Binding In Rat Brain Research Articles

P-645 - Repeated noise stress alters metabotropic glutamate receptor binding in rat brain

P-645 - Repeated noise stress alters metabotropic glutamate receptor binding in rat brain

Long-term effects of chronic ethanol on muscarinic receptor binding in rat brain.

Effects of chronic ethanol treatment (CET) on muscarinic acetylcholine receptor (mAChR) binding properties were investigated via quantitative autoradiography in rats maintained on an ethanol-containing liquid diet for 28 weeks and withdrawn from ethanol for 8 weeks before harvesting of tissues. Controls received an identical diet in which sucrose was substituted isocalorically for ethanol. Maximal binding of the radiolabeled mAChR antagonist quinuclidinyl benzilate ([3H]QNB) was not reduced in hippocampal area CA1, dentate gyrus, neocortex, striatum, or thalamus, suggesting that CET results in no significant mAChR loss in these regions. Binding affinities of the cholinergic agonist carbachol to mAChRs were unaffected by CET in each of these regions, as determined by competitive displacement of [3H]QNB labeling. These results suggest that CET-induced functional deficits in brain cholinergic responses are not due to direct effects of CET on mAChR binding properties.

Effects of chlorpyrifos on high-affinity choline uptake and [3H]hemicholinium-3 binding in rat brain.

High, subcutaneous doses of the organophosphorus insecticide chlorpyrifos (CPF) in adult male rats can be well-tolerated despite extensive and persistent acetylcholinesterase (AChE) inhibition. We propose that changes in acetylcholine synthesis could modulate the toxicity associated with extensive AChE inhibition following CPF exposure. High-affinity choline uptake (HACU, the rate-limiting step in acetlcholine synthesis) and binding to [3H]-hemicholinium-3 (HC-3, a specific ligand for the choline transporter) were chosen as indicators of acetylcholine synthesis. Female, Sprague-Dawley rats (220-280 g) were treated with either vehicle (peanut oil, 2 ml/kg, sc) or CPF (280 mg/kg, 2 ml/kg, sc), examined daily for clinical signs of toxicity, and sacrificed 1, 2, or 7 days later for neurochemical measurements (AChE inhibition, muscarinic receptor binding using [3H]quinuclidinyl benzilate (QNB) and [3H]cis-methyldioxolane (CD) as ligands, HACU and [3H]HC-3 binding) in frontal cortex. Despite extensive AChE inhibition (90-93%) at all time points, relatively minor degrees of overt toxicity were noted in CPF-treated rats. Binding to the non-selective muscarinic antagonist [3H]QNB was reduced (10-34%), whereas binding to the putative m2-selective agonist [3H]CD was increased (15-23%) at all three time points. HACU was reduced (20%) in crude synaptosomes prepared from CPF-treated rats 1 day following exposure but no significant changes were noted at 2 or 7 days after treatment. CPF-oxon, the active oxidative metabolite of CPF, was a weak inhibitor of HACU in vitro (IC50 > 200 microM). Binding to [3H]HC-3 was reduced in a dose-related manner 1 day after CPF exposure. Kinetic analyses of [3H]HC-3 binding 1 day after CPF (280 mg/kg) indicated a significant reduction in density (Bmax: control, 187 +/- 18 fmol/mg protein; CPF, 104 +/- 12 fmol/mg protein) with no apparent change in binding affinity (Kd: control, 25 +/- 3 nM; CPF, 19 +/- 3 nM). These results suggest that a reduction in HACU/acetylcholine synthesis may contribute, along with compensatory changes in cholinergic receptors, to the diminished toxicity following extensive AChE inhibition by CPF.

Inositol hexakisphosphate binding sites in rat heart and brain

1. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol hexakisphosphate (InsP6) are produced in response to stimulation of cardiac alpha 1-adrenoceptors. While the role of Ins(1,4,5)P3 and Ins(1,4,5)P3 receptors is well-defined in many tissues including brain, the functional role of the putative InsP6-InsP6 receptor system in cardiac function is less clear. Using quantitative autoradiography, this study examined the characteristics and regional localization of [3H]-InsP6 binding sites in rat heart and compared the affinity of a range of inositol polyphosphates for [3H]-InsP6 and [3H]-Ins(1,4,5)P3 binding sites in heart and brain. 2. [3H]-InsP6 bound to a single, high affinity site in sections of rat heart (KD ranging from 22 +/- 1.9 nM in right atria to 35 +/- 2.6 nM in the interventricular septum, n = 7). The maximal number of binding sites (Bmax) ranged from 5.1 +/- 0.48 to 12 +/- 1.8 pmol mg-1 protein in left atrium and left ventricle, respectively. Inositol phosphates inhibited binding of [3H]-InsP6 with the order of potency: InsP6 > Ins(1,4,5)PS3 > inositol 1,3,4,5-tetrakisphosphate > or = inositol pentakisphosphate > Ins(1,4,5)P3 > > inositol mono- and bisphosphates, consistent with the labelling of an InsP6 binding site. 3. The Ins(1,4,5)P3 analogue, Ins(1,4,5)PS3, originally investigated as a putative selective radioligand for the Ins(1,4,5)P3 receptor, was a potent inhibitor of [3H]-InsP6 binding in all heart regions (K1 = 170-260 nM). The K1 of Ins(1,4,5)PS3 for the inhibition of [3H]-Ins(1,4,5)P3 binding in rat brain (60-220 nM) was similar to that observed for the inhibition of [3H]-InsP6 binding in heart, suggesting that Ins(1,4,5)PS3 is not a specific ligand for either Ins(1,4,5)P3 or InsP6 receptor binding sites. 4. Previous studies have detected [3H]-InsP6 binding in mitochondrial and sarcoplasmic reticulum fractions of heart and links between InsP6 and cardiac mitochondrial Ca2+ regulation have been proposed, suggesting further studies are warranted to determine the functional role(s) of InsP6 and InsP6 receptor binding sites in cardiac tissue.

Chronic antipsychotic treatment alters glycine-stimulated NMDA receptor binding in rat brain

In this study the chronic effect of antipsychotic drugs (APDs) on N-methyl- d-aspartate (NMDA) receptor binding was evaluated. Rats were treated for 21 days with i.p. injections of haloperidol (0.5 mg/kg), pimozide (0.5 mg/kg), clozapine (20 mg/kg), risperidone (1 mg/ kg) or water vehicle (1 ml/kg). Brain tissue sections underwent different [ 3H]MK-801 assay conditions. Following a short preincubation wash, there were no effects of APDs on either unenhanced or agonist enhanced [ 3H]MK-801 binding. Following a prolonged preincubation wash, APDs resulted in a reduction in both unenhanced binding and glycine enhanced binding. The attenuation of glycine stimulation in the APD treated animals was selective as neither NMDA nor spermidine enhanced binding was significantly affected. The present data suggest specific actions at the glycine regulatory site on the NMDA receptor as part of the chronic effects of APDs.

Visualization and characterization of group 2 and 3 metabotropic glutamate receptors using [ 35s]GTPγS binding in rat brain

Visualization and characterization of group 2 and 3 metabotropic glutamate receptors using [ 35s]GTPγS binding in rat brain

Orphanin FQ: Receptor binding and analog structure activity relationships in rat brain

A tritiated form of orphanin FQ (a heptadecapeptide also known as Nociceptin) has been prepared. This radioligand (33 Ci/mmole) was used to develop a radioreceptor assay using rat brain homogenates. Binding was observed to be saturable, and analyses of the binding data indicate the presence of a single binding site with a dissociation constant of 5 ± 1.1 nM and Bmax of 535 ± 85 fmoles/mg protein. Thirty-four analogues of orphanin FQ, including a complete alanine “scan” of orphanin FQ, and truncation analogues from both the N- and C- terminals were synthesized and tested. The data obtained indicate that the N-terminus plays a more critical role in binding than the C-terminus and that residues 1, 2, 4, and 8 are essential for binding.

Loreclezole modulates [ 35S] t-butylbicyclophosphorothionate and [ 3H]flunitrazepam binding via a distinct site on the GABA A receptor complex

The allosteric modulation of [ 35S] t-butylbicyclophosphorothionate ([ 35S]TBPS) and [ 3H]flunitrazepam binding was utilized to evaluate the actions of loreclezole at the GABA A receptor complex in the rat brain. Loreclezole was observed to allosterically inhibit the binding of [ 35S]TBPS in a dose-dependent manner with micromolar potency (IC 50 = 1 μM). Loreclezole was found to have an additive effect on neuroactive steroid modulation of [ 35S]TBPS binding, but merely potentiated the effect of Ro5-4864 (4″-chlorodiazepam) modulation of [ 35S]TBPS binding. These observation suggest that loreclezole modulates [ 35S]TBPS binding through a site independent of the neuroactive steroid and Ro5-4864 sites on the GABA A receptor complex. The enhancement of [ 3H]flunitrazepam binding to the benzodiazepine receptor by loreclezole as well as the effect of loreclezole on CL218872/[ 3H]flunitrazepam dose-response curves suggest that loreclezole does not act through the benzodiazepine site on the GABA A receptor complex, nor does it selectively modulate benzodiazepine receptor subtypes. The potency of loreclezole as an inhibitor of [ 35S]TBPS binding in rat brain was regionally dependent and GABA-sensitive. Loreclezole modulation of [ 35S]TBPS binding showed greater potency and GABA sensitivity in the cerebellum and thalamus when compared to other brain regions such as the cortex, hippocampus and striatum. This finding is consistent with previous reports of the selectivity of loreclezole for GABA A receptor complex's containing β 2 and β 3 subunits. These β subunit isoforms predominate in the cerebellum and thalamus. Collectively the evidence suggests that loreclezole modulates [ 35S]TBPS and [ 3H]flunitrazepam binding through a site distinct from benzodiazepine, neuroactive steroid, Ro5-4864 and GABA sites on the GABA A receptor complex.

Naltrexone-induced changes in mu-opioid-receptor binding in neonatal rat brain following chronic prenatal hypoxia.

Naltrexone-induced changes in mu-opioid-receptor binding in neonatal rat brain following chronic prenatal hypoxia.

Identification of opioid receptor-like (ORL1) peptide-stimulated [35S]GTP gamma S binding in rat brain.

Recent reports have identified an endogenous peptide ligand for the opioid receptor-like (ORL1) receptor. In the present study, ORL1 peptide-stimulated [35>]GTP gamma S binding was assessed in rat cortical membranes and brain sections to localize ORL1 receptor-activated G-proteins. In membrane assays, with 20 microM GDP, ORL1 peptide stimulated [35S]GTP gamma S binding by approximately two-fold with an ED50 value of 20 nM. ORL1 peptide-stimulated [35S]GTP gamma S binding was unaffected by opioid or other G-protein-coupled receptor antagonists. In brain sections, ORL1 peptide-stimulated [35S]GTP gamma S binding was identified in regions including cortex, amygdala, hypothalamus, thalamus and brain stem. The anatomical distribution of ORL1 peptide-stimulated [35S]GTP gamma S binding suggests its involvement in cognition, emotion and homeostasis.

Washable endogenous substances and regional heterogeneity in agonist enhanced [ 3H]MK-801 binding in rat brain

NMDA receptor/ion channel function is modulated through a number of distinct sites that regulate channel opening. Published studies report widely varying results in modulatory site agonist effects due to assay conditions and technique. Also, NMDA receptor regulation at these sites by endogenous substances remains poorly characterized. The objectives of the present study in Sprague-Dawley rat forebrain sections were: (i) determine the contribution of various prewash variables on agonist stimulation of the NMDA receptor, (ii) compare regional differences in functional glycine, spermidine and NMDA binding sites under optimized prewash conditions, and (iii) define the influence of endogenous substances at each modulatory site by analyzing changes in binding at different prewash durations. We demonstrate that prewash conditions have a critical influence on [ 3H]MK-801 binding in rat tissue sections and that this effect was differentially expressed across brain regions. An extended prewash duration caused a regionally specific decrease in unenhanced [ 3H]MK-801 binding, while a short prewash caused a regionally specific biphasic effect on enhanced [ 3H]MK-801 binding. After prolonged prewash, binding was restored to previous (unwashed) binding levels with exogenously added glycine, NMDA, or spermidine alone or combinations of agonists. These data suggest that washable endogenous substances contribute to the full functionality of the NMDA receptor and the regional heterogeneity in [ 3H]MK-801 binding is dependent on the interaction of receptor protein subtypes and the presence of one or more endogenous substances.

P-907 - Effect of amygdaloid kindling on histamine H1 receptor binding in rat brain.

P-907 - Effect of amygdaloid kindling on histamine H1 receptor binding in rat brain.

Effects of Chlorpyrifos on High-Affinity Choline Uptake and [3H]Hemicholinium-3 Binding in Rat Brain

Effects of Chlorpyrifos on High-Affinity Choline Uptake and [³H]Hemicholinium-3 Binding in Rat Brain

Effects of chronic ethanol treatment on inositol 1,4,5-trisphosphate receptors and inositol 1,3,4,5-tetrakisphosphate receptors in rat brain.

In this study, the effects of chronic ethanol treatment on inositol 1,4,5-trisphosphate (IP3) and inositol 1,3,4,5-tetrakisphosphate (IP4) specific binding in rat brain was investigated. In the cerebellum, chronic, but not acute, ethanol treatment caused a decrease in the number IP3 receptors. The effect of chronic ethanol treatment on IP3 and IP3 receptor mRNA levels was also studied in order to determine the mechanisms responsible for the decrease in IP3 receptor binding. Chronic ethanol treatment did not change IP3 levels, indicating that the decrease of IP3 receptors is not caused by the alteration of IP3 levels. Also IP3 receptor mRNA levels had no change after chronic ethanol treatment. These findings suggest that the decrease in IP3 receptors in the cerebellum could be caused by either a decrease in the translation of IP3 receptor mRNA or an increase in proteolysis of IP3 receptors. In contrast, chronic ethanol treatment had no effect on the Bmax or Kd of IP4 specific binding in the cerebellum. It is speculated that a change at the level of the IP3 receptor in the cerebellum may be associated with the development of adaptation and tolerance to chronic ethanol exposure.

P-508 - Effects of intraperitoneal administration of 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (6R-BH4) on the monoaminergic receptor binding in rat brain.

P-508 - Effects of intraperitoneal administration of 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (6R-BH4) on the monoaminergic receptor binding in rat brain.

P-127 - Effects of ethanol treatment on inositol 1,4,5-trisphosphate receptor binding in rat brain.

P-127 - Effects of ethanol treatment on inositol 1,4,5-trisphosphate receptor binding in rat brain.

Modest reduction of benzodiazepine binding in rat brain in vivo induced by antisense oligonucleotide to GABA A receptor γ 2 subunit subtype

The GABA A (γ-aminobutyric acid-A) receptor γ 2 subunit subtype is functionally integral part of the benzodiazepine binding site of the GABA A receptor complex, important for benzodiazepine pharmacology. We have evaluated the possibility of specifically reducing benzodiazepine receptor binding properties in vivo using phosphorothioate antisense oligodeoxynucleotides to inhibit the expression of GABA A receptor γ 2 subinit subtype. Intracerebroventricular infusions of an antisense oligonucleotide reduced benzodiazepine receptor radioligand binding by 9–15% in specific rat brain regions.

Characterization of the pharmacology and regional distribution of (S)‐[3H]‐5‐fluorowillardiine binding in rat brain

1. This study examined the binding of the new radioligand (S)-[3H]-5-fluorowillardiine to rat brain synaptic membranes. Specific binding represented greater than 80% of the total binding and was increased by 10% in the presence of 100mM potassium thiocyanate (KSCN). 2. In the absence of KSCN, (S)-[3H]-5-fluorowillardiine identified two binding sites with KD1=22.5 nM, Bmax1=1.4 pmol mg(-1) protein and KD2=1.5 microM, Bmax2=10.8 pmol mg(-1) protein. In the presence of 100 mM KSCN the affinities of both the binding sites were increased, yielding values of KD1=6.9 nM and KD2=0.4 microM KSCN was without effect on the Bmax values. 3. (S)-[3H]-5-fluorowillardiine binding was displaced by non-NMDA receptor ligands with the rank order of potency: 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX) > domoate > (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionice acid (AMPA) = L-glutamate > 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) > kainate >> (R)-5-fluorowellardiine. In contrast, both N-methyl-D-aspartate (NMDA) and the metabotropic glutamate receptor agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) were inactive. 4. By use of quantitative autoradiography the regional distribution of (S)-[3H]-5-fluorowillardiine binding in rat brain was assessed. The highest levels of binding were in the dentate gyrus and the CA1 region of the hippocampus. Lower levels of binding were detected in the cerebral cortex, olfactory system, lateral septum, caudate putamen and nucleus accumbens. 5. We conclude that the pharmacological profile and regional distribution of (S)-[3H]-5-fluorowillardiine binding is consistent with its specific interaction with AMPA receptors.

Chronic postnatal phencyclidine treatment on [ 3HI](+) pentazocine binding in juvenile rat brain

Chronic phencyclidine (PCP) administrations in postnatal rats produce long-term behavioral changes. Here we report the effects of repeated postnatal PCP treatment on [3H](+)pentazocine binding in juvenile male rats. Saturation analyses of the binding data showed no significant difference in any of the binding characteristics between chronic PCP-treated rats and saline-treated controls suggesting that mechanisms other than alterations in or, binding underlie the behavioral effects of repeated postnatal PCP administration in immature rats.

Regional differences in the effects of chronic ethanol administration on [3H]zolpidem binding in rat brain.

A strong association has been observed between [3H]zolpidem binding and the presence of gamma-aminobutyric acid (GABAA) receptor mRNA for alpha 1-, beta 2-, and gamma 2-subunits in specific brain regions. This correlates with observed sensitivity of individual neurons to zolpidem and ethanol in these same regions. Previous studies using homogenate binding approaches showed small alterations in [3H]zolpidem binding levels after chronic ethanol exposure. This study was undertaken to ascertain if there is regional specificity of the effects of chronic ethanol administration on [3H]zolpidem binding levels. Chronic ethanol administration induced small, but significant alterations in [3H]zolpidem (5 nM) binding in the inferior colliculus, substantia nigra, and the medial septum. [3H]Zolpidem binding was increased in the inferior colliculus and substantia nigra, and decreased in the medial septum. No significant differences in [3H]zolpidem binding were noted in any other brain area analyzed, including the cortex and cerebellum. These findings show that chronic ethanol administration has small effects on [3H]zolpidem binding, although they occur in a site-specific and bidirectional manner. Moreover, there is no correlation between changes in [3H]zolpidem binding and alterations in GABAA receptor subunit expression.