levels. The decrease of striatal NT binding sites was less than expected from the decrease of DA content in this nucleus, suggesting only partial localization of NT receptors on nigrostriatal DAergic projections. These data also suggest that under severe DA denervation, treatment with D1 or D2 DA agonists does not modulate NT receptors and DAT density.     

Topographic nonoverlapping distribution of D1 and D2 dopamine receptors in the amygdaloid nuclear complex of the rat brain.

The distribution of D1 and D2 dopamine (DA) receptors in the nuclei and subnuclear zones of the rat amygdaloid complex was mapped using quantitative light microscopic autoradiography. [125I]iodosulpiride and [125I]SCH 23982 (in the presence of 50 nM ketanserin) were used to label D2 and D1 DA receptors, respectively. The DA receptor subtypes exhibited a topographic, nonoverlapping distribution which generally conformed to the cytoarchitectonic boundaries of the component nuclei and subnuclear zones of the amygdaloid complex. The highest density of [125I]iodosulpiride binding sites was observed in the main intercalated cell group and the central amygdaloid nucleus where a medial to lateral gradient of binding sites was localized to its subnuclear zones. [125I]SCH 23982 binding sites were localized in the main intercalated cell group and the basolateral amygdaloid nucleus with a uniform low density in the central nucleus. The functional topography of mesoamygdaloid DA neurons may therefore be mediated, in part, at the level of DA receptor subtypes. The pattern of distribution of [125I]iodosulpiride binding sites in subdivisions of the central amygdaloid nucleus and bed nucleus of the stria terminalis suggests that the functions of the "extended amygdala," a major system of the functional organization of the basal forebrain, may be regulated by DA afferents at multiple key sites of D2 receptor action.     

Characterization of NK-1 receptors in guinea pig and rat brain membranes with NK-1 peptides and a non-peptide antagonist.

The major finding of the present investigation is the demonstration of different NK-1 receptors in rat and guinea pig brain membranes with CP 96345 (non-peptide NK-1 antagonist) and R-544 (NK-1 peptide antagonist). We used [3H][Sar9,Met(O2)11]SP, the highly selective ligand for NK-1 receptor to compare NK-1 binding sites in rat and guinea pig brain membranes. Scatchard analysis revealed the existence of a single population of [3H][Sar9,Met(O2)11]SP binding sites in both preparations. The affinity and the maximal number of binding sites were found closely similar in rat (Kd 2 nM, Bmax = 37 fmol/mg protein) and guinea pig brain membranes (Kd = 3 nM, Bmax = 25 fmol/mg of protein). The order of potency of neurokinins to inhibit [3H][Sar9,Met(O2)11]SP binding from rat brain (SP > NKA > NKB) was found different of that observed on guinea pig brain (SP > NKB > NKA). Results obtained with [Sar9,Met(O2)11]SP, [beta Ala8]NKA(4-10) and [MePhe7]NKB suggest that selective agonists cannot discriminate between NK-1 receptors of different species. Using the non-peptide antagonist CP 96345 and the tripeptide R-544, we found that these two NK-1 antagonists discriminate between rat and guinea pig [3H][Sar9,Met(O2)11]SP binding sites.     

Postnatal development of striatal dopamine function. I. An examination of D1 and D2 receptors, adenylate cyclase regulation and presynaptic dopamine markers

We have characterized the postnatal development from 1 to 7 weeks after birth in rat striatal homogenates of D1 and D2 dopamine (DA) receptor sites, adenylate cyclase (AC) enzyme activity coupled to DA receptor function, guanine nucleotide binding sites and presynaptic markers of DA terminal function. D1 receptor density, expressed per unit of membrane protein, does not increase over this developmental interval, while maximum DA-stimulated AC activity per mg membrane protein increases 50-100%. D1 agonist affinity for D1 receptor sites doubles by 7 weeks of age but is consistently reduced by guanine nucleotide during development. Guanine nucleotide stimulation of AC develops a biphasic dose-response curve after 3 weeks of age. Between 2 and 4 weeks postnatal age there is a rapid increase in AC catalytic component activity as manifested by the capacity of forskolin or manganese ion to stimulate AC in presence of guanine nucleotide and DA. Reversible [3H]GppNHp (guanyldiphosphonateimidophosphate) binding to striatal homogenates is dependent on Mg2+, inhibited by Ca2+ and GppNHp analogues, and occurs in about a 300-fold excess over D1 sites. Presynaptic markers of dopaminergic function indicate a 7-fold increase in tissue DA levels, a 2-fold reduction in DA turnover and no apparent change in density of DA uptake sites, assayed by [3H]mazindol binding. Subcomponents of D1 and D2 DA receptors have distinct postnatal developmental profiles. Striatal D1 sites do not change significantly during development, but D2 receptors and GTP inhibition of AC increase and appear, respectively, at 3-4 weeks of age, at the same time as the massive matrix innervation of striatum by DA terminals.     

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

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

Localization of nigrostriatal dopamine receptor subtypes and adenylate cyclase

Quantitative autoradiography using [3H]-SCH 23390, [3H]-sulpiride and [3H]-forskolin was used to assess the effects of single and combined neurotoxin lesions of the nigrostriatal pathway in the rat brain on dopamine (DA) receptor subtypes and adenylate cyclase (AC), respectively. Ibotenic acid (IA) lesions of the caudate-putamen (CPu) resulted in near total loss of both [3H]-SCH 23390 and of [3H]-forskolin binding in the ipsilateral CPu and substantia nigra reticulata (SNR). [3H]-sulpiride binding in the CPu was only partially removed by this same lesion, and nigral [3H]-sulpiride binding was virtually unchanged. 6-Hydroxydopamine (6-OHDA) and IA lesions of the substantia nigra compacta (SNC) did not affect [3H]-SCH 23390 or [3H]-forskolin binding, but largely removed [3H]-sulpiride binding in the SNC. A 6-OHDA lesion of the nigrostriatal pathway followed by an ipsilateral IA injection of the CPu failed to further reduce [3H]-sulpiride binding in the CPu. These results demonstrate that postsynaptic DA receptors in the CPu are of both the D1 and D2 variety; however, a portion of D2 receptors in the CPu may be presynaptic on afferent nerve terminals to this structure. D1 receptors in the SNR are presynaptic on striatonigral terminals, whereas the D2 receptors of the SNC are autoreceptors on nigral DA neurons. The existence of presynaptic D2 receptors on nigrostriatal DA-ergic terminals could not be confirmed by this study. Co-localization of D1 receptors and AC occurs in both the CPu and SNR.     

Differential response of striatal dopamine and muscarinic cholinergic receptor subtypes to the loss of dopamine: I. Effects of intranigral or intracerebroventricular 6-hydroxydopamine lesions of the mesostriatal dopamine system

Quantitative autoradiography was utilized to examine the response of the dopamine (DA) and muscarinic cholinergic system within the striatum to lesions of the mesostriatal DA system following intranigral 6-hydroxydopamine (6-OHDA) injections. In addition, the response of DA system was examined in the striatum of animals treated with low, medium, or high doses of 6-OHDA made intracerebroventricularly (icv). Three weeks following removal of the mesostriatal DA fibers with intranigral 6-OHDA, there was an almost complete depletion of DA and [3H]mazindol binding throughout the striatum. The resulting increase in D2 receptors labeled with [3H]spiroperidol (27%) was most evident in the lateral striatum and topographically correlated with an increase in choline uptake sites labeled with [3H]hemicholinium-3 (20%). There was a smaller but significant decrease in D1 receptors labeled with [3H]SCH 23390 (15-18%) that was not topographically related to changes in [3H]spiroperidol or [3H]hemicholinium-3 binding. All doses of icv 6-OHDA produced a significant loss of DA and of [3H]mazindol binding as compared to vehicle injections that was more pronounced in the medial than in the lateral striatum. No increase in D1 receptors was observed with any dose of 6-OHDA and greater than 90% loss of DA and [3H]mazindol resulted in an increase in D2 receptors in the lateral striatum and a reduction in D1 receptors in the dorsal striatum. These data are consistent with the evidence that there is independent regulation of the two subtypes of the DA receptor. Moreover, the distribution and regulation of the subtypes of the muscarinic receptor were independent. Muscarinic M2 receptors ([3H]N-methylscopolamine in presence of excess pirenzepine) showed a lateral to medial gradient (highest laterally) that was related to the pattern of choline uptake sites and D2 receptors. Loss of DA resulted in a reduction in M2 receptors (24-30%) that was correlated with the increase in choline uptake sites. In contrast, M1 ([3H]pirenzepine) receptors showed a reverse gradient from the M2 receptor and a smaller reduction following loss of DA.     

Dopamine D2 and D3 receptors in human putamen, caudate nucleus, and globus pallidus

Because radioactive raclopride and radioactive (+)-4-propyl-9-hydroxynaphthoxazine ((+)-PHNO) are used to image dopamine (DA) D2 and D3 receptors in the striatum and globus pallidus in humans, the present study examined the proportions of D2 and D3 receptors in postmortem tissues from these regions. Conflicting results were obtained when using a single concentration of remoxipride to occlude D2 receptors or using a single concentration of U99194A or FAUC 365 to occlude D3 receptors. However, using a range of concentrations of FAUC 365, a D3-selective antagonist, to inhibit the binding [(3)H]raclopride or [(3)H]-(+)-PHNO to D3 receptors at low concentrations (1-10 nM) and to inhibit ligand binding to D2 receptors at higher concentrations (100-2000 nM), it was possible to measure the proportion of D2 and D3 receptors in the tissues. This method revealed that these two radioligands detected only D2 receptors in the dorsal putamen and the dorsal caudate nucleus, but detected a mixed population of two-thirds D2 and one-third D3 DA receptors in the ventral putamen, the ventral caudate, and the globus pallidus. The present findings are in good agreement with the known gene expression data for D2 and D3 receptors in these human brain regions.     

Increased affinity of dopamine for D(2) -like versus D(1) -like receptors. Relevance for volume transmission in interpreting PET findings

Evidence indicates that dopamine (DA) mainly acts as a volume transmission (VT) transmitter through its release into the extracellular fluid where the D(1) -like and D(2) -like receptors are predominantly extrasynaptic. It was therefore of interest to compare the affinities of the two major families of DA receptors. [(3)H] raclopride /DA and [(3)H] SCH23390/DA competition assays compared the affinity of DA at D(2) -like and D(1) -like receptors in rat dorsal striatal membrane preparations as well as in membrane preparations from CHO cell lines stably transfected with human D(2L) and D(1) receptors. The IC(50) values of DA at D(2) -like receptors in dorsal striatal membranes and CHO cell membranes were markedly and significantly reduced compared with the IC(50) values of DA at D(1) -like receptors. These IC(50) values reflect differences in both the high and low affinity states. The K(iH) value for DA at [(3)H] raclopride-labeled D(2) -like receptors in dorsal striatum was 12 nM, and this can help explain PET findings that amphetamine-induced increases in DA release can produce an up to 50% decrease of [(11)C] raclopride binding in the dorsal striatum in vivo. These combined results give indications for the existence of striatal D(2) -like receptor-mediated DA VT at the local circuit level in vivo. The demonstration of a K(iH) value of 183 nM for DA at D(1) antagonist-labeled D(1) -like receptors instead gives a likely explanation for the failure of a reduction of D(1) -like receptor binding after amphetamine-induced DA release in PET studies using the D(1) -like antagonist radioligands [(11)C] SCH23390 and [(11)C] NNC. It seems difficult to evaluate the role of the extrasynaptic D(1) receptors in VT in vivo with the PET radioligands available for this receptor.     

Developmental and age-related changes in D1-dopamine receptors and dopamine content in the rat striatum

The relationship between the postnatal development of dopaminergic (DAergic) nerve endings and the maturation of D1 DA receptors in the rat striatum was analyzed by measuring the content of DA and dihydroxyphenylacetic acid (DOPAC), two biochemical markers of DAergic nerve terminal proliferation, and the ontogenetic changes in [3H]SCH 23390 binding sites. DA-stimulated adenylate cyclase (AC) activity was also measured in order to characterize the coupling of [3H]SCH 23390 binding sites to the responses mediated by the activation of D1 DA receptors. Striatal levels of DA and DOPAC, as well as the density and affinity of [3H]SCH 23390 binding sites and DA-stimulated AC activity were also measured in senescent rats. The striatal content of DA increased slowly after birth, reaching adult levels by postnatal day 60 and remaining constant through adulthood and senescence (up to 20 months of age). The density of [3H]SCH 23390 binding sites increased 14-fold from birth to postnatal day 35, when a peak value was reached, whereas a significant decrease was observed in the striatum of aged rats. In contrast, the affinity of D1 DA receptors for [3H]SCH 23390 remained unchanged from birth through senescence. The stimulation of cyclic AMP formation induced by 100 microM DA increased 4-fold from birth to postnatal day 14, when the maximal responsiveness to DA was observed and then returned to adult levels. No significant alterations were observed in the Km values during development, whereas the stimulatory effect of 100 microM DA on AC activity was significantly decreased in senescent rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dopaminergic abnormalities in amygdaloid nuclei in major depression: a postmortem study.

BACKGROUND: A deficiency of mesolimbic dopamine (DA) is a leading candidate for the etiology of certain symptoms of depression (e.g., anhedonia and loss of motivation). Here we show amounts of dopaminergic proteins in the amygdala, a key brain structure involved in the integration of emotions and stress, in subjects with major depression and in psychiatrically normal control subjects. METHODS: The specific binding of [(125)I]RTI 55 to the DA transporter, [(3)H]SCH 23390 to the D1 receptor and [(125)I]epidepride to D2/D3 receptors were measured in the right amygdaloid complex in postmortem brains from 11 subjects with major depression and 11 matched control subjects. RESULTS: The binding of [(125)I]RTI 55 to DA transporter was significantly lower in the basal and central amygdaloid nuclei, whereas the binding of [(125)I]epidepride to D2/D3 receptors was significantly higher in the basal, central, and lateral amygdaloid nuclei in major depression compared with control subjects. No difference in the binding of [(3)H]SCH 23390 to D1 receptors was observed. CONCLUSIONS: Given that DA depletion in rats can induce a reduction in the DA transporter and an upregulation of D2/D3 receptors, our data are consistent with the hypothesis that major depression is associated with a deficiency of mesolimbic DA.     

3H]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.     

Differential response of striatal dopamine and muscarinic cholinergic receptor subtypes to the loss of dopamine. II. Effects of 6-hydroxydopamine or colchicine microinjections into the VTA or reserpine treatment.

In the previous paper it was demonstrated that striatal dopamine (DA) D1 and D2 receptor subtypes and muscarinic M1 and M2 receptor subtypes show differing responses to lesions of the mesostriatal DA system. To examine this differential regulation further rats were given unilateral injections of 6-hydroxydopamine (6-OHDA) or colchicine into the ventral tegmental area (VTA), or treated chronically with reserpine or saline. Two weeks later the animals were tested for their behavioral response to a subthreshold dose of apomorphine and 24 h later their brains were removed and processed for quantitative autoradiography or for analysis of DA levels by high-performance liquid chromatography. The 6-OHDA-lesioned animals showed a supersensitive rotational response to apomorphine. The loss of DA, loss of DA uptake sites, regulation of DA D1 and D2 receptors and regulation of the muscarinic cholinergic system was similar to the previous paper. Injection of colchicine in the VTA resulted in incomplete loss of striatal DA (50%), [3H]mazindol binding (50%), and no behavioral supersensitivity to apomorphine. There was a small loss of presynaptically located D2 receptors (13%). Similar to the 6-OHDA lesions there was a loss of D1 (12%) and M1 receptors. Reserpine treatment produced an 86% decrease in DA levels, an enhanced stereotyped responsiveness to apomorphine, and an increase of both D2 (28%) and D1 receptors (26%). There was a loss of muscarinic M1 but not M2 receptors. Thus removal of DA terminals or blockade of transport of proteins in the mesostriatal axons can lead to a reduction in D1 receptor density in the striatum. In contrast, loss of DA without removal of DA terminals leads to a significant up-regulation of the D1 receptor. D2 receptors show increases following removal of DA or of DA terminals. Alteration in the muscarinic cholinergic system following damage to the mesostriatal DA system is a complex response not mimicked by either reserpine or colchicine treatment.     

Retrograde degeneration of nigrostriatal neurons induced by intrastriatal 6-hydroxydopamine injection in rats.

Quantitative receptor autoradiography was used to assess the effects of unilateral intrastriatal injections of 6-hydroxydopamine (6-OHDA) on the distribution of D1 and D2 dopamine (DA) receptors and of DA uptake sites in the mesostriatal pathway. [3H]Mazindol-labeled DA uptake sites were reduced both in the striatum (-97%) and in the substantia nigra pars compacta (SNpc) (-88%) on the injected side. There were also significant decreases of dopamine uptake sites in the nucleus accumbens (NAc) (-73%) and in the ventral tegmental area (VTA) (-70%). Changes in [3H]mazindol binding were also found within the contralateral VTA (-30%) and SNpc (-13%) but not in the contralateral-striatum. [3H]SCH23390-labeled D1 receptors were significantly reduced in the dorsomedial (-18%) and ventromedial (-14%) aspects of the striatum ipsilateral to the side of the lesions. In contrast, the concentration of [3H]spiperone-labeled D2 receptors was not altered. There were also significant decreases in D1 (-18%) and of D2 (-27%) receptors in the SNpc and of D1 (-10%) in the SN pars reticulata (SNpr). These results suggest that oxyradical-induced damage in striatal DA terminals could lead to retrograde changes in the SNpc. In addition, the data indicate that unilateral striatal damage can result in bilateral changes in the SNpc, thus confirming the interdependence of the two nigrostriatal pathways in rats.     

The discovery of PD 89211 and related compounds: selective dopamine D4 receptor antagonists

The dopamine (DA) D2 family of receptors consists of the D2, D3, and D4 receptors. The DA D4 receptor is of interest as a target for drugs to treat schizophrenia based upon its high affinity for the atypical antipsychotic clozapine and its localization to the limbic and cortical regions of the brain. As part of a program to identify novel DA D4 receptor antagonists, a high-volume screen using the Parke-Davis compound library was initiated. This led to the discovery of PD 89211 (benzenemethanol, 2-chloro-4-[4-[(1H-benzimidazol-2-yl)methyl]-1-piperzinyl]) that displaced [3H]spiperone binding to hD4.2 with an affinity (Ki) of 3.7 nM. PD 89211 exhibited high selectivity for the DA D4.2 receptor (> 800-fold) as compared to other hDA receptor subtypes, rat brain serotonin, and adrenergic receptors. In vitro, PD 89211 had D4 receptor antagonist activity reversing quinpirole-induced [3H]thymidine uptake in CHOpro5 cells (IC50 = 2.1 nM). Limited structure-activity relationship (SAR) studies indicated that compounds with a 4-chloro-, 4-methyl-, and 3-chloro- substituents on the phenyl ring retained high affinity for D4 receptors, while those with a 4-methoxy- and no substituent had less affinity. While all clinically effective antipsychotics increase DA synthesis (DOPA accumulation) in rodents, PD 89211 did not increase DA synthesis in the DA-enriched striatum, indicating no effect on DA turnover and low propensity for exhibiting motor side effects. However, it did increase catecholamine synthesis in rat hippocampus, as did clozapine. Moreover, PD 89211 selectivity increased catecholamine synthesis in the hippocampus of wild type but not in mice lacking D4 receptors, suggesting that one function of D4 receptors may be to modulate DA/norepinephrine (NE) turnover in this brain area known to possess D4 receptors. The discovery of compounds like PD 89211 provides a tool to help in understanding the function of DA D4 receptors in the CNS.