Drastic decrease in dopamine receptor levels in the striatum of acetylcholinesterase knock-out mouse

BackgroundThe acetylcholinesterase knock-out mouse lives to adulthood despite 60-fold elevated acetylcholine concentrations in the brain that are lethal to wild-type animals. Part of its mechanism of survival is a 50% decrease in muscarinic and nicotinic receptors and a 50% decrease in adrenoceptor levels.HypothesisThe hypothesis was tested that the dopaminergic neuronal system had also adapted.MethodsRadioligand binding assays measured dopamine receptor level and binding affinity in the striatum. Immunohistochemistry of brain sections with specific antibodies visualized dopamine transporter. Effects on the intracellular compartment were measured as cAMP content, PI-phospholipase C activity.ResultsDopamine receptor levels were decreased 28-fold for the D₁-like, and more than 37-fold for the D₂-like receptors, though binding affinity was normal. Despite these huge changes in receptor levels, dopamine transporter levels were not affected. The intracellular compartment had normal levels of cAMP and PI-phospholipase C activity.ConclusionSurvival of the acetylcholinesterase knock-out mouse could be linked to adaptation of many neuronal systems during development including the cholinergic, adrenergic and dopaminergic. These adaptations balance the overstimulation of cholinergic receptors caused by high acetylcholine concentrations and thus maintain homeostasis inside the cell, allowing the animal to live.     

A novel affinity purification of D-1 dopamine receptors from rat striatum

When rat striatal membranes were pretreated with the sulfhydryl (-SH) modifying reagent, N-ethylmaleimide (NEM) in the presence of the D-1-specific agonist, SKF R-38393, the D-1 dopamine receptor was completely protected from NEM-mediated inactivation. The D-1 receptors, solubilized from these membranes with 1% sodium cholate in the presence of phospholipids, bound with high efficiency (greater than 90%) to mercury-agarose columns. The bound receptors were eluted from the affinity column with a -SH reducing agent, beta-mercaptoethanol. Upon removal of beta-mercaptoethanol from the eluted fractions by inclusion chromatography, the receptor was reconstituted into phospholipid vesicles and assayed for ligand binding activity. The affinity purified receptor exhibited saturable and specific binding of the D-1-specific ligand 125I-SCH 23982, with a Kd of 1.6 nM comparable to that measured in intact membranes and solubilized extracts. The binding capacity of these receptors for 125I-SCH 23982 was 11,000 pmol/mg protein, representing greater than an 8000-fold purification over the starting membrane preparation. The purity of the affinity eluted receptors was estimated to be 78%. The purified receptors retained the pharmacological properties of membrane-bound receptors, including the ability to distinguish between active and inactive enantiomers of specific dopaminergic antagonists. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining revealed the presence of two major polypeptides of 74 and 54 kDa. These two polypeptides were absent in those affinity eluted fractions which did not display 125I-SCH 23982-binding activity and also were not detected in preparations obtained from membranes which were NEM-treated in the absence of D-1-specific agonist. The molecular weights of these polypeptides were similar to those of membrane-bound D-1 receptors, when labeled with a D-1-specific photo-affinity ligand, 125I-8-hydroxy-3-methyl-1-(4-azidphenyl)-2,3,4,5-tetrahydro-1H-3-b enzazepine. These two polypeptides may represent glycosylated and deglycosylated forms of the D-1 dopamine receptor.     

Distinct target size of dopamine D-1 and D-2 receptors in rat striatum

Frozen rat striatal tissue was exposed to 10 MeV electrons from a linear accelerator. Based on the theory of target size analysis, the molecular weights of dopamine D-1 receptors (labelled by 3H-piflutixol) and dopamine D-2 receptors (labelled by 3H-spiroperidol) were 79,500 daltons and 136,700 daltons, respectively. The size of the dopamine-stimulated adenylate cyclase was 202,000 daltons. The estimated molecular sizes were deduced by reference to proteins with known molecular weights which were irradiated in parallel. The results showed that the molecular entities for 3H-piflutixol binding and 3H-spiroperidol binding were not identical. The present results do not allow conclusions as to whether D-1 and D-2 receptors are two distinct proteins in the membrane, or whether the receptors are located on the same protein. In the latter case the binding of 3H-spiroperidol needs the presence of a second molecule.     

CB1-independent inhibition of dopamine transporter activity by cannabinoids in mouse dorsal striatum

Cannabinoid drugs are known to affect dopaminergic neurotransmission in the basal ganglia circuitry. In this study, we used in vitro and in vivo techniques to investigate whether cannabinoid agonists and antagonist could affect dopaminergic transmission in the striatum by acting at the dopamine transporter. Incubation of striatal synaptosomes with the cannabinoid agonists WIN55,212-2 or methanandamide decreased dopamine uptake (IC(50) = 2.0 micromol/L and 3.1 micromol/L, respectively). A similar inhibitory effect was observed after application of the inactive WIN55,212-2 isomer, S(-)WIN55,212-3. The CB(1) antagonist AM251 did not reverse WIN55,212-2 effect but rather mimicked it. WIN55,212-2 and AM251 partially displaced the binding of the cocaine analog [(3)H]WIN35,428, thus acting as dopamine transporter pseudo-substrates in the high micromolar range. High-speed chronoamperometry measurements showed that WIN55,212-2 (4 mg/kg, i.p.) caused significant release of endogenous dopamine via activation of CB(1) receptors, followed by a reduction of dopamine clearance. This reduction was CB(1)-independent, as it was mimicked by S(-)WIN55,212-3. Administration of AM251 (1 and 4 mg/kg, i.p.) increased the signal amplitude and reduced the clearance of dopamine pressure ejected into the striatum. These results indicate that both cannabinoid agonists and antagonists inhibit dopamine transporter activity via molecular targets other than CB(1) receptors.     

Sub-second dopamine detection in human striatum

Fast-scan cyclic voltammetry at carbon fiber microelectrodes allows rapid (sub-second) measurements of dopamine release in behaving animals. Herein, we report the modification of existing technology and demonstrate the feasibility of making sub-second measurements of dopamine release in the caudate nucleus of a human subject during brain surgery. First, we describe the modification of our electrodes that allow for measurements to be made in a human brain. Next, we demonstrate in vitro and in vivo, that our modified electrodes can measure stimulated dopamine release in a rat brain equivalently to previously determined rodent electrodes. Finally, we demonstrate acute measurements of dopamine release in the caudate of a human patient during DBS electrode implantation surgery. The data generated are highly amenable for future work investigating the relationship between dopamine levels and important decision variables in human decision-making tasks.     

Comparison of alterations in tyrosine hydroxylase,dopamine levels,and dopamine uptake in the striatum of the weaver mutant mouse

Previous reports have shown that among the markers for the nigro-striatal dopamine (DA) system measured in the striatum, dopamine uptake seems to be more severely affected than the others in the weaver mutant mouse. In the present study we examined DA levels, tyrosine hydroxylase (TH) activity, and high-affinity DA uptake to determine if the DA uptake is most affected when all the measurements are made in the same striatal homogenate in the same laboratory. We found that the DA uptake activity was most altered (93% lower) compared to DA levels (68% lower) and TH activity (64% lower). The DA uptake was so low in the weaver that we could not obtain reliable kinetic parameters. For TH activity we found that the Vmax was 36% lower while the Km forl-tyrosine was 92% higher in the weaver striatum. This lower affinity for substrate suggests that the TH enzyme itself may be altered in the nigro-striatal system of the weaver mutant mouse.Special issue dedicated to Dr. Morris H. Aprison.     

Changes in gene expression within the nucleus accumbens and striatum following sexual experience

Sexual experience, like repeated drug use, produces long-term changes including sensitization in the nucleus accumbens and dorsal striatum. To better understand the molecular mechanisms underlying the neuroadaptations following sexual experience, we employed a DNA microarray approach to identify genes differentially expressed between sexually experienced and sexually naive female hamsters within the nucleus accumbens and dorsal striatum. For 6 weeks, a stimulus male was placed in the home cage of one-half of the hormonally primed, ovariectomized female hamsters. On the seventh week, the two experimental groups were subdivided, with one half paired with a stimulus male. In comparison with sexually naive animals, sexually experienced hamsters receiving a stimulus male on week 7 exhibited an increase in a large number of genes. Conversely, sexually experienced female hamsters not receiving a stimulus male on week 7 exhibited a reduction in the expression of many genes. For directional changes and the categories of genes regulated by the experimental conditions, data were consistent across the nucleus accumbens and dorsal striatum. However, the specific genes exhibiting changes in expression were disparate. These experiments, among the first to profile genes regulated by female sexual behavior, will provide insight into the mechanisms by which both motivated behaviors and drugs of abuse induce long-term changes in the mesolimbic and nigrostriatal dopamine pathways.     

RGS mRNA expression in rat striatum: modulation by dopamine receptors and effects of repeated amphetamine administration

Single injections of cocaine, amphetamine, or methamphetamine increased RGS2 mRNA levels in rat striatum by two- to fourfold. The D1 dopamine receptor-selective antagonist SCH-23390 had no effect by itself but strongly attenuated RGS2 mRNA induction by amphetamine. In contrast, the D2 receptor-selective antagonist raclopride induced RGS2 mRNA when administered alone and greatly enhanced stimulation by amphetamine. To examine the effects of repeated amphetamine on RGS2 expression, rats were treated with escalating doses of amphetamine (1.0-7.5 mg/kg) for 4 days, followed by 8 days of multiple daily injections (7.5 mg/kg/2 h x four injections). Twenty hours after the last injection the animals were challenged with amphetamine (7.5 mg/kg) or vehicle and killed 1 h later. In drug-naive animals, acute amphetamine induced the expression of RGS2, 3, and 5 and the immediate early genes c-fos and zif/268. RGS4 mRNA levels were not affected. Prior repeated treatment with amphetamine strongly suppressed induction of immediate early genes and RGS5 to a challenge dose of amphetamine. In sharp contrast, prior exposure to amphetamine did not reduce the induction of RGS2 and RGS3 mRNAs to a challenge dose of amphetamine, indicating that control of these genes is resistant to amphetamine-induced tolerance. These data establish a role for dopamine receptors in the regulation of RGS2 expression and suggest that RGS2 and 3 might mediate some aspects of amphetamine-induced tolerance.     

Reductions in binding and functions of D2 dopamine receptors in the rat ventral striatum during amphetamine sensitization

Rats receiving amphetamine (5 mg/kg, i.p. once daily) for 14 continuous days develop behavioral sensitization to a subsequent amphetamine challenge (1 mg/kg) at withdrawal days 8 to 10. The present study was aimed at investigating whether there are changes in binding or functions of striatal D2 dopamine receptors in amphetamine-sensitized rats. The results indicated that the Bmax value of D2 receptors in the ventral striatum decreased 40% and 52% 7 and 10 days after amphetamine withdrawal, respectively, without changes in their binding affinities (Kd). During this withdrawal period, the D(2/3) receptor agonist-induced (a) locomotor activation (bromocriptine, 5 mg/kg, i.p. or quinpirole, 1 mg/kg, i.p.) and (b) inhibition of forskolin-enhanced adenylyl cyclase activity (bromocriptine, 50 or 150 microM) in the ventral striatum were both suppressed as compared with saline controls. The decreases in D2 receptor function were unrelated to the coupled G-proteins, since none of the G alpha i-3, G alpha o or G alpha q in the ventral striatum exhibited quantitative differences between control and amphetamine sensitized rats. Collectively, these results demonstrate that intermittent amphetamine administration for a period of 14 days leads to diminished D2 receptor expression and functions in the ventral striatum at late withdrawal periods. The decrease of D2 receptors might reflect cellular mechanisms underlying the expression of amphetamine sensitization.     

The dopamine neuron synaptic map in the striatum

1. Download : Download high-res image (165KB)
2. Download : Download full-size image

     

Changes in the kinetics of dopamine release and uptake have differential effects on the spatial distribution of extracellular dopamine concentration in rat striatum

The objective of this study was to examine whether the limited diffusion distance of dopamine in rat striatum produces spatial heterogeneity in the extracellular dopamine concentration on a dimensional scale of a few micrometers. Such heterogeneity would be significant because it would imply that the concentration of dopamine at a given receptor depends on the receptor's ultrastructural location. Spatially resolved measurements of extracellular dopamine were performed in the striatum of chloral hydrate-anesthetized rats with carbon fiber microdisk electrodes. Dopamine was monitored during electrical stimulation of the nigrostriatal pathway before and after administration of drugs that selectively affect the kinetics of evoked dopamine release and dopamine uptake. The effects of nomifensine (20 mg/kg), L-DOPA (250 mg/kg), and alpha-methyl-p-tyrosine (250 mg/kg) on the amplitude of the stimulation responses were examined. The outcome of these experiments was compared with predictions derived from a mathematical model that combines diffusion with the kinetics of release and uptake. The results demonstrate that the extracellular dopamine concentration is spatially heterogeneous on a micrometer scale and that changing the kinetics of dopamine release and uptake has different effects on this spatial distribution. The impact of these results on brain neurochemistry is considered.     

Changes of antioxidants levels in two maize lines following atrazine treatments.

Growth and antioxidants levels of shoot of 10-d-old maize lines (Zea mays L. Hybrid 351 and Giza 2) differentially responded to atrazine treatment at the recommended field dose (RFD) during the following 20 d. Atrazine significantly reduced shoot fresh and dry weights but significantly accumulated H(2)O(2), lipid peroxides and carbonyl groups in Giza 2 during the whole experiment; an effect that prolonged with either elapse of time or increasing the herbicide dose. Meanwhile, ascorbic acid (AsA) and reduced glutathione (GSH) contents were significantly decreased along with significant inhibitions in activities of superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.7), guaiacol peroxidase (GPX; EC 1.11.1.7), and glutathione-S-transferase (GST; EC 2.5.1.18). Similar responses were observed in Hybrid 351 only during the first 12 d, and seemed to be overcome thereafter. These findings indicate an induced oxidative stress in maize following atrazine treatments. Such state appeared to be counterbalanced in Hybrid 351 but continued in Giza 2 concluding Giza 2 as more susceptible to atrazine than Hybrid 351. Therefore, the differential susceptibility of Giza 2 to atrazine is related to deficiency in antioxidant levels.