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.     

Purification of the D-2 dopamine receptor from bovine striatum

The D-2 dopamine receptor has been purified 21500 fold from bovine striatal membranes. Solubilized receptor preparation was partially purified by affinity chromatography on a haloperidol adsorbent followed by gel filtration on a Sephacryl S-300 column. The fractions eluted from this column which contained the ligand binding activity were further chromatographed on wheat germ agglutinin conjugated to Sepharose. The resulting receptor preparation displays a major polypeptide band of an apparent molecular weight of 92 kDa, and exhibits a specific binding activity of 2490 pmol spiperone per mg protein. This purified receptor preparation can reabsorb specifically to the haloperidol affinity column indicating that the 92 kDa polypeptide represents the ligand binding unit of the D-2 dopamine receptor.     

Solubilization of the D-1 dopamine receptor from rat striatum

The D-1 dopamine receptor was extracted from rat striatal membranes with 0.7% sodium cholate and 1 M NaCl. Pretreatment of the membranes with a D-1 specific agonist, inclusion of crude phospholipids in the solubilization buffer, and subsequent removal of the detergent led to a maximal extraction of 48% of the receptor binding sites. The D-1 antagonist, [125I]SCH 23982, bound to single class of sites with a Kd of 1.8 nM and a Bmax of 1.65 pmol/mg protein. The solubilized receptors retained the ability to discriminate between active and inactive enantiomers of agonists and antagonists selective for the D-1 receptor.     

Nanomolar concentrations of kynurenic acid reduce extracellular dopamine levels in the striatum

Precise regulation of dopaminergic activity is of obvious importance for the physiology and pathology of basal ganglia. We report here that nanomolar concentrations of the astrocyte-derived neuroinhibitory metabolite kynurenic acid (KYNA) potently reduce the extracellular levels of striatal dopamine in unanesthetized rats in vivo. This effect, which is initiated by the KYNA-induced blockade of alpha7 nicotinic acetylcholine receptors, highlights the functional relevance of glia-neuron interactions in the striatum and indicates that even modest increases in the brain levels of endogenous KYNA are capable of interfering with dopaminergic neurotransmission.     

Norepinephrine transporter (NET) knock-out upregulates dopamine and serotonin transporters in the mouse brain

The noradrenaline, serotonin and dopamine transporters are three main transporters, which are the target of the antidepressant drugs. In the present study we demonstrate that the life-long deletion of the noradrenaline transporter (NET) induced up-regulation of two other monoamine transporters, dopamine and serotonin (DAT and SERT, respectively). An increase in the binding of [³H]paroxetine to the SERT and [³H]GBR12935 to the DAT was observed in various brain regions of NET-KO mice, without alterations of mRNA encoding these transporters, as measured by in situ hybridization. This important finding impacts the interpretation of previous data indicating the supersensitizity of NET-KO mice for psychostimulants or stronger effect of citalopram in behavioral tests. While using the NET-KO mice in various psychopharmacological studies is very important, one has to be aware that these mice lack NET from the earliest period of their existence, thus compensatory alterations do take place and have to be considered when it comes to interpretation of the obtained results.     

Modulation of dopamine D1 receptor binding by neurotensin in the rat striatum

The effect of neurotensin on binding characteristics of dopamine D1 receptors was examined in the rat striatal membranes through radioreceptor assay. Neurotensin or its analogs were added to incubation medium of[³H]SCH 23390 saturation or dopamine/[³H]SCH 23390 inhibition experimental systems. Neurotensin did not modulate D1 antagonist binding but converted a part of D1 agonist high affinity binding sites to a low affinity state. Neurotensin_8–13 had the same potency as neurotensin itself, whereas neurotensin_1–8 had only weak activity in modulating D1 agonist binding. GTP and neurotensin had the same effect on D1 agonist binding. However, when both neurotensin and GTP were added, the result was the same as with either alone.

These data suggest that neurotensin modulates the functional state of D1 receptors probably via a GTP binding protein in the rat striatum.     

Adenosine and dopamine receptor interactions in striatum and caffeine-induced behavioral activation

This review will examine how dopamine, a monoamine neurotransmitter, and adenosine, a neuromodulator, regulate behavioral activation, primarily as reflected by locomotor activity, in rodents. Complex interactions among 2 major types of adenosine receptors (A1AR and A2AAR) and 2 dopamine receptors (D1R and D2R) occur due to physical interactions that alter their ligand-binding properties and subsequent effects on common postreceptor signaling molecules. The output from these interactions in striatum modulates neurotransmission and subsequently influences spontaneous locomotor activity. Caffeine is a nonselective adenosine receptor antagonist that blocks 2 major types of adenosine receptors, A1AR and A2AAR, in the brain. Pharmacologic manipulation of these receptors with drugs such as caffeine offers potential therapeutic benefit for treatment of Parkinson disease.     

Affinity chromatography of the D1 dopamine receptor from rat corpus striatum

The D1 dopamine receptor from rat corpus striatum has been purified 200-250-fold by using a newly developed biospecific affinity chromatography matrix based on a derivative of the D1 selective antagonist SCH 23390. This compound, (RS)-5-(4-aminophenyl)-8-chloro-2,3,4,5-tetrahydro-3-methyl-1H-3-benz azepin-7-o l (SCH 39111), possesses high affinity for the D1 receptor and, when immobilized on Sepharose 6B through an extended spacer arm, was able to adsorb digitonin-solubilized D1 receptors. The interaction between the solubilized receptor and the affinity matrix was biospecific. Adsorption of receptor activity could be blocked in a stereoselective fashion [SCH 23390 greater than SCH 23388; (+)-butaclamol greater than (-)-butaclamol]. The elution of [3H]SCH 23390 activity from the gel demonstrated similar stereoselectivity for antagonist ligands. Agonists eluted receptor activity with a rank order of potency consistent with that of a D1 receptor [apomorphine greater than dopamine greater than (-)-epinephrine much greater than LY 171555 greater than serotonin]. SCH 39111-Sepharose absorbed 75-85% of the soluble receptor activity, and after the gel was washed extensively, 35-55% of the absorbed receptor activity could be eluted with 100 microM (+)-butaclamol with specific activities ranging from 250 to 450 pmol/mg of protein. The affinity-purified receptor retains the ligand binding characteristics of a D1 dopamine receptor. This affinity chromatography procedure should prove valuable in the isolation and molecular characterization of the D1 dopamine receptor.     

Specific involvement of neurotensin type 1 receptor in the neurotensin-mediated in vivo dopamine efflux using knock-out mice

Abstract Neurotensin is a tridecapeptide neurotransmitter known to be involved in psychiatric disorders, various physiological processes and several different neurobiological mechanisms, including modulation of accumbal dopamine release. Two neurotensin extracellular binding sites, namely NT1- and NT2-receptor (NT1R and NT2R), have been cloned from the rat brain. These receptors are distinguishable by their different in vitro pharmacological properties but the available pharmacological tools have weak in vivo potency and specificity. The use of genetically engineered knock-out mice has provided a powerful alternative to the classical pharmacological approach to investigate their respective roles. In this study, using in vivo differential pulse amperometry, we show that, in wild-type mice, neurotensin application into the ventral tegmental area dose-dependently evokes dopamine efflux in the nucleus accumbens. This neurotensin-mediated efflux is dramatically decreased in mice lacking NT1R while it is unaffected in NT2R-deleted mice. This finding indicates that a large part of the dopamine efflux evoked by neurotensin in the nucleus accumbens of wild-type mice is mediated via NT1R present in the ventral tegmental area.     

Expression of D2 dopamine receptor in the mouse brain

The neurotransmitter, dopamine, binds to dopamine receptor (DR), and is involved in several functions of the brain, such as initiation and execution of movement, emotion, prolactin secretion, etc. Of all the five DRs, D2 dopamine receptor has maximal affinity for dopamine. D2 has a short isoform, D2S, and a long isoform D2L. D2L is longer than D2S by 29 amino acid residues. We studied the expression of the gene and protein of D2 receptor in the cerebral and cerebellar cortices of the brain of new born, developing, adult, and old male mice to find out: (i) at what stage of development, expression of the gene peaks and (ii) if it undergoes any changes as the animal ages, which may account for the neurodegenerative changes and symptoms of Parkinson's and other diseases seen in old age. RT-PCR and Western blot studies show that peak expression of D2 gene occurs in the cerebral and cerebellar cortices around 15-day after birth. We speculate that the majority of dopaminergic synapses are established and possibly become functional in the brain around 15-day after birth. The expression of D2 receptor is upregulated in the cerebral cortex in old mice. However, it is down-regulated in the cerebellar cortex.     

Pregnenolone sulfate induces NMDA receptor dependent release of dopamine from synaptic terminals in the striatum

Neuromodulators that alter the balance between lower-frequency glutamate-mediated excitatory and higher-frequency GABA-mediated inhibitory synaptic transmission are likely to participate in core mechanisms for CNS function and may contribute to the pathophysiology of neurological disorders such as schizophrenia and Alzheimer's disease. Pregnenolone sulfate (PS) modulates both ionotropic glutamate and GABA(A) receptor mediated synaptic transmission. The enzymes necessary for PS synthesis and degradation are found in brain tissue of several species including human and rat, and up to 5 nM PS has been detected in extracts of postmortem human brain. Here, we ask whether PS could modulate transmitter release from nerve terminals located in the striatum. Superfusion of a preparation of striatal nerve terminals comprised of mixed synaptosomes and synaptoneurosomes with brief-duration (2 min) pulses of 25 nM PS demonstrates that PS increases the release of newly accumulated [3H]dopamine ([3H]DA), but not [14C]glutamate or [3H]GABA, whereas pregnenolone is without effect. PS does not affect dopamine transporter (DAT) mediated uptake of [3H]DA, demonstrating that it specifically affects the transmitter release mechanism. The PS-induced [3H]DA release occurs via an NMDA receptor (NMDAR) dependent mechanism as it is blocked by D-2-amino-5-phosphonovaleric acid. PS modulates DA release with very high potency, significantly increasing [3H]DA release at PS concentrations as low as 25 pM. This first report of a selective direct enhancement of synaptosomal dopamine release by PS at picomolar concentrations via an NMDAR dependent mechanism raises the possibility that dopaminergic axon terminals may be a site of action for this neurosteroid.     

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.     

Strain‐specific proportion of the two isoforms of the dopamine D2 receptor in the mouse striatum: associated neural and behavioral phenotypes

Genetic variability in the proportion of the two alternative dopamine D2 receptor (D2R) mRNA splice variants, D2R‐long (D2L) and D2R‐short (D2S), influence corticostriatal functioning and could be implicated in liability to psychopathology. This study compared mesostriatal D2L/D2S ratios and associated neural and behavioral phenotypes in mice of the DBA/2J and C57BL/6J‐inbred strains, which differ for schizophrenia‐ and addiction‐like phenotypes. Results showed that DBA/2J mice lack the striatal predominance of D2L that has been reported in the rat and in C57BL/6J mice and confirmed in the latter strain by this study. Only C57BL/6J mice showed enhanced striatal c‐Fos expression under D1R and D2/3R co‐stimulation, indicating synergistic interaction between the subtypes of DA receptors. Instead, DBA/2J mice were characterized by opposing effects of D2/3R and D1R stimulation on striatal c‐Fos expression, in line with a more pronounced influence of D2S isoform, and did not express stereotyped climbing under D1R and D2/3R co‐stimulation, as reported for D2L?/? mice. Finally, strain‐specific modulation of c‐Fos expression by D1R and D2/3R co‐stimulation was selectively observed in striatal compartments receiving inputs from the prefrontal cortex and involved in the control of motivated behaviors. These results show differences in tissue‐specific D2R splicing in mice with intact genotypes and support a role for this phenotype in individual variability of corticostriatal functioning and in liability to psychopathology.     

The effect of prolyl oligopeptidase inhibition on extracellular acetylcholine and dopamine levels in the rat striatum

Prolyl oligopeptidase (PREP, EC 3.4.21.26) inhibitors have potential as cognition enhancers, but the mechanism of action behind the cognitive effects remains unclear. Since acetylcholine (ACh) and dopamine (DA) are known to be associated with the regulation of cognitive processes, we investigated the effects of two PREP inhibitors on the extracellular levels of ACh and DA in the rat striatum using in vivo microdialysis. KYP-2047 and JTP-4819 were administered either as a single systemic dose (50 μmol/kg～17 mg/kg i.p.) or directly into the striatum by retrodialysis via the microdialysis probe (12.5, 37.5 or 125 μM at 1.5 μl/min for 60 min). PREP inhibitors had no significant effect on striatal DA levels after systemic administration. JTP-4819 significantly decreased ACh levels both after systemic (by ～25%) and intrastriatal (by ～30-50%) administration. KYP-2047 decreased ACh levels only after intrastriatal administration by retrodialysis (by ～40-50%) when higher drug levels were reached, indicating that higher brain drug levels are needed to modulate ACh levels than to inhibit PREP. This result does not support the earlier hypothesis that the positive cognitive effects of PREP inhibitors in rodents would be mediated through the cholinergic system. In vitro specificity studies did not reveal any obvious off-targets that could explain the observed effect of KYP-2047 and JTP-4819 on ACh levels, instead confirming the concept that these compounds have a high selectivity towards PREP.     

Differential expression of the mouse D2 dopamine receptor isoforms

We have identified and characterized the cDNAs corresponding to the mouse D2 dopamine receptors. We show that in the mouse the D2 dopamine receptor is found in two forms, generated by alternative splicing of the same gene, mRNA distribution analysis of areas expressing the D2 receptors shows that the larger form is the most abundant, except in the brain stem where the shorter form is predominant. Membranes of mammalian cells transiently transfected with both forms of D2 receptor bind [3H]spiperone with a high affinity.     

Localization of melatonin receptor 1 in mouse retina and its role in the circadian regulation of the electroretinogram and dopamine levels

Melatonin modulates many important functions within the eye by interacting with a family of G-protein-coupled receptors that are negatively coupled with adenylate cyclase. In the mouse, Melatonin Receptors type 1 (MT(1)) mRNAs have been localized to photoreceptors, inner retinal neurons, and ganglion cells, thus suggesting that MT(1) receptors may play an important role in retinal physiology. Indeed, we have recently reported that absence of the MT(1) receptors has a dramatic effect on the regulation of the daily rhythm in visual processing, and on retinal cell viability during aging. We have also shown that removal of MT(1) receptors leads to a small (3-4 mmHg) increase in the level of the intraocular pressure during the night and to a significant loss (25-30%) in the number of cells within the retinal ganglion cell layer during aging. In the present study we investigated the cellular distribution in the C3H/f(+/+) mouse retina of MT(1) receptors using a newly developed MT(1) receptor antibody, and then we determined the role that MT(1) signaling plays in the circadian regulation of the mouse electroretinogram, and in the retinal dopaminergic system. Our data indicate that MT(1) receptor immunoreactivity is present in many retinal cell types, and in particular, on rod and cone photoreceptors and on intrinsically photosensitive ganglion cells (ipRGCs). MT(1) signaling is necessary for the circadian rhythm in the photopic ERG, but not for the circadian rhythm in the retinal dopaminergic system. Finally our data suggest that the circadian regulation of dopamine turnover does not drive the photopic ERG rhythm.     

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.     

The dopamine neuron synaptic map in the striatum

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