Functional damage of dopamine nerve terminals following intrastriatal kainic acid injection

The release of [³H] dopamine ([³H]DA) previously taken up into rat striatal slices was studied one week after a monolateral intrastriatal injectuion of kainic acid (KA). Different releasing stimuli (electrical pulses, veratrine, high-K⁺) were applied. The electrically evoked release in the KA-lesioned striata was drastically reduced with respect to the unlesioned contralateral striata. In contrast, KA had no effect on the release of [³H]DA evoked by veratrine or high-K⁺. In unlesioned striatal slices, depolarized with 15 mM KCl, apomorphine reduced and (−)sulpiride increased the release of [³H]DA. The effect of apomorphine was antagonized by (−)sulpiride indicating the presence of an autoreceptor system similar to that seen in unlesioned striata stimulated electrically. However, the effects of apomorphine and of (−)sulpiride were dramatically reduced in K⁺-depolarized slices prepared from KA-lesioned striata. The results suggest that the axon terminals in KA-treated areas remain intact in several of their properties but may be damaged in some critical processes.     

The effects of chronic continuous versus intermittent levodopa treatments on striatal and extrastriatal D1 and D2 dopamine receptors and dopamine uptake sites in the 6-hydroxydopamine lesioned rat — an autoradiographic study

The effects of chronic ‘continuous’ infusion and ‘intermittent’ modes of levodopa/carbidopa administration on apomorphine induced circling behaviour, DA uptake sites (labelled with [³H]mazindol) and D₁ and D₂ DA receptor binding (labelled with [³H]SCH 23390 and [³H]sulpiride, respectively) were investigated in rats with unilateral 6-OHDA lesions of the medial forebrain bundle. The circling behaviour in response to apomorphine was greatly enhanced following chronic ‘intermittent’ but not ‘continuous’ levodopa treatments. Following the ‘intermittent’ regime, the lower dose of apomorphine induced a period of intense circling with delayed onset and rapid offset, than in rats given either ‘continuous’ infusion of levodopa or saline. The 6-OHDA lesion itself induced gross depletion of [³H]mazindol binding in all striatal subregions, NAc and OT, but not frontal cortex. [³H]Sulpiride binding in the ventrolateral striatal quadrant was increased on the denervated side and this correlated with the peak contralateral turns in response to 0.5 mg/kg apomorphine challenge. This asymmetry in striatal [³H]sulpiride binding was reduced in both groups of rats receiving levodopa. [³H]sulpiride binding in the NAc and OT and [³H]SCH 23390 binding in the striatum, NAc, OT and SNr were unaffected by DA denervation or either regime of levodopa treatments. ‘Continuous’ infusion and not ‘intermittent’ injections of levodopa reduced [³H]mazindol binding in the striatal subregions and the frontal cortex on both the denervated and intact sides. The potentiation of the behavioural response to apomorphine by chronic ‘intermittent’ levodopa treatment does not correspond with the levodopa induced alterations in striatal or extrastriatal DA receptors. In the same group of animals the narrowing of the duration of response to the lower dose of apomorphine may mimic the fluctuations in response to levodopa, seen clinically in long-term levodopa treated parkinsonian patients.     

Opposite presynaptic regulations by glutamate through NMDA receptors of dopamine synthesis and release in rat striatal synaptosomes

Purified striatal synaptosomes were superfused continuously with L-[3,5-³H]tyrosine to measure simultaneously the synthesis ([³H]water formed during the conversion of [³H]tyrosine into [³H]DOPA) and the release of [³H]dopamine ([³H]DA). Glutamate (10⁻³ M) and NMDA (10⁻³ M, in the absence of Mg²⁺) stimulated the release of [³H]DA, but they reduced the efflux of [³H]water. This reduction of [³H]DA synthesis was blocked by 2-amino-5-phosphonovalerate indicating the involvement of NMDA receptors. Although D,L--amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate (AMPA) and kainate stimulated the release of [³H]DA, they did not affect its synthesis. The glutamate-evoked inhibition of [³H]DA synthesis was prevented when synaptosomes were superfused continuously with adenosine adenosine deaminase plus quinpirole, a treatment which markedly reduces the phosphorylation of tyrosine hydroxylase by cAMP dependent protein kinase. The opposite effects of glutamate on [³H]DA synthesis and release were mimicked by ionomycin (10⁻⁶ M). It is proposed that both an activation of a cyclic nucleotide phosphodiesterase and a dephosphorylation of tyrosine hydroxylase linked to the influx of calcium through NMDA receptors is responsible for the inhibition of dopamine synthesis by glutamate and that calcineurin could play a critical role in these processes.     

Rats bred for enhanced apomorphine susceptibility have elevated tyrosine hydroxylase mRNA and dopamine D2-receptor binding sites in nigrostriatal and tuberoinfundibular dopamine systems

From a Wistar population two rat lines were generated using as criterion the behavioral response to the dopamine agonist apomorphine. Rats of the apomorphine-susceptible (apo-sus) line revealed a vigorous gnawing response to apomorphine administration while the other rat line, the apomorphine-unsusceptible (apo-unsus) line, was selected for lack of response to the drug. In the present study using the 12th and 13th generation of these genetically selected lines, we have investigated whether this difference in apomorphine responsiveness was correlated with changes in dopamine neurochemistry. Therefore, we measured tyrosine hydroxylase (TH), the rate limiting enzyme in dopamine synthesis, as well as dopamine D₁ and D₂ receptor mRNA levels in discrete brain regions by in situ hybridization. Dopamine (D₂/D₃) receptor binding was assessed with [¹²⁵I]iodosulpride in a membrane binding assay and by quantitative autoradiography on tissue sections. [³H]SCH 23390 was used to analyze D₁ receptor binding. Apo-sus rats displayed significantly higher TH mRNA levels in the A₉ cell group of the substantia nigra pars compacta and in the A₁₂ cell group of the arcuate nucleus. No difference was found in the A₁₀ cell group of the VTA and the A₆ cell group of the locus coeruleus. The density ofD_2/3 binding sites as well as D₁ receptor mRNA levels in the striatal projection area of the A₉ substantia nigra neurons, were significantly elevated in apo-sus rats. Dopamine D₂ receptor mRNA and D₁ receptor binding levels in caudate putamen and nucleus accumbens, however, were similar in rats of both lines. In conclusion, high apomorphine susceptibility is related to a potentially enhanced dopamine responsiveness selective for the nigrostriatal and tuberoinfundibular pathways.     

Effects of thiopental, halothane and isoflurane on the calcium-dependent and -independent release of GABA from striatal synaptosomes in the rat

The effects of the anesthetic agents thiopental, halothane and isoflurane on the release of GABA induced by depolarization and/or reversal of the GABA carrier were investigated in a synaptosomal preparation obtained from the rat striatum. Veratridine (1 μM) and KCl (9 mM) elicited a significant Ca²⁺-dependent release of [³H]GABA. The KCl-evoked release was not significantly modified in the presence of nipecotic acid (10⁻⁵ M), a selective blocker of the neuronal GABA carrier. The [³H]GABA release was significantly decreased by ω-conotoxin (10⁻⁷ M, a blocker of the N voltage-dependent Ca²⁺ channels, but was affected by neither nifedipine (10⁻⁴ M) nor ω-Aga-IVA (10⁻⁷ M) which block the L and Ca²⁺ channels, respectively. Thiopental application (10⁻⁵ to 10⁻³ M) was followed by a dose-related, significant, decrease in both the veratridine and KCl-induced releases, whether nipecotic acid was present or not. In contrast, halothane and isoflurane (1–3%) failed to alter [³H]GABA release. Altogether, these results suggest that reduction of the depolarization-evoked GABA release might contribute to thiopental anesthesia, but this seems unlikely for volatile anesthetics.     

Differential modulation of NMDA-stimulated [H]dopamine release from rat striatum by neuropeptide Y and σ receptor ligands

Although the identity of the endogenous ligands for sigma (σ) receptors is unknown, neuropeptide Y (NPY) has been named as a possible candidate for a natural transmitter at these receptors. Using a superfusion system, we compared the effect of NPY on NMDA-stimulated [³H]dopamine release in rat striatum to that of the σ agonists (+)-pentazocine and BD737. In contrast to (+)-pentazocine- or BD737-mediated inhibition of release, NPY enhanced release. However, the same σ antagonists (BD1008, DuP734, haloperidol and DTG) that reverse (+)-pentazocine- or BD737-mediated inhibition, as well as a Y receptor antagonist, PYX-1, all reversed the enhancement. PYX-1 also reversed the (+)-pentazocine- and BD737-mediated inhibition of release. Peptide YY (PYY) and [Leu³¹,Pro³⁴]NPY did not mimic the effect of NPY. NPY_13–36 enhanced release to the same extent as NPY but the effect was not reversed by σ antagonists. Our findings are consistent with the potential role of NPY as an endogenous ligand for a subtype of σ receptor with characteristics different from Y₁, Y₂ and Y₃ receptors but sensitive to PYX-1.     

Differential serotonergic inhibition of in vitro striatal [3H]acetylcholine release in prenatally cocaine-exposed male and female rats

Previous research indicates that prenatal cocaine (pCOC)-exposure results in greater 5-HT₃ agonist-induced inhibition of electrically evoked [³H]acetylcholine (ACh) overflow in rat striatal slices. The present study examines the effects of fluoxetine (FLU)-induced and exogenous serotonin (5-HT) on electrically evoked ACh release from striatal slices prepared from adult male and female (in periods of diestrus or proestrus) rats exposed to saline or cocaine in utero. Additionally, we assessed the impact of monoaminergic receptor stimulation on evoked ACh release by superfusion with selective 5-HT₂, 5-HT₃ and D₂ receptor antagonists in the presence of FLU-induced and exogenous 5-HT and measuring the capacity of these drugs to reverse inhibitory effects of 5-HT. Given our previous findings of accentuated inhibition of ACh release by 5-HT₃ agonism in striata of pCOC-exposed adult rats, we hypothesized that superfusion of endogenous and exogenous 5-HT would lead to greater suppression of evoked ACh release in this group of animals. Our results indicated that ACh release from slices of all prenatal saline (pSAL) rats was inhibited comparably by FLU (10 μM)-elicited increases in endogenous 5-HT or by increases elicited with application of exogenous 5-HT (5 μM). Robust FLU-mediated inhibition of ACh release was evident in slices from pCOC male and pCOC diestrus female rats vs. their respective PSAL control groups. Superfusion of striatal slices with 5-HT (5 μM) produced a pattern of ACh inhibition similar to that produced by FLU; however, the magnitude of ACh inhibition was consistently greater than that observed with FLU. Inhibition of ACh overflow by FLU was blocked by co-superfusion with ketanserin, a 5-HT₂ receptor antagonist, ICS-205,930, a 5-HT₃ receptor antagonist or sulpiride, a D₂ receptor antagonist. Conversely, serotonergic inhibition of ACh overflow was only blocked by a high concentration of ICS-205,930 (5 μM) and was completely reversed by sulpiride (1 μM). Collectively, these findings demonstrate serotonergic modulation of cholinergic neurons varying as a function of prenatal treatment, sex and, for females, phase of estrous. Inhibition of ACh release by 5-HT appears to be mediated by a complex relationship between 5-HT₂, 5-HT₃ and D₂ receptor regulation, as the blockade of any of these receptors reversed the inhibitory effects of FLU on ACh release. Conversely, in the case of exogenous 5-HT-induced inhibition, only blockade of D₂ receptors and high concentrations of the 5-HT₃ receptor antagonists were capable of reversing monoaminergic inhibition. These data support the hypothesis that the enhanced serotonergic modulation of ACh neurons in pCOC-exposed animals is largely mediated by dopamine (DA) and reflect a major biochemical persistence of neurodevelopmental adaptations elicited by early cocaine exposure.     

Striatal dopamine innervation and receptor density: regional effects of the weaver mutation

Mice homozygous for the autosomal recesive gene weaver (wv) exhibit a regionally specific depletion of forebrain dopamine (DA). DA is reduced approximately 70% in the dorsal striatum of homozygotes (wv/wv) relative to heterozygous (+/wv) controls while DA content in ventral striatum is relatively unchanged. The goal of the present study was to determine the regional effects of the weaver mutation on striatal DA receptors and DA uptake sites using quantitative autoradiography. Catecholamine histofluorescence was used to examine midbrain DA-containing cell bodies. Compared to behaviorally normal (+/-) littermates, the binding of [³H]spiroperidol to D₂ sites was significantly increased in the dorsal but not ventral striatum of wv/wv mice. Binding of the D₁ ligand, [³H]SCH23390, was significantly decreased throughout the striatum of wv/wv mice. The binding of [³H]mazindol to DA uptake sites was dramatically reduced in all wv/wv striatal regions except the ventrolateral portion. Compared to +/-littermates, wv/wv mice had far fewer fluorescent cell bodies in the substantia nigra and a less pronounced reduction of ventral tegmental area fluorescent somata. These findings support the hypothesis that heterogeneities exist in the genetic control of the mesotelencephalic DA system. The results underscore the usefulness of the weaver mouse in the study of mesostriatal sub-systems, receptor regulation, and potentially as a model of human neuropathologies that affect distinct populations of cells in the mesotelecephalic system.     

Electrically induced release of [H]dopamine from slices obtained from different rat brain cortex regions. Evidence for a widespread dopaminergic innervation of the neocortex

Slices obtained from the deeper layers of the rat dorsal frontal, parietal and occipital brain cortex were incubated in vitro with6.25 × 10⁻⁷ M [³H]dopamine (DA), and subsequently superfused and electrically stimulated, while held on quick transfer electrodes, and changes in the efflux of³H and of the individual amines measured. The separation of the amines, with quantitative recoveries, was performed by chromatography on cation-exchange resins eluted sequentially with water,1 NHCl and 6 M urea in1 N HCl. When no drugs were used, the prestimulation efflux was entirely formed by deaminated metabolites, while following stimulation there was an increase in the efflux of deaminated metabolites, and considerable amounts of [³H]-noradrenaline (NA) now appeared. No DA was present in the pre- or poststimulation medium. Similar results were obtained in all the regions studied. When the slices were incubated with10⁻⁵ M desmethylimipramine (DMI),10⁻⁴ M nialamide and 10⁻⁴ M tropolone, before and during incubation with [³H]DA, it was observed that, prior to stimulation, the efflux was composed of deaminated metabolites, DA and 3-methoxytyramine (MTA), and following the electrical stimulus there was an increased release of DA, NA and deaminated compounds (in order of decreasing release), while no change in that of MTA was evident. The stimulus-induced release of DA was greatest from frontal slices, intermediate from parietal, and lowest from occipital ones. DMI-resistant uptake of [³H]DA also diminished when passing from frontal to occipital. These findings are interpreted as due to the presence of dopaminergic axon terminals in all the regions studied, but with a density that diminishes in a rostrocaudal direction.     

Dopamine D1 receptors are involved in the modulation of D2 receptors induced by cholecystokinin receptor subtypes in rat neostriatal membranes

The action of cholecystokinin octapeptide (CCK-8) on rat neostriatal dopamine (DA) D₂ receptors was evaluated in membrane binding experiments. 0.1 nM of CCK-8 inreased theK_d value of the D₂ agonist [³H]N-propylnorapomorphine (NPA) binding sites by 42%. The CCK_B antagonist PD134308 blocked this action. Kinetic analysis demostrated that this effect of CCK-8 was related to a reduction by 45% of the association rate constant of [³H]NPA. In contrast, 1 nM of CCK-8 decreased theK_H and theK_L values of DA for the D₂ antagonist [³H]raclopride binding sites by 56% and 50%, respectively. Both the CCK_A antagonist L364718 and the CCK_B antagonist PD134308 blocked this effect. The D₁ antagonist SCH23390 counteracted the CCK-8 induced decrease in theK_H and theK_L values of DA, and allowed 1 nM of CCK-8 to produce a significant increase in the IC₅₀ value of NPA for the [³H]raclopride binding sites. These results indicate that CCK-8 can reduce the affinity of the neostriatal D₂ agonist binding sites, but increase the affinity of D₂ receptors for DA. D₁ receptors may exert a switching role in the modulation of the neostriatal D₂ receptors by the CCK receptors.     

Release of endogenous dopamine and cholecystokinin from rat striatal slices: effects of amphetamine and dopamine antagonists

The release of immunoreactive cholecystokinin (CCK) and dopamine was monitored simultaneously from superfused rat striatal slices. Exposure of the tissue to medium containing elevated of dopamine (10⁻⁷ and 10⁻⁶)M), the dopamine agonist pergolide (10⁻⁷, M), the D₂-antagonist sulpride (1 μM) or the D₁-antagonist (SCH 23390) had no significant effect on basal overflow or on evoked release of CCK. On the other hand, preincubation of striatal slices withd-amphetamine (10⁻⁵ M) enhanced basal and veratrine-stimulated dopamine release but markedly suppressed evoked CCK release. Sulpiride blocked this action of amphetamine whereas SCH 23390 was ineffective. The data suggests that whereas it is difficult to observe any effects of exogenous dopamine agonists or antagonists on evoked CCK release, endogenously released dopamine appears to interact with D₂-receptors to suppress evoked CCK release from rat striatal slices.     

The resolution of dopamine and beta 1- and beta 2-adrenergic-sensitive adenylate cyclase activities in homogenates of cat cerebellum, hippocampus and cerebral cortex.

The stimulation of adenylate cyclase by dopamine and various β-adrenergic agonists has been investigated in homogenates from 3 areas of cat brain: the cerebral cortex, cerebellum and hippocampus. The purpose of the study was to determine whether the β-adrenergic receptors coupled to adenylate cyclase could be classified as either β₁ and β₂ subtypes in the different regions studied.

The stimulation of adenylate cyclase by the β-adrenergic agonist, (−)isoproterenol (5 × 10⁻⁶M), was completely blocked by the specific β-adrenergic antagonist, (−)alprenolol (10⁻⁵ M), but not by the dopaminergic antagonist, fluphenazine (10⁻⁵ M), whereas the stimulation of adenylate cyclase by (−)epinephrine (10⁻⁴ M) was blocked to varying extents by these two drugs in each of the 3 regions studied. The (−)epinephrine effect was always blocked in the combined presence of (−)alprenolol and fluphenazine. The adenylate cyclase stimulation by (−)epinephrine which is not blocked by (−)alprenolol was due to interaction of (−)epinephrine with a dopaminergic-sensitive adenylate cyclase which has been characterized in cerebral cortex, hippocampus and cerebellum.

Regional differences in the affinity of β-adrenergic-sensitive adenylate cyclase for various agonists were investigated in the presence of fluphenazine (10⁻⁵ M). In the cerebellum the potency order was (±)protokylol> (±)hydroxybenzylisoproterenol> (±)isoproterenol> (−)epinephrine> (±)salbutamol> (−)norepinephrine, indicating the presence of a β₂-adrenergic receptor. In the cerebral cortex the potency order was (−)isoproterenol> (±)protokylol> (±)hydroxybenzylisoproterenol> (−)epinephrine= (−)norepinephrine((±)salbutamol being inactive). A similar pattern was found in the hippocampus indicating the presence of a β₁-adrenergic receptor in these two regions. (±)Salbutamol was a partial agonist in the cerebellum and a competitive antagonist in the cerebral cortex.

The ratio of the antagonist potencies of (±)practolol and (±)butoxamine preferential β₁- and β₂-adrenergic antagonists respectively, to block the stimulation of adenylate cyclase was 25 in the cerebellum, compared to 0.5 in the cerebral cortex and 1.6 in the hippocampus. These results confirm the presence of a β₂ subtype of receptor coupled to adenylate cyclase in the former and β₁ subtypes in the latter two regions. The comparison between the affinities of a series of β-adrenergic agonists and antagonists for the β-adrenergic receptors coupled with an adenylate cyclase in cerebral cortex and cerebellum with their affinities for well characterized β₂-adrenergic receptors in lung and β₁-adrenergic receptor in heart substantiated this conclusion.     

Detection of dopamine receptors in homogenates of rat hippocampus and other brain areas

The binding of [³H]spiperone (24–26pM) specifically to the dopaminergic (DAergic) D₂ site has been investigated in homogenates of rat hippocampus, pineal gland and cerebellum. It was found that specific binding in hippocampal homogenates was heat-labile and for a part pH-sensitive. Like striatal tissue, binding to the dopaminergic D₂ site in hippocampal and cerebellar homogenates was greatly reduced by (pre)incubation with the iron chelatoro-phenanthroline. The binding characteristics in hippocampus and cerebellum have been compared to those in the striatum. In all tissues tested, low (74–138 times fewer than in striatum) but significant numbers of DAergic D₂ sites were found. The DAergic D₂site in the hippocampus, as far as measured in cerebellum and pineal gland exhibited similar binding characteristics to that in the striatum.     

Quantitative autoradiography of the dopamine uptake complex in rat brain using [H]GBR 12935: binding characteristics

The dopamine uptake complex was examined in the rat central nervous system using [³H]GBR 12935 and in vitro quantitative autoradiography to determine all binding data. [³H]GBR 12935 labels two unique binding sites, the dopamine uptake complex and a piperazine acceptor site. These two sites differ in their pharmacologic properties, anatomical distributions, densities, and response to lesions. Using appropriate binding conditions, [³H]GBR 12935 can be used to specifically label the dopamine uptake complex. [³H]GBR 12935 labeled a single binding site with characteristics of the dopamine uptake complex when mazindol (25 μM) was used as a blank. The specific binding and autoradiographic appearance of [³H]GBR 12935 to the dopamine uptake complex was improved by including trans-flupentixol (0.75 μM) to displace binding to a previously desrribed piperazine acceptor site, recently determined to be a site on cytochrome P450IID1. Binding was saturable and reversible to the dopamine uptake complex. The equilibrium dissociation constant (1.4 ± 0.7nM), maximal number of binding sites (6.0 ± 1.3pmol/mg protein), and Hill coefficient (1.1 ± 0.1) of [³H]GBR 12935 in rat striatum using mazindol to define non-specific binding was not significantly altered by the inclusion of trans-flupentixol (0.75 μM). Using GBR 12909 as a blank produced a greater maximal number of binding sites (8.4 ± 2.3pmol/mg protein), but no significant difference in the equilibrium dissociation constant (1.6 ± 0.3nM) or Hill coefficient (1.1 ± 0.1). A series of drugs that bind to the dopamine uptake complex displaced [³H]GBR 12935 in a rank order consistent with other binding and behavioral studies of this complex. The rank order of these drugs was GBR 12909 > mazindol > nomifensine > benztropine > desipramine > amphetamine > dopamine; all these drugs displayed a Hill coefficient near one and were best modeled as a single site. Cocaine and WIN 35, 428 (a cocaine congener) were unique in their competition for [³H]GBR 12935 binding, displaying biphasic curves, low Hill coefficients, and were best modeled as two site fits. Lesioning of the dopaminergic median forebrain bundle resulted in a dramatic loss of the dopamine uptake complex in the striatum, nucleus accumbens, olfactory tubercle, and substantia nigra. Other dopaminergic projection areas were decreased to a lesser extent. Striatal ibotenate lesions did not decrease the density of the dopamine uptake complex, despite a large decrease in the dopamine D₁ receptor. [³H]GBR 12935 can be used as an effective ligand to label the dopamine uptake complex for quantitative autoradiographic studies. It offers a number of advantages over previous autoradiographic assays for this complex including high specificity (> 95% specific binding in rat striatum), high sensitivity (detection of mazindol displaceable sites in the cerebral cortex), low background (comparable to film background), and low cost. This assay also supports the existence of two binding sites for cocaine on the dopamine uptake complex. The exact nature and differences between these two cocaine sites remains to be determined.     

Dopamine D2 receptor-mediated G-protein activation in rat striatum: functional autoradiography and influence of unilateral 6-hydroxydopamine lesions of the substantia nigra.

Unilateral 6-hydroxydopamine (6-OHDA) lesions of substantia nigra pars compacta (SNPC) neurons in rats induce behavioural hypersensitivity to dopaminergic agonists. However, the role of specific dopamine receptors is unclear, and potential alterations in their transduction mechanisms remain to be evaluated. The present study addressed these issues employing the dopaminergic agonist, quinelorane, which efficaciously stimulated G-protein activation (as assessed by [³⁵S]GTPγS binding) at cloned hD₂ (and hD₃) receptors. At rat striatal membranes, dopamine stimulated [³⁵S]GTPγS binding by 1.9-fold over basal, but its actions were only partially reversed by the selective D₂/D₃ receptor antagonist, raclopride, indicating the involvement of other receptor subtypes. In contrast, quinelorane-induced stimulation (48% of the effect of dopamine) was abolished by raclopride, and by the D₂ receptor antagonist, L741,626. Further, novel antagonists selective for D₃ and D₄ receptors, S33084 and S18126, respectively, blocked the actions of quinelorane at concentrations corresponding to their affinities for D₂ receptors. Quinelorane potently induced contralateral rotation in unilaterally 6-OHDA-lesioned rats, an effect abolished by raclopride and L741,626, but not by D₃ and D₄ receptor-selective doses of S33084 and S18126, respectively. In functional ([³⁵S]GTPγS) autoradiography experiments, quinelorane stimulated G-protein activation in caudate putamen and, to a lesser extent, in nucleus accumbens and cingulate cortex of naive rats. In unilaterally SNPC-lesioned rats, quinelorane-induced G-protein activation in the caudate putamen on the non-lesioned side was similar to that seen in naive animals (50% stimulation), but significantly greater on the lesioned side (80%). This increase was both pharmacologically and regionally specific since it was reversed by raclopride, and was not observed in nucleus accumbens or cingulate cortex. In conclusion, the present data indicate that, in rat striatum, the actions of quinelorane are mediated primarily by D₂ receptors, and suggest that behavioural hypersensitivity to this agonist, induced by unilateral SNPC lesions, is associated with an increase in D₂, but not D₃ or D₄, receptor-mediated G-protein activation.     

Binding of [3H]-dihydroalprenolol and [3H]-acetobutolol to human blood platelets is not related to occupancy of beta-adrenoceptors

Binding of {³H}-dihydroalprenolol to human platelet lysates is inhibited by (±)-propranolol and (±)-butoxamine, but less effectively by (±) practolol. (−)-Isoprenaline causes no significant inhibition of binding where stimulation of adenylate cyclase can be shown. Binding of {³H}-acetobutolol is also inhibited by (±)-propranolol. “Specific” binding of {³H}-dihydroalprenolol and {³H}-acetobutolol defined by (±) propranolol shows a non-classical saturation curve. 50% maximal binding is observed in the range 15 – 25 mM. The extent of “specific” binding is 2-fold greater for {³H}-dihydroalprenolol. Similar and rapid rates of binding of {³H}-dihydroalprenolol are observed at 4°C and 20°C. No stereoselectivity is observed for inhibition of {³H}-dihydroalprenolol binding by (+) and (−)-propranolol. Binding of {³H}-dihydroalprenolol and {³H}-acetobutolol may relate to the lipophilic character of these radioligands and does not represent interaction with β-adrenoceptors.     

δ1-OPIOID receptor-mediated controlofacetylcholine (ACh) release in human neocortex slices

In slices of human neocortex, prelabelled with [³H]-choline, the release of [³H]-acetylcholine reflects the evoked release of endogenous acetylcholine whichwaselicited by the same electrical stimulation paradigm. [³H]-Acetylcholine releasewasdepressed by the δ-opioid receptor agonist -Pen₂- -Pen₅-enkephalin. When the nerve endings were depolarized byelevatingextracellular potassium the evoked [³H]-acetylcholine release was similarlydepressed by -Pen₂- -Pen₅-enkephalin in theabsence, but notin the presence, of tetrodotoxin which blocks action potential propagation.Therefore, the δ-opioid receptor inhibiting [³H]-acetylcholine release should notbe located tocholinergic nerve terminals, but rather to interneurons. The somatostatin₂receptorpartial agonist octreotide per se did not influence action potential-evoked [³H]-acetylcholine release, but prevented the inhibition of release of [³H]-acetylcholine by -Pen₂- -Pen₅-enkephalin.Similarly, the δ₁-opioid receptor antagonist 7-benzylidenenaltrexon perse did notinfluence [³H]-acetylcholine release, but prevented of the inhibition ofrelease by -Pen₂- -Pen₅-enkephalin.

From the presentfindings we conclude : (1) The evoked release of [³H]-acetylcholine from humanneocortex slices reflects the release of endogenous acetylcholine.(2) It is inhibited in an indirectmanner by opioid receptors of the δ₁-subtype,which (3) are not localized oncholinergic axon terminals but on soma and dendrites ofsomatostatin-containing interneurons,where they inhibit somatostatin release. (4) Theseinterneurons innervate cholinergic nerveendings in the human neocortex and appear to facilitateacetylcholine release via somatostatin₂ receptors.     

Calcium-dependent displacement of haloperidol-sensitive σ receptor binding in rat hippocampal slices following tissues depolarization

To evaluate the possible existence of an endogenous ligand for the haloperidol-sensitive σ receptor, we developed an in vivo competition assay to measure endogenous ligand release. Depolarization of in vitro hippocampal slices by either veratridine or potassium reduced [³H]ditolylguanidine binding in a calcium-dependent and transient manner. None of the drugs or ion substitutions directly affected [³H]ditolylguanidine binding to rat brain membranes. Veratridine-induced depolarization also reduced the binding of [³H](+)3-(3-hydroxyphenyl)-N-(1-propyl)piperidine, another σ radioligand, in a calcium-dependent manner. Radioligand displacement was not associated with alteration in σ receptor dissociation kinetics or receptor degradation in the hippocampal slice. In contrast, KCl depolarization had no effect on [³H]ditolylguanidine binding to σ receptors in liver slices. The results suggest that a calcium-dependent, depolarization-induced reduction in σ receptor binding may have been caused by the release of an endogenous σ ligand in rat hippocampal tissue.     

Stimulation of both D1 and D2 dopamine receptors appears necessary for full expression of postsynaptic effects of dopamine agonists: a neurophysiological study

The abilities of 4 dopamine agonists to inhibit the tonic single unit activity of substantia nigra dopamine neurons and stimulate tonic activity of globus pallidus neurons were compared to study the agonists' effects on pre- and postsynaptic dopamine receptors, respectively. The agonists studied were apomorphine and pergolide, which interact with both D₁ and D₂ receptors, and the selective D₂ agonists quinpirole and RU 24926. Drugs were administered systematically. The 4 dopamine agonists were equipotent and equiefficacious at inhibiting the firing rates of dopamine neurons. In contrast, their effects on pallidal cells were not identical; apomorphine and pergolide induced significantly greater increases in pallidal cell activity than did quinpirole and RU 24926. In addition, pretreatment with a small dose of quinpirole did not attenuate the excitatory effect of apomorphine on globus pallidus cell activity, as low doses of apomorphine have previously been shown to do. Possible mechanisms underlying the differences in efficacy between the non-selective and D₂ selective dopamine agonists in the globus pallidus were investigated. Coadministering quinpirole with apomorphine did not significantly attenuate the effect of apomorphine, suggesting that quinpirole is not a partial agonist at postsynaptic dopamine receptors. In addition, prazosin pretreatment did not attenuate the stimulatory effect of pergolide on firing rates of pallidal cells, indicating that the greater efficacy of the non-selective agonists was not due to concurrent stimulation of ₁ adrenergic receptors and dopamine receptors. However, the effect of quinpirole on pallidal cell activity was significantly potentiated by pretreatment with the D₁ agonist RS-SKF 38393 but not its inactive enantiomer S-SKF 38393. These results suggest that concurrent D₁ and D₂ receptor stimulation may be necessary for the full expression of postsynaptic receptor-mediated effects of dopamine and dopamine agonists in the basal ganglia.     

Presynaptic D2 dopaminergic receptors mediate inhibition of excitatory synaptic transmission in rat neostriatum

The effect of dopamine (DA) on excitatory synaptic transmission was studied in rat neostriatal neurons using intracellular- and whole-cell voltage clamp-recording methods. Depolarizing excitatory postsynaptic potentials (EPSPs) were evoked by cortical stimulation. Superfusion of DA (0.01–10 μM) reversibly decreases EPSP in a concentration-dependent manner and with a estimated IC₅ of 0.3 μM. In addition, the inhibitory effect induced by DA at a low concentratiion (0.1 μM) was antagonized by sulpiride (1–10 nM), a selective D₂ dopaminergic receptor antagonist. However, D₁ dopaminergic receptor antagonist SKF-83566 (1–5 μM) did not affect the blocking effect by DA 0.1 μM. Based on these findings, we conclude that DA at a low concentration ( 0.1 μM) reduced the excitatory response of neostriatal neurons following cortical stimulation via the activation of D₂, but not D₁ dopaminergic receptors, located on the terminals of corticostriatal neurons.