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The nonsteroidal anti-inflammatory drug (NSAID) niflumic acid, a fenamate in structure, has many molecular targets, one of them being specific subtypes of the main inhibitory ligand-gated anion channel, the GABA A receptor. Here, we report on the effects of other fenamates and other classes of NSAIDs on brain picrotoxinin-sensitive GABA A receptors, using an autoradiographic assay with [35S]TBPS as a ligand on mouse brain sections. We found that the other fenamates studied (flufenamic acid, meclofenamic acid, mefenamic acid and tolfenamic acid) affected the autoradiographic signal at low micromolar concentrations in a facilitatory-like allosteric fashion, i.e., without having affinity to the [35S]TBPS binding site. Unlike niflumic acid that shows clear preference for inhibiting cerebellar granule cell layer GABA A receptors, the other fenamates showed little brain regional selectivity, indicating that their actions are not receptor-subtype selective. Of the non-fenamate NSAIDs studied at 100 μM concentration, diclofenac induced the greatest inhibition of the binding, which is not surprising as it has close structural similarity with the potent fenamate meclofenamic acid. Using two-electrode voltage-clamp assays on Xenopus oocytes, the effect of niflumic acid was found to be dependent on the β subunit variant and the presence of γ2 subunit in rat recombinant α1β and α1βγ2 GABA A receptors, with the β1 allowing the niflumic acid inhibition and β3 the stimulation of the receptor-mediated currents. In summary, the fenamate NSAIDs constitute an interesting class of compounds that could be used for development of potent GABA A receptor allosteric agonists with other targets to moderate inflammation, pain and associated anxiety/depression. [ABSTRACT FROM AUTHOR]

The ATP-gated ion channel P2X7 has emerged as a potential central nervous system (CNS) drug target based on the hypotheses that pro-inflammatory cytokines such as IL-1β that are released by microglia, may contribute to the etiology of various disorders of the CNS including depression. In this study, we identified two closely related P2X7 antagonists, JNJ-54232334 and JNJ-54140515, and then tritium labeled the former to produce a new radioligand for P2X7. JNJ-54232334 is a high affinity ligand for the rat P2X7 with a pK i of 9.3±0.1. In rat cortical membranes, [ 3 H] JNJ-54232334 reached saturable binding with equilibrium dissociation ( K d ) constant of 4.9±1.3 nM. The compound displayed monophasic association and dissociation kinetics with fast on and off rates. In rat brain sections, specific binding of [ 3 H] JNJ-54232334 was markedly improved compared to the previously described P2X7 radioligand, [ 3 H] A-804598. In P2X7 knockout mouse brain sections, [ 3 H] A-804598 bound to non-P2X7 binding sites in contrast to [ 3 H] JNJ-54232334. In rat or wild type mouse brain sections [ 3 H] JNJ-54232334 bound in a more homogenous and region independent manner. The ubiquitous expression of P2X7 receptors was confirmed with immunohistochemistry in rat brain sections. The partial displacement of [ 3 H] A-804598 binding resulted in the underestimation of the level of ex vivo P2X7 occupancy for JNJ-54140515. Higher levels of P2X7 ex vivo occupancy were measured using [ 3 H] JNJ-54232334 due to less non-specific binding. In summary, we describe [ 3 H] JNJ-54232334 as a novel P2X7 radioligand, with improved properties over [ 3 H] A-804598. [ABSTRACT FROM AUTHOR]

The neuronal α7 nicotinic acetylcholine receptor is a homo-pentameric ligand-gated ion channel that is a promising drug target for cognitive deficits in Alzheimer׳s disease and schizophrenia. We have previously described 11 C-NS14492 as a suitable agonist radioligand for in vivo positron emission tomography (PET) occupancy studies of the α7 nicotinic receptor in the pig brain. In order to investigate the utility of the same compound for in vitro studies, 3 H-NS14492 was synthesized and its binding properties were characterized using in vitro autoradiography and homogenate binding assays in pig frontal cortex. 3 H-NS14492 showed specific binding to α7 nicotinic receptors in autoradiography, revealing a dissociation constant ( K d ) of 2.1±0.7 nM and a maximum number of binding sites ( B max ) of 15.7±2.0 fmol/mg tissue equivalent. Binding distribution was similar to that of another selective ligand 125 I- α -bungarotoxin ( 125 I-BTX) in autoradiography, and unlabeled NS14492 displaced 125 I-BTX with an inhibition constant ( K i ) of 23 nM. 3 H-NS14492 bound to α7 nicotinic receptors in homogenized pig frontal cortex with a K d of 0.8±0.3 nM and a B max of 30.2±11.6 fmol/mg protein. This binding assay further revealed the K i rank order for a number of α7 nicotinic receptor agonists, and positive allosteric modulators (PAMs). Further, we saw increased binding of 3 H-NS14492 to pig frontal cortex membranes when co-incubated with PNU-120596, a type II PAM. Taken together, these findings show that 3 H-NS14492 is a useful new in vitro radioligand for the pig α7 nicotinic receptor. [ABSTRACT FROM AUTHOR]

The loss of cholinergic neurons and synapses relates to the severity of dementia in several neurodegenerative pathologies; and the vesicular acetylcholine transporter (VAChT) provides a reliable biomarker of cholinergic function. We recently characterized and 11 C-labeled a new VAChT inhibitor, (−)-TZ659. Here we report the in vitro and ex vivo characterization of (−)-TZ659. A stably transfected PC12 A123.7 cell line which expresses human VAChT (hVAChT) was used for the in vitro binding characterization of (−)-[ 3 H]TZ659. A saturated binding curve was obtained with K d =1.97±0.30 nM and B max =3240±145.9 fmol/mg protein. In comparison, a PC12 A123.7 cell line that expresses mutant hVAChT showed decreased binding affinity ( K d =15.94±0.28 nM). Competitive binding assays using a panel of other CNS ligands showed no inhibition of (−)-[ 3 H]TZ659 binding. On the other hand, binding inhibitions were observed only using VAChT inhibitors ( K i =0.20–31.35 nM). An in vitro assay using rat brain homogenates showed that (−)-[ 3 H]TZ659 had higher binding in striatum than in cerebellum, with a target: non-target ratio>3.46. Even higher ex vivo striatum-to-cerebellum ratios (9.56±1.11) were observed using filtered homogenates of brain tissue after rats were injected intravenously with (−)-[ 11 C]TZ659. Ex vivo autoradiography of (−)-[ 11 C]TZ659 confirmed high striatal uptake, with a consistently high striatum-to-cerebellum ratio (2.99±0.44). In conclusion, (−)-TZ659 demonstrated high potency and good specificity for VAChT in vitro and in vivo . These data suggest that (−)-[ 11 C]TZ659 may be a promising PET tracer to image VAChT in the brain. [ABSTRACT FROM AUTHOR]

Dopamine D 3 receptors are a major target for drug discovery programs related to psychiatric disorders such as schizophrenia. The ability of a compound to occupy significant levels of D 3 receptors is important for achieving therapeutic efficacy in both pre-clinical and clinical settings. Here we attempt to characterise antipsychotic drug-effects at D 3 receptors by measuring receptor occupancy via ex-vivo [ 3 H]7-OH-DPAT autoradiography, and further validating this outcome via analysis of Fos-like immunoreactivity (Fos-LI) in the rat major islands of Calleja (ICjM), a brain structure with high D 3 expression. Rats were treated subcutaneously with haloperidol (0.04 mg/kg), clozapine (20 mg/kg) and olanzapine (0.63 mg/kg), the selective D 2 antagonist L-741626 (2.5 mg/kg) and the selective D 3 antagonist SB-277011-A (10 mg/kg). Doses were based on levels of D 2 occupancy considered clinically relevant (60–80%). When measuring D 3 occupancy, clozapine and SB-277011-A displayed meaningful levels of occupancy (60% and 77%, respectively), haloperidol and olanzapine showed limited occupancy (16% and 27%, respectively), whereas L-741626 showed no occupancy. There were no significant changes in ICjM Fos-LI after L-741626 and haloperidol treatment, minor but significant increases after olanzapine treatment, whereas highly significant increases were seen with SB-277011-A and clozapine. Additionally, pre-treating clozapine with the D 1 antagonist SCH23390 caused a significant, albeit non-complete, reduction in Fos-LI, highlighting the D 1 agonist property of clozapine. In conclusion, it appears that drugs occupying >50% D 3 receptors produce robust increases in ICjM Fos-LI. This study may help to identify the appropriate D 3 receptor antagonists that have the potential to be tested in the clinic. [ABSTRACT FROM AUTHOR]

Abstract: The MCHR1 is an interesting pharmacological and pharmaceutical target, due to its involvement in pathologies as diabetes, gut inflammation and adiposity. in vivo PET-studies of the MCHR1 in energy homeostasis and diabetes could be of great value for deeper understanding of endocrinological hormone status and consequential pharmacological interactions. Furthermore, PET-tracers would facilitate compound dose selection of MCHR1 antagonists for treatment. Therefore, we developed two potential PET-tracers, [11C]SNAP-7941 and [18F]FE@SNAP, for the in vivo visualization of this receptor. Aim of this study was a preclinical in vitro evaluation of both unlabeled ligands. Therefore, a comparative autoradiographic investigation on CNS (coronal rat brain and 4 different human brain regions) and peripheral tissues (rat tongue as target and rat testes as non-target region) was conducted. Competition experiments, using the two radioligands [125I]-MCH and [125I]-S36057, were performed with selective and specific MCHR1 ligands as PMC-3886, a MCHR1 agonist, SNAP-7941 and FE@SNAP, two MCHR1 antagonists. Additionally, immunohistochemical staining with a specific MCHR1 antibody was performed. Specific binding was found in all tissues known to express the MCHR1 as human and rat CNS and peripheral rat tongue tissue. No specific binding was found in the non-target region of rat testes. MCHR1 antibody staining complemented the outcome of the autoradiographic experiments. The compounds SNAP-7941 and FE@SNAP were generally comparable with PMC-3886. Hence, the in vitro autoradiographic study of the unlabeled compounds SNAP-7941 and FE@SNAP further qualifies the potential of the PET-tracers [11C]SNAP-7941 and [18F]FE@SNAP as useful MCHR1 PET-tracers. [Copyright &y& Elsevier]

Abstract: Several methodological approaches suggest that receptor heteromers exist in cell systems, but their presence in physiological tissue is widely contentious. We describe a novel method to determine if heterodimers exist in brain tissue sections using autoradiographic binding comparisons from single and double gene knockout mice, where tissues either have a full receptor complement and can form heterodimers, or are incapable of making heterodimers. We have tested this model, which we have named Knockout Subtraction Autoradiography, to determine if heterodimerisation of the kappa (KOP) and delta opioid (DOP) receptors occurs, as evidence from binding studies in cell systems suggest they are present in the brain. Using labeling of putative KOP receptor/DOP receptor heterodimers with either [3H]bremazocine or with [3H]naltrindole, two ligands which were used to provide evidence suggesting that these opioid receptor subtypes heterodimerize, we have applied a subtraction equation model based on the principle that receptor gene double knockout of either MOP receptor/KOP receptor (DOP receptor expression only) or MOP receptor/DOP receptor (KOP receptor expression only) produces tissue incapable of making the KOP receptor/DOP receptor heterodimer. We have shown in most brain regions that the labeling fits a simple additive model of monomer labeling, but that in a few brain regions opioid receptor heterodimerization does occur. The data does not support the conclusion that KOP receptor/DOP receptor heterodimerisation is widespread in the central nervous system, but does indicate that this novel methodology can detect heterodimerisation, when ligands with distinct binding affinities for monomer and heterodimer forms exist. [Copyright &y& Elsevier]

Abstract: NMDA receptor antagonists, particularly these targeting the GluN2B subunit are of therapeutic interest for the treatment of severe mood disorders. The receptor occupancy profiles of several NMDA receptor antagonists (30mg/kg, s.c.) were compared in mouse hippocampus by ex vivo autoradiography using [3H]MK-801, a non-selective NMDA channel blocker, and [3H]ifenprodil a selective GluN2B antagonist. Subcutaneous administration of ketamine ((RS)-2-(2-Chlorophenyl)-2-(methylamino)cyclohexanone) and memantine (3,5-dimethyladamantan-1-amine) inhibited [3H]MK-801 but not [3H]ifenprodil binding in mouse hippocampus. Ketamine reached maximal occupancy of [3H]MK-801 binding sites after 15min and rapidly cleared from the brain with no significant level of occupancy measured at the 1h time point. Memantine significantly occupied [3H]MK-801 binding sites throughout the 6h time course. The selective GluN2B antagonist CP101,606 ((1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol) and Ro 25-6981 ((αR,βS)-α-(4-Hydroxyphenyl)-β-methyl-4-(phenylmethyl)-1-piperidinepropanol maleate) inhibited [3H]ifenprodil but not [3H]MK-801 binding and significant levels of occupancy (above 50%) were measured throughout the 6h time course. These data highlight the unique quick pulse target engagement profile of ketamine compared to other NMDA receptor antagonists. [Copyright &y& Elsevier]

Abstract: Pregabalin, a synthetic branched chain γ-amino acid with anticonvulsant, anxiolytic, and analgesic activities, has been shown to bind with high affinity to the voltage-gated calcium channel α2δ subunit. Given the broad therapeutic utility of pregabalin, a series of experiments was undertaken to determine the potency, selectivity, and specificity of pregabalin''s receptor-binding profile at α2δ-1 and α2δ-2 subunits of voltage-gated calcium channels along with 38 widely studied receptors and channels. Receptor autoradiography was used to assess regional-binding density of pregabalin throughout the rat spinal cord and brain. In addition, a series of studies using in vivo electrophysiological recordings of γ-aminobutyric acid (GABA)A- and GABAB-evoked currents was undertaken to determine the interaction of pregabalin with GABAergic receptor subtypes. Together, the results of these studies demonstrate potent and selective binding of pregabalin to α2δ-1 and α2δ-2 subunits in native and recombinant human and porcine systems. Pregabalin did not interact with any of the 38 receptors and ion channels evaluated, and a variety of central nervous system (CNS)-targeted therapeutic drugs did not show activity at the α2δ subunits of voltage-gated calcium channels. Receptor autoradiography demonstrated extensive [3H]-pregabalin binding throughout the CNS, with high-level binding in the cortex, hippocampus, cerebellum, dorsal horn of the spinal cord, and amygdala. Finally, receptor-binding and electrophysiological techniques failed to show evidence of an interaction between pregabalin and GABAA or GABAB receptors. These studies suggest that the clinical effects of pregabalin are likely due to direct and selective interactions with α2δ-1 and α2δ-2 subunits of voltage-gated calcium channels. [Copyright &y& Elsevier]

Abstract: MePPEP ((3R,5R)-5-(3-methoxy-phenyl)-3-((R)-1-phenyl-ethylamino)-1-(4-trifluoromethyl-phenyl)-pyrrolidin-2-one) is an inverse agonist shown to be an effective PET ligand for labeling cannabinoid CB1 receptors in vivo. [11C]MePPEP and structurally related analogs have been reported to specifically and reversibly label cannabinoid CB1 receptors in rat and non-human primate brains, and [11C]MePPEP has been used in human subjects as a PET tracer. We have generated [3H]MePPEP, an ortholog of [11C]MePPEP, to characterize the molecular pharmacology of the cannabinoid CB1 receptor across preclinical and clinical species. [3H]MePPEP demonstrates saturable, reversible, and single-site high affinity binding to cannabinoid CB1 receptors. In cerebellar membranes purified from brains of rat, non-human primate and human, and cells ectopically expressing recombinant human cannabinoid CB1 receptor, [3H]MePPEP binds cannabinoid CB1 receptors with similar affinity with K d values of 0.09nM, 0.19nM, 0.14nM and 0.16nM, respectively. Both agonist and antagonist cannabinoid ligands compete [3H]MePPEP with predicted rank order potency. No specific binding is present in autoradiographic sections from cannabinoid CB1 receptor knockout mouse brains, demonstrating that [3H]MePPEP selectively binds cannabinoid CB1 receptors in native mouse tissue. Furthermore, [3H]MePPEP binding to anatomical sites in mouse and rat brain is comparable to the anatomical profiles of [11C]MePPEP in non-human primate and human brain in vivo, as well as the binding profiles of other previously described cannabinoid CB1 receptor agonist and antagonist radioligands. Therefore, [3H]MePPEP is a promising tool for translation of preclinical cannabinoid CB1 receptor pharmacology to clinical PET ligand and cannabinoid CB1 receptor inverse agonist therapeutic development. [Copyright &y& Elsevier]

Abstract: The present study was undertaken to investigate the mechanistic role of l-prolyl-l-leucyl-glycinamide (PLG) in modulating agonist binding to the dopamine D2L receptor. Competition and displacement assays indicate that the photoaffinity-labeling peptidomimetics of PLG, 3(R)-[(4(S)-(4-azido-2-hydroxy-benzoyl) amino-2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide hydrochloride (1a) and 3(R)-[(4(S)-(4-azido-2-hydroxy-5-iodo-benzoyl)amino-2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide hydrochloride (1b) bind at the same site as PLG. Autoradiography was used to establish the covalent binding of [125I]-1b to a∼51kDa protein in bovine striatal membranes. Western blot analysis with a dopamine D2L-specific antibody, in combination with autoradiography, following a two-dimensional gel separation, suggested this∼51kDa protein to be the dopamine D2L receptor. Further evidence for binding of 1b to dopamine D2L was provided by samples immunoprecipitated with the D2L antibody. These samples were analyzed by western blotting in parallel with autoradiography of [125I]-1b labeled protein. Both methods revealed bands at∼51kDa. Furthermore, PLG is shown to compete with 1b for binding to the dopamine D2L receptor as determined by autoradiography, as well as competition experiments with PLG and 1a. Collectively, these findings suggest the successful development of a photoaffinity-labeling agent, compound 1b, that has been used to elucidate the interaction of PLG specifically with the dopamine D2L receptor. [Copyright &y& Elsevier]

Abstract: The present study describes the optimisation of an autoradiography assay that provides a means to measure the in vitro potency of melanin-concentrating hormone receptor 1 (MCH1) antagonists in native tissues and their ex vivo receptor occupancy. Initial localisation studies demonstrated that the MCH1 receptor radioligand [125I]-S36057 bound to rat caudate putamen with specific binding of consistently >60%. In vitro, the MCH1 receptor antagonists GW3430, SNAP-94847 and 4′-{[1-(cyclopropylmethyl)piperidin-4-ylidene] [5-fluoro-6-(trifluoromethyl)-1H-benzimidazol-2-yl]methyl}biphenyl-3-carbonitrile (referred to as Compound A) exhibited concentration dependent inhibition of the specific binding of [125I]-S36057, with a rank order of affinity of SNAP-94847>Compound A>GW3430. In an ex vivo occupancy assay, Compound A dosed orally to rats caused a concentration dependent inhibition of the specific binding of [125I]-S36057 to rat caudate putamen. The occupancy reached 87±11% at 30 mg/kg and the estimated ED50 was 9.3 mg/kg, which was equivalent to a free plasma concentration of 40 nM. As MCH has been reported to play a role in the regulation of the sleep cycle, the effect of Compound A on sleep parameters was investigated. However Compound A, at exposures that achieved near maximal receptor occupancy, failed to demonstrate any effects on the sleep/wake pattern in telemetered rats. We conclude that our ex vivo receptor occupancy assay is suitable for selecting centrally penetrant MCH1 receptor antagonists and that, despite high levels of receptor occupancy, the selective MCH1 receptor antagonist Compound A failed to elicit any changes in sleep electroencephalogram (EEG) parameters. [Copyright &y& Elsevier]

Abstract: The adenosine A2A receptor in the basal ganglia is involved in the control of movement and plays a role in movement disorders such as Parkinsonism. Developing ligands to evaluate that receptor by noninvasive methods such as positron emission tomography has a high priority. In vitro radioligand binding guides the selection of ligands for in vivo application. This study measured the binding of the adenosine A2A receptor antagonist [3H]MSX-2 (3-(3-hydroxypropyl)-8-m-methoxystyryl)-7-methyl-1-propargylxanthine) to rat, mouse and pig brain by autoradiography. Other studies measured binding to membranes from PC12 pheochromocytoma cells. Those binding parameters were compared to those of the adenosine A2A receptor antagonist [3H]ZM241385 (4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino)ethyl)phenol), the adenosine A2A receptor agonist [3H]CGS21680 (2-[p-(2-carboxyethyl)-phenethylamino]-5′-N-ethylcarboxamidoadenosine) and the unselective adenosine receptor agonist [3H]NECA (5′N-ethylcarboxamido)adenosine). The potency order (K d) in the three species was [3H]ZM241385<[3H]MSX-2<[3H]NECA<[3H]CGS21680. The density of [3H]MSX-2 binding sites was greater in the striatum than in the cortex. Preliminary ex vivo experiments showed that by 10min after iv injection, [3H]MSX-2 and [3H]CGS21680 crossed the blood–brain barrier to the extent of almost 1% ID/g brain tissue, but [3H]NECA and [3H]ZM241385 to only 0.2% ID/g. The prior administration of unlabeled ZM241385 significantly lowered brain uptake of [3H]MSX-2. In conclusion, [3H]MSX-2 has a high affinity and sufficient selectivity for the adenosine A2A receptor. It penetrates the blood–brain barrier. Sensitivity to photoisomerization is a limitation. Further investigations assess its suitability as a ligand for imaging the brain adenosine A2A receptor. [Copyright &y& Elsevier]

Abstract: Exposure to chronic stress has been found to decrease cannabinoid CB1 receptor expression in the hippocampus; however, the specificity of this phenomenon to specific subfields of the hippocampus has not been characterized. To this extent, male Sprague-Dawley rats were exposed to 21 days of restraint stress (6 h/day), after which autoradiographical analysis of cannabinoid CB1 receptor binding site densities were examined in the CA1, CA3 and dentate gyrus subfields of the hippocampus. Chronic stress was found to produce a significant reduction in cannabinoid CB1 receptor binding in the dentate gyrus, while increasing cannabinoid CB1 receptor binding in the CA3. There was no effect of chronic stress on cannabinoid CB1 receptor binding in the CA1, or two other proximal regions, the retrosplenial cortical gyrus and the laterodorsal thalamus. Given the role of hippocampal cannabinoid CB1 receptor activity in the maintenance of synaptic integrity and neuroplasticity in the hippocampus, these data suggest that changes in cannabinoid CB1 receptor activity following stress may contribute to stress-induced modulation of these processes. [Copyright &y& Elsevier]

Abstract: We examined neuroadaptive changes in γ-aminobutyric acid (GABA)B receptor binding following cocaine self-administration and its withdrawal in several rat brain structures using a “yoked” procedure and a quantitative autoradiographic analysis. In order to estimate the distribution of GABAB receptors in several brain areas, we used ([S-(R⁎,R⁎)]-[3-[[1-(3,4-diclorophenyl)ethyl]amino]-2-hydroxypropyl]([3,4-3H]-cyclohexylmethyl) phosphinic acid) ([3H]CGP 54626), a GABAB receptor antagonist. The binding of [3H]CGP 54626 in the nucleus accumbens subareas and the amygdala was decreased by ca. 20% in rats that actively self-administered cocaine. Similar decreases in the nucleus accumbens were seen in the animals passively receiving cocaine. These animals also showed a reduction in GABAB receptor binding in the prefrontal and frontal cortices, septum and dorsal striatum. The [3H]CGP 54626 binding in several rat brain areas was decreased after 10-day withdrawal from self-administered cocaine. In summary, the decreases in the GABAB receptor binding seem to reflect the effects of chronic administration of cocaine per se and not the motivated process of reinforced responding. Furthermore, withdrawal from cocaine self-administration evoked changes in the GABAB receptor binding in several rat brain areas that may indicate neurobiological adaptations. [Copyright &y& Elsevier]

Abstract: The relationship between the abundances of agonist and antagonist-binding sites for monoamine receptors is poorly established. Therefore, we used quantitative autoradiography to investigate the distribution and concentration of binding sites for histamine H3 receptor ligands in cryostat sections of pig brain. As in other species, binding of the histamine H3 receptor agonist [3H]Nα-methylhistamine was highly heterogeneous in the pig brain, with highest B max in the substantia nigra, followed by the nucleus accumbens and caudate, intermediate binding in frontal cortex, diencephalon, and mesencephalon, and absent specific binding in cerebellum: the affinity of [3H]Nα-methylhistamine was close to 1 nM in all regions of pig brain. Thus, the saturation binding parameters for this H3 receptor agonist in pig brain were similar to the earlier reports in rat, guinea pig, and human. The distribution of histamine H3 receptors labeled with the receptor antagonist [125I]iodophenpropit in adjacent cryostat sections from the same group of pigs was very similar to that of [3H]Nα-methylhistamine. However, the B max of the receptor antagonist was 40% higher in the basal ganglia than was the B max of the receptor agonist. The K d for the receptor antagonist ligand was close to 0.9 nM in all regions. These results suggest that histamine H3 receptor agonist-binding sites, i.e. those linked to intracellular G-protein, comprise a subset of the total receptor antagonist-binding sites in the basal ganglia, as has been reported for dopamine D2 receptors. [Copyright &y& Elsevier]

Abstract: Imidazoline I2 binding sites are heterogeneous in nature and have been observed in the brain of a number of species. Development of specific imidazoline I2 radioligands, such as [3H]2-BFI and [3H]BU224, that have a high affinity for the imidazoline I2 binding site, has enabled the central distribution of these sites to be mapped. Extensive studies have been conducted on the rat brain with a number of radioligands. However, to date a comprehensive analysis of imidazoline I2 ligand binding in mouse brain has not been completed. In the present work we describe levels of [3H]2-BFI specific binding found throughout the mouse brain. [3H]2-BFI (2 nM) showed discrete regional distribution which was readily displaced by saturating concentrations of the specific imidazoline I2 ligand BU224. The highest levels of [3H]2-BFI specific binding were found in the dorsal raphe, paraventricular thalamus and nucleus accumbens. Moderate levels were found throughout the lining of the aqueduct, lateral ventricle, lateral 4th ventricle, 4th ventricle, 3rd ventricle, but not the dorsal 3rd ventricle. Based on the loss of [3H]idazoxan binding in brain homogenates from monoamine oxidase-A and B (MAO-A and MAO-B) deficient mice it has been suggested that imidazoline I2 binding sites are predominantly on MAO. Consistent with this hypothesis the regional distribution of [3H]2-BFI shows some overlap with that previously reported for MAO. However, in the rat imidazoline I2 binding sites have been shown to be heterogeneous in nature and it is likely [3H]2-BFI is binding to multiple imidazoline I2 binding sites within mouse brain. [Copyright &y& Elsevier]

Abstract: Quantitative autoradiography has been used to assess whether [3H]paracetamol (3 μM) binds specifically to any area of the murine brain and spinal cord and to investigate whether paracetamol (1–100 μM) competes for binding to the nociceptin opioid peptide (NOP) receptor or to the nitrobenzylthioinosine (NBTI)-sensitive adenosine transporter in the brains of mice. [3H]paracetamol binding was homogenous and, although there was some indication of specific binding overall, this binding in most individual regions failed to reach statistical significance. However, thoracic segments of the spinal cord were found to have significantly higher specific binding than cervical and lumbar regions. Paracetamol did not significantly compete for binding to the NOP receptor or to the NBTI-sensitive adenosine transporter, showing that it does not mediate its effect via these sites. Although paracetamol did bind specifically to the murine brain and spinal cord, the binding was not region-specific, suggesting binding is not related to any particular neurotransmitter system. [Copyright &y& Elsevier]

The binding of the adenosine A2A receptor antagonist [3H] 4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol ([3H]ZM 241385) to mouse brain and spinal cord was investigated. In brain homogenates, single-site binding was observed with a Bmax of 299±28 fmol mg−1 protein and a Kd of 0.75±0.08 nM. In autoradiographic studies, there was a high density of specific binding of [3H]ZM 241385 in the striatum, with a very low density in the cortex and no binding elsewhere in the brain or in the spinal cord. All specific binding of [3H]ZM 241385 was lost in genetically modified mice lacking the adenosine A2A receptor, confirming the selectivity of this radioligand. [Copyright &y& Elsevier]

Chronic treatment with nicotine differentially alters behavior in adolescent rats compared to adult rats. It is not known, however, whether the effects of nicotine on the neurochemical pathways with which it interacts differ in adolescents vs. adults. In the current study, the effects of a 7-day treatment with nicotine on nicotinic, dopaminergic, and serotonergic neurochemistry were examined in the caudate putamen and nucleus accumbens in periadolescent vs. adult male rats. Nicotine treatment increased dopamine transporter densities and decreased serotonin transporter densities in periadolescent rats. There was no change in nicotinic acetylcholine receptor densities or dopamine D1 or D2 receptor densities in nicotine-pretreated periadolescent rats. In adult rats pretreated with nicotine, there was an increase in nicotinic acetylcholine densities, but no change in dopamine transporter, dopamine D1 or D2 receptor, or serotonin transporter densities. Overall, these findings show that periadolescent rats have neurochemical adaptations to nicotine different from adult rats. These alterations may explain, at least in part, the differential behavioral effects of chronic nicotine in adult and adolescent male rats. [Copyright &y& Elsevier]

[Ethyl-3H][8aR,12aS,13aS]-5,8,8a,9,10,11,12,12a,13,13a-decahydro-3-methoxy-12-(ethylsulfonyl)-6H-isoquino[2,1-g][1,6]naphthyridine ([ethyl-3H]RS-79948-197) was evaluated for α2-adrenoceptor autoradiography in brain sections from wild-type mice and α2A- and α2ABC-adrenoceptor knockout mice. Receptor numbers were 83% lower in cortex and 28% lower in caudate putamen of α2A-knockout mice than in wild-type mice. No specific binding was seen in α2ABC-knockout mice. [Ethyl-3H]RS-79948-197 saturation binding parameters were compared to those of [3H]2-(2,3-dihydro-2-methoxy-1,4-benzodioxan-2-yl)-4,5-dihydro-1H-imidazoline ([3H]RX821002) and [methyl-3H]17α-hydroxy-20α-yohimban-16β-carboxylic acid methyl ester ([methyl-3H]rauwolscine). [Ethyl-3H-]RS-79948-197 detected a larger number of both α2A- and α2B/C-adrenoceptors than [3H]RX821002, while [methyl-3H]rauwolscine only underestimated the number of α2A-adrenoceptors. Oxymetazoline and prazosin competed for [ethyl-3H]RS-79948-197 binding with the expected rank order of affinities. Higher than necessary [ethyl-3H]RS-79948-197 concentrations resulted in a rapid increase in non-specific binding. Slow dissociation kinetics, high specific radioactivity and high α2-adrenoceptor affinity (slightly lower for the α2A-adrenoceptor than for the other subtypes) confer [ethyl-3H]RS-79948-197 distinct advantages compared to [3H]RX821002 for detection of α2-adrenoceptor subtypes in a mixed α2-adrenoceptor population. [Copyright &y& Elsevier]

Analogues of 3,4-dimethyl-4-(3-hydroxyphenyl)piperidines are high affinity inverse agonists for μ-, δ- and κ-opioid receptors. To characterize inverse agonist binding, we synthesized a high specific activity radioligand from this series, [3H]LY515300 (3-[1-((3-cyclohexyl-[3,4-3H2])-3(R,S)-hydroxypropyl)-3(R),4(R)-dimethylpiperidin-4-yl]phenol). In membranes expressing cloned human opioid receptors, [3H]LY515300 binding was saturable and exhibited low nonspecific binding. [3H]LY515300 bound with high affinity to the μ- (Kd=0.07 nM), δ- (Kd=0.92 nM) and κ-(Kd=0.45 nM) opioid receptors. High affinity [3H]LY515300 binding to all opioid receptors was Na+-dependent, a characteristic of inverse agonists. Displacement by standard opioid compounds yielded Ki values consistent with their known opioid receptor affinities. Autoradiographic localization of specific [3H]LY515300 binding in rat and guinea pig brain was high in areas known to express high levels of opioid (particularly μ-opioid receptor) binding sites including the caudate, nucleus accumbens, and nucleus tractus solitarius. Thus, [3H]LY515300 is the first radiolabeled opioid receptor inverse agonist useful for the study of opioid receptors in cell lines and native tissues. [Copyright &y& Elsevier]

Levetiracetam (2S-(2-oxo-1-pyrrolidinyl)butanamide, KEPPRA®), a novel antiepileptic drug, has been shown to bind to a specific binding site located in brain (levetiracetam binding site [Eur. J. Pharmacol. 286 (1995) 137]). However, [3H]levetiracetam displayed only micromolar affinity for these sites making it an unsuitable probe for further characterization. The present study describes the binding properties of an analogue of levetiracetam: [3H]ucb 30889, (2S)-2-[4-(3-azidophenyl)-2-oxopyrrolidin-1-yl]butanamide. [3H]ucb 30889 binds reversibly to specific binding sites in rat brain. Kinetics at 4 °C were biphasic with half-times of association and dissociation of, respectively, 3 and 4 min for the fast component and 47 and 61 min for the slow component. [3H]ucb 30889 saturation binding curves were compatible with the labelling of a homogenous population of binding sites having a Bmax of 4496±790 fmol/mg protein (mean±S.D., n=5) and a Kd of 62±20 nM (mean±S.D., n=5), a 20-fold increase in affinity compared to [3H]levetiracetam. Competition binding curves with ligands known to interact with levetiracetam binding sites and tissue distribution restricted to the brain indicated that [3H]ucb 30889 and [3H]levetiracetam bind to the same site. Although levetiracetam binding sites and GABAA (γ-aminobutyric acid) receptors share some ligands such as pentobarbital and pentylenetetrazol, experiments performed with [35S]TBPS (tert-butyl-bicyclo[2.2.2]phosporothionate), a probe for the GABAA Cl− channel do not support the hypothesis that levetiracetam binding sites are part of the GABAA receptor complex. Preliminary autoradiography studies in rat brain revealed that [3H]ucb 30889 labels specific sites in all brain regions and that this binding is concentration-dependently displaced by levetiracetam. [Copyright &y& Elsevier]

Neurochemical changes were examined in dopamine D1 receptor knockout (D1−/−), dopamine D3 receptor knockout (D3−/−) and dopamine D1/D3 receptor double knockout (D1−/−D3−/−) mice. The level of dopamine D1- and D2-like receptors and γ-aminobutyric acid (GABAA) receptor was assessed by ligand autoradiography and dopamine D1- and D2 receptor, enkephalin, dynorphin and substance P transcripts measured by in situ hybridization. D1−/− mice had normal GABAA receptor levels, reduced dynorphin and substance P, and increased enkephalin mRNA and dopamine D2-like binding. D1−/−D3−/− mice evidenced decreased dynorphin and substance P but normal enkephalin expression, whereas dopamine D2-like and GABAA receptor binding were increased. Major changes occur in substance P and dynorphin expression in D1−/− mice and these changes are unaffected by loss of dopamine D3 receptors. Upregulated dopamine D2-like binding and enkephalin in D1−/− mice may be due to decreased dopamine turnover. Upregulated enkephalin in D1−/− mice is dependent on functional dopamine D3 receptors. [Copyright &y& Elsevier]

μ-opioid receptor knockout mice have been reported to show loss of some δ-opioid receptor function. We hypothesised that this is due to some δ-opioid receptors being uncoupled from G-proteins in the absence of the μ-opioid receptor. To address this possibility, we have used quantitative autoradiography to determine the binding of three δ-opioid receptor agonist ligands ([3H]deltorphin I, [3H] [R-Atc3, Ile5,6]deltorphin II, [3H] 4-[(alpaR)-alpha-((2S,5R)-4-propyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC-121)) and the δ-opioid receptor antagonist, [3H]naltrindole in the presence and absence of a GTP analogue, guanylylimidodiphosphate (GMPPNP) in the brains of mice lacking the μ-opioid receptor gene. Guanylylimidodiphosphate caused a decrease in the binding of the agonist ligands (to differing extents) and an increase in binding for the antagonist in wild-type controls. Overall, there were no major differences in the effects of guanylylimidodiphosphate for the agonist ligands in μ-knockout mice although a few structures showed a smaller sensitivity to the effects of this GTP analogue most notably for [3H]naltrindole. These findings suggest that in the majority of brain regions, G-protein coupling is unaltered in μ-opioid receptor knockout mice. [Copyright &y& Elsevier]

BU224 (2-(4,5-dihydroimidaz-2-yl)-quinoline) is a selective imidazoline I2 binding site ligand characterised in both competition binding assays and functional studies. However, in some studies, BU224 has been reported to have a different functional effect from that seen with another selective imidazoline I2 binding site ligand 2-BFI (2-(2-benzofuranyl)-2-imidazoline). This effect may reflect differing efficacies of the ligands or a difference in their brain distribution. The present study has investigated the distribution of the tritiated form of BU224 in rat brain and correlated this distribution with other imidazoline I2 binding site ligands, [3H]idazoxan and [3H]2-BFI. Saturation studies revealed binding of [3H]BU224 was of high affinity and saturable. The central distribution of [3H]BU224 was similar to that previous reported for imidazoline I2 binding site in rat brain. Autoradiography revealed that the highest levels of binding were in the arcuate nucleus, interpeduncular nucleus, area postrema, pineal gland and ependymal cell layer lining the ventricles. Correlation analysis of the binding distribution with our previous published studies revealed a highly significant correlation between [3H]BU224 and both [3H]idazoxan (r=0.94) and [3H]2-BFI (r=0.96). These data indicate [3H]BU224 labels the same population of imidazoline I2 binding site in rat brain as seen with [3H]idazoxan and [3H]2-BFI. Therefore, the differences in functional effects observed with these compounds may reflect agonist and antagonist properties. [Copyright &y& Elsevier]

Vesicular acetylcholine transporter plays a crucial role in the cholinergic system, and its alterations is implicated in several neurodegenerative disorders. We recently developed a PET imaging tracer [18F]VAT to target VAChT in vivo with high affinity and selectivity. Here we report in vitro characterization of [3H]VAT, a tritiated counterpart of [18F]VAT. Using human VAChT-rich cell membrane extracts, a saturated binding curve was obtained for [3H]VAT with K d = 6.5 nM and B max = 22.89 pmol/mg protein. In the [3H]VAT competition-binding assay with a panel of CNS ligands, binding inhibition of [3H]VAT was observed using VAChT ligands, the K i values ranged from 5.41 to 33.3 nM. No inhibition was detected using a panel of other CNS ligands. In vitro [3H]VAT autoradiography of rat brain sections showed strong signals in the striatum, moderate to high signals in vermis, thalamus, cortex, and hippocampus, and weak signals in cerebellum. Strong [3H]VAT ARG signals were also observed from striatal sections of normal nonhuman primates and human brains. Competitive ARG study with human striatal sections demonstrated strong ARG signals of [3H]VAT in caudate and putamen were blocked significantly by either VAChT ligand TZ659 or (−)-vesamicol, but not by the σ 1 receptor ligand Yun-122. ARG study also indicated that signal in the striatal sections from PSP human brains was lower than normal human brains. These data provide solid evidence supporting [18F]VAT as a suitable PET radiotracer for quantitative assessment of VAChT levels in vivo. [Display omitted] [ABSTRACT FROM AUTHOR]

Rovatirelin is a newly synthetized thyrotropin-releasing hormone (TRH) analog. This study aimed to investigate the effect of rovatirelin on motor function using rolling mouse Nagoya (RMN), a mouse model of hereditary ataxia, and compare it with that of taltirelin, which is clinically used to treat spinocerebellar degeneration in Japan. We also examined the effect of rovatirelin on glucose metabolism in various brain regions of RMN using autoradiography (ARG). Rovatirelin (1, 3, 10, and 30 mg/kg) dose-dependently reduced the fall index in RMN, and its effect was more potent than that of taltirelin (3, 10, 30, and 100 mg/kg). No attenuation of the effect was observed by repeated daily administration for 2 weeks. Furthermore, the reduction in the fall index by rovatirelin persisted for 2 weeks after completing treatment. In the ARG study, rovatirelin induced a significantly elevated uptake of glucose in the prefrontal cortex, nucleus accumbens shell, nucleus accumbens core, striatum, anterior cingulate cortex, secondary motor area, pretectal area, ventral tegmental area, black pars compacta, locus coeruleus, nucleus cerebellaris middle nucleus, medial nucleus of the vestibular nerve, fourth/fifth lobule, and third lobule. Furthermore, rovatirelin increased cerebellar mRNA level of brain derived neurotrophic factor. These results suggest that rovatirelin activates the cerebellum and other parts of the central nervous system to improve motor function in spinocerebellar ataxia (SCA) model animals, and its action is more potent than that of taltirelin. Therefore, rovatirelin can be a potential alternative to the traditionally used therapeutics for SCA. [ABSTRACT FROM AUTHOR]