Density Of Receptor Binding Sites Research Articles

Hyperstabilization of T cell microvilli contacts by chimeric antigen receptors.

T cells typically recognize their ligands using a defined cell biology-the scanning of their membrane microvilli (MV) to palpate their environment-while that same membrane scaffolds T cell receptors (TCRs) that can signal upon ligand binding. Chimeric antigen receptors (CARs) present both a therapeutic promise and a tractable means to study the interplay between receptor affinity, MV dynamics and T cell function. CARs are often built using single-chain variable fragments (scFvs) with far greater affinity than that of natural TCRs. We used high-resolution lattice lightsheet (LLS) and total internal reflection fluorescence (TIRF) imaging to visualize MV scanning in the context of variations in CAR design. This demonstrated that conventional CARs hyper-stabilized microvillar contacts relative to TCRs. Reducing receptor affinity, antigen density, and/or multiplicity of receptor binding sites normalized microvillar dynamics and synapse resolution, and effector functions improved with reduced affinity and/or antigen density, highlighting the importance of understanding the underlying cell biology when designing receptors for optimal antigen engagement.

Autoradiography of 3H-pirenzepine and 3H-AFDX-384 in Mouse Brain Regions: Possible Insights into M1, M2, and M4 Muscarinic Receptors Distribution.

Autoradiography helps to determine the distribution and density of muscarinic receptor (MR) binding sites in the brain. However, it relies on the selectivity of radioligands toward their target. 3H-Pirenzepine is commonly believed to label predominantly M1MR, 3H-AFDX-384 is considered as M2MR selective ligand. Here we performed series of autoradiographies with 3H-AFDX-384 (2 nM), and 3H-pirenzepine (5 nM) in WT, M1KO, M2KO, and M4KO mice to address the ligand selectivity. Labeling with 3H-pirenzepine using M1KO, M2KO, and M4KO brain sections showed the high selectivity toward M1MR. Selectivity of 3H-AFDX-384 toward M2MR varies among brain regions and depends on individual MR subtype proportion. All binding sites in the medulla oblongata and pons, correspond to M2MR. In caudate putamen, nucleus accumbens and olfactory tubercle, 77.7, 74.2, and 74.6% of 3H-AFDX-384 binding sites, respectively, are represented by M4MR and M2MR constitute only a minor portion. In cortex and hippocampus, 3H-AFDX-384 labels almost similar amounts of M2MR and M4MR alongside significant amounts of non-M2/non-M4MR. In cortex, the proportion of 3H-AFDX-384 binding sites attributable to M2MR can be increased by blocking M4MR with MT3 toxin without affecting non-M4MR. PD102807, which is considered as a highly selective M4MR antagonist failed to improve the discrimination of M2MR. Autoradiography with 3H-QNB showed genotype specific loss of binding sites. In conclusion: while 3H-pirenzepine showed the high selectivity toward M1MR, 3H-AFDX-384 binding sites represent different populations of MR subtypes in a brain-region-specific manner. This finding has to be taken into account when interpreting the binding data.

Antidepressant activity of zinc: Further evidence for the involvement of the serotonergic system.

The present study sought to further evaluate the role of the serotonergic system especially the postsynaptic 5-HT1A receptors (5-HT1AR) in the mechanism of antidepressant action of zinc. Messenger RNA (mRNA), protein level, and 5-HT1AR density as well as the rate of monoamine (dopamine, DA, and serotonin) metabolism in the prefrontal cortex (PFC) and hippocampus (Hp) of rats subjected to acute and chronic (21days) zinc (5mg Zn/kg) treatment were measured. Acute or chronic zinc treatment did not induce any changes in 5-HT1AR mRNA levels in the PFC or Hp of rats. However, chronic zinc treatment induced increases in both 5-HT1AR protein levels and density of 5-HT1A receptor binding sites in the Hp of rats. Chronic zinc treatment also increased tissue levels of serotonin metabolite and turnover in the rat Hp. On the other hand, DA, DOPAC, HVA tissue levels increased while DOPAC/DA and 3MT/DA decreased in the PFC of rats after chronic zinc treatment. Acute treatment induced increases only in tissue levels of DOPAC, and DOPAC/DA. Our results confirm that the antidepressant effects of zinc are mediated in concert with the modulation of the serotonergic system including postsynaptic 5-HT1ARs and allude to a possible involvement of dopaminergic neurotransmission in this action.

Expression of Adrenoceptor Subtypes in Preterm Piglet Heart Is Different to Term Heart

Preterm delivery increases the risk of inadequate systemic blood flow and hypotension, and many preterm infants fail to respond to conventional inotrope treatments. If the profile of cardiac adrenoceptor subtypes in the preterm neonate is different to that at term this may contribute to these clinical problems. This study measured mRNA expression of β1, β2, α1A, α2A and α2B-adrenoceptor subtypes by real time PCR in term (113d), preterm (91d) and preterm piglets (91d) exposed to maternal glucocorticoid treatment. Abundance of β-adrenoceptor binding sites in the left ventricle was measured using saturation binding assays. Relative abundance of β1-adrenoceptor mRNA in untreated preterm hearts was ∼50% of term abundance in both left and right ventricles (P<0.001). Trends in receptor binding site density measurements supported this observation (P = 0.07). Glucocorticoid exposure increased β1-adrenoceptor mRNA levels in the right ventricle of preterm hearts (P = 0.008) but did not alter expression in the left ventricle (P>0.1). Relative abundance of α1A-adrenoceptor mRNA was the same in preterm and term piglet hearts (P = >0.1) but was reduced by maternal glucocorticoid treatment (P<0.01); α2A-adrenoceptor mRNA abundance was higher in untreated and glucocorticoid exposed preterm piglet hearts than in term piglets (P<0.001). There was no difference between male and female piglets in mRNA abundance of any of the genes studied. In conclusion, there is reduced mRNA abundance of β1-adrenoceptors in the preterm pig heart. If this lower expression of β-adrenoceptors occurs in human preterm infants, it could explain their poor cardiovascular function and their frequent failure to respond to commonly used inotropes.

Upregulation of CB1 receptor binding in the ventromedial prefrontal cortex promotes proactive stress-coping strategies following chronic stress exposure

Accumulating evidence has revealed that dysregulation of the endocannabinoid system could contribute to the development of major depression. Studies carried out post-mortem in depressed suicide victims have revealed increased CB1 receptor binding site density in the prefrontal cortex (PFC). Accordingly, exposure of rodents to chronic unpredictable stress (CUS) results in phenotypic changes that mirror those of human depression, including increased CB1 receptor binding site density in the PFC. Our goal in these studies was to examine the effects of CUS on the density of CB1 receptor binding sites in the rodent medial PFC and to explore the role of this alteration in the behavioral changes invoked by CUS. Rodents exposed to CUS exhibited increased CB1 receptor maximal binding site density (Bmax) within the ventromedial PFC, but not the dorsomedial PFC. To determine whether this change in the ventromedial PFC is an adaptive response, or alternatively, a consequence of chronic stress that contributes to the adoption of passive coping, we examined whether local CB1 receptor blockade within the ventromedial PFC following CUS would significantly alter behaviors in the forced swim test (FST). CUS exposure significantly increased passive coping in the FST, and this was further augmented by discrete ventromedial PFC microinfusions of the CB1 receptor antagonist AM251 prior to swim stress. Moreover, local CB1 receptor blockade reduced active coping responses in CUS-exposed rats. These findings suggest that the increase in CB1 receptor Bmax observed in the ventromedial PFC of rodents exposed to CUS maintains proactive coping strategies following chronic stress exposure.

Genetic variation in 5-hydroxytryptamine transporter expression causes adaptive changes in 5-HT4 receptor levels

Genetic variation in 5-HT transporter (5-HTT) expression is a key risk factor for psychiatric disorder and has been linked to changes in the expression of certain 5-HT receptor subtypes. This study investigated the effect of variation in 5-HTT expression on 5-HT₄ receptor levels in both 5-HTT knockout (KO) and overexpressing (OE) mice using autoradiography with the selective 5-HT₄ receptor radioligand, [³H]SB207145. Compared to wild-type (5-HTT⁺/⁺) controls, homozygous 5-HTT KO mice (5-HTT⁻/⁻) had reduced 5-HT₄ receptor binding site density in all brain regions examined (35-65% of 5-HTT⁺/⁺). In contrast, the density of 5-HT₄ receptor binding sites was not significantly different between heterozygous 5-HTT KO mice (5-HTT⁻/⁺) and 5-HTT⁺/⁺ mice. The 5-HT synthesis inhibitor p-chlorophenylalanine (250 mg/kg twice daily for 3 d) abolished the difference in 5-HT₄ binding between 5-HTT⁻/⁻ and 5-HTT⁺/⁺ mice in all brain regions. Compared to wild-type (WT) littermate controls, 5-HTT OE mice had increased 5-HT₄ binding density across all brain regions, except amygdala (118-164% of WT) and this difference between genotypes was reduced by the 5-HTT inhibitor, fluoxetine (20 mg/kg twice daily, 3 d). Together, these findings suggest that variation in 5-HTT expression causes adaptive changes in 5-HT₄ receptor levels which are directly linked to alterations in 5-HT availability.

Impact of RNA editing on functions of the serotonin 2C receptor in vivo.

Transcripts encoding 5-HT2C receptors are modified posttranscriptionally by RNA editing, generating up to 24 protein isoforms. In recombinant cells, the fully edited isoform, 5-HT2C-VGV, exhibits blunted G-protein coupling and reduced constitutive activity. The present studies examine the signal transduction properties of 5-HT2C-VGV receptors in brain to determine the in vivo consequences of altered editing. Using mice solely expressing the 5-HT2C-VGV receptor (VGV/Y), we demonstrate reduced G-protein coupling efficiency and high-affinity agonist binding of brain 5-HT2C-VGV receptors. However, enhanced behavioral sensitivity to a 5-HT2C receptor agonist was also seen in mice expressing 5-HT2C-VGV receptors, an unexpected finding given the blunted G-protein coupling. In addition, mice expressing 5-HT2C-VGV receptors had greater sensitivity to a 5-HT2C inverse agonist/antagonist enhancement of dopamine turnover relative to wild-type mice. These behavioral and biochemical results are most likely explained by increases in 5-HT2C receptor binding sites in the brains of mice solely expressing 5-HT2C-VGV receptors. We conclude that 5-HT2C-VGV receptor signaling in brain is blunted, but this deficiency is masked by a marked increase in 5-HT2C receptor binding site density in mice solely expressing the VGV isoform. These findings suggest that RNA editing may regulate the density of 5-HT2C receptor binding sites in brain. We further caution that the pattern of 5-HT2C receptor RNA isoforms may not reflect the pattern of protein isoforms, and hence the inferred overall function of the receptor.

Chronic stress differentially regulates cannabinoid CB1 receptor binding in distinct hippocampal subfields

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.

Regional alterations in the endocannabinoid system in an animal model of depression: effects of concurrent antidepressant treatment

It has been suggested that disturbances in endocannabinoid signaling contribute to the development of depressive illness; however, at present there is insufficient evidence to allow for a full understanding of this role. To further this understanding, we performed an analysis of the endocannabinoid system in an animal model of depression. Male rats exposed to chronic, unpredictable stress (CUS) for 21 days exhibited a reduction in sexual motivation, consistent with the hypothesis that CUS in rats induces depression-like symptoms. We determined the effects of CUS, with or without concurrent treatment with the antidepressant imipramine (10 mg/kg), on CP55940 binding to the cannabinoid CB(1) receptor; whole tissue endocannabinoid content; and fatty acid amide hydrolase (FAAH) activity in the prefrontal cortex, hippocampus, hypothalamus, amygdala, midbrain and ventral striatum. Exposure to CUS resulted in a significant increase in CB(1) receptor binding site density in the prefrontal cortex and a decrease in CB(1) receptor binding site density in the hippocampus, hypothalamus and ventral striatum. Except in the hippocampus, these CUS-induced alterations in CB(1) receptor binding site density were attenuated by concurrent antidepressant treatment. CUS alone produced a significant reduction in N-arachidonylethanolamine (anandamide) content in every brain region examined, which was not reversed by antidepressant treatment. These data suggest that the endocannabinoid system in cortical and subcortical structures is differentially altered in an animal model of depression and that the effects of CUS on CB(1) receptor binding site density are attenuated by antidepressant treatment while those on endocannabinoid content are not.

Serotonin-2 (5-HT2) Receptor-Mediated Signal Transduction in Human Ciliary Muscle Cells: Role in Ocular Hypotension

The aim of this study was to characterize the serotonin (5-hydroxytryptamine; 5-HT) receptors that mediate phosphoinositide (PI) hydrolysis and intracellular Ca2+ ([Ca2+]i) mobilization in isolated cells of human ciliary muscle (h-CM) from multiple donors using a variety of agonists and antagonists. An additional aim was to visualize the mRNAs and receptor binding sites for 5-HT2 receptors in human ciliary body (h-CB), CM, and other tissues by reverse transcriptase polymerase chain reaction and quantitative autoradiography techniques, respectively, and to correlate with ocular hypotensive activity of such compounds. CBs isolated from several donor eyes revealed the presence of 5-HT(2A2C) receptor mRNAs. [3H]-5-HT and [3H]-ketanserin autoradiography on sections of human eyes revealed a high density of 5-HT2 receptor binding sites in the iris, ciliary epithelium, and longitudinal CM. In isolated h-CM cells, the agonists alpha-methyl-5-HT (EC50=63+/-17 nM), 5-HT (EC50=85+/-16 nM), (R)-DOI (EC50=165+/-47 nM), and 5-methoxy alpha-methyl tryptamine (EC50=1200+/-270 nM) differentially stimulated PI turnover. These agonists also mobilized [Ca2+]i in h-CM cells with the following potencies (EC50s): 5-methoxy-tryptamine=42+/-11 nM; alpha-methyl-5-HT=36+/-11 nM; (R)-DOI=120 nM; 5-HT=130+/-36 nM; MK-212=470 nM; mCPP>1 microM; BW723C86=1766 nM. The agonist-induced [Ca2+]i mobilization in h-CM cells was potently blocked by the 5-HT2A-selective antagonist M-100907 (IC50=1.2+/-0.4 nM) but less potently by the antagonists for 5-HT2B (RS-127445, IC50>10 microM) and 5-HT2C (RS-102221, IC50=5.8+/-2.3 microM) receptors. In conclusion, h-CB, h-CM, and CM cells express mRNAs and proteins for 5-HT2 receptor subtypes, of which the predominant functionally active subtype is the 5-HT2A receptor, as defined by agonist and antagonist activities. These receptors may be responsible for mediating the intraocular pressure reduction observed in recent literature with a number of 5-HT2 agonists, such as (R)-DOI, alpha-methyl-5HT, and AL-34662.

Distribution of mRNA and binding sites of adrenoceptors and muscarinic receptors in the rat heart

Since there exist some obscurities in the expression of mRNAs and their receptors in the heart, we have investigated the gene expression (mRNA levels) of adrenoceptors (α 1A-, α 1B-, β 1-, β 2-, β 3-) and muscarinic receptors (M 2) and the density of receptor binding sites (α 1A-, α 1B-, β 1-, β 2-adrenoceptors, muscarinic receptors). Moreover, the heart regions consist of tissue rich in ganglion cells (that are of importance in heart neural circuits) and those virtually free of them (myocytes). Therefore, we have examined the differences in the distribution of mRNAs/receptor binding sites in the atrial samples of the heart rich in ganglion cells vs. those are virtually free of them. Binding sites and mRNAs of muscarinic receptors and α 1B-adrenoceptors differ in their distribution in different heart regions. The mRNAs for β 1- and β 2-adrenoceptors were almost equally distributed herein, while the amount of β-adrenoceptors significantly differs in the heart regions. The α 1A- and β 3-adrenoceptors mRNAs were also found in all investigated heart regions, but at significantly lower level and have not shown region differences. This is a new finding, especially to β 3-adrenoceptors, as they were not regularly found in each heart regions. α 1B-adrenoceptors have similar distribution of their mRNAs and binding sites in some heart parts. Thus, we can conclude that there are noticeable differences in the presence of receptors in heart regions that contain ganglion cells in comparison to those are virtually free of them.

Acute zolpidem administration produces pharmacodynamic and receptor occupancy changes at similar doses

Zolpidem is chemically unrelated to classical benzodiazepines but has demonstrated relatively high affinity binding to the α 1 GABA A receptor. To assess pharmacodynamic and neurochemical effects of zolpidem, open-field behavior, pentylenetetrazole-induced seizure threshold and benzodiazepine receptor binding in vitro were evaluated in the same animal following a single dose of zolpidem. Zolpidem (2, 5 and 10 mg/kg), lorazepam (2 mg/kg) or vehicle was administered intraperitoneally in male CD-1 mice. Behavioral activity, assessed by three open-field parameters, was decreased following the two highest doses of zolpidem (5 and 10 mg/kg), and reached significance at the 10 mg/kg dose. Locomotor activity was also decreased significantly by lorazepam as expected. Pentylenetetrazole-induced seizure threshold was increased with the administration of 2 and 10 mg/kg zolpidem as well as with lorazepam. Apparent affinity ( K D) of [ 3H]flunitrazepam, a non-selective ligand, for the benzodiazepine receptor in cortical membrane preparations was not significantly changed, while receptor number ( B max) was decreased at all doses of zolpidem, reaching significance at the 10 mg/kg dose. These results confirm that the behavioral effects of zolpidem are similar to those of classical benzodiazepines. In addition, zolpidem had no significant effect on the affinity of the benzodiazepine receptor for [ 3H]flunitrazepam, but did decrease the density of receptor binding sites.

Sexually dimorphic effects of the Lhx7 null mutation on forebrain cholinergic function

It has been reported recently that mice lacking both alleles of the LIM-homeobox gene Lhx7, display dramatically reduced number of forebrain cholinergic neurons. In the present study, we investigated whether the Lhx7 mutation affects male and female mice differently, given the fact that gender differences are consistently observed in forebrain cholinergic function. Our results show that in adult male as well as female Lhx7 homozygous mutants there is a dramatic loss of choline acetyltransferase immunoreactive forebrain neurons, both projection and interneurons. The reduction of forebrain choline acetyltransferase immunoreactive neurons in Lhx7 homozygous mutants is accompanied by a decrease of acetylcholinesterase histochemical staining in all forebrain cholinergic neuron target areas of both male and female homozygous mutants. Furthermore, there was an increase of M1-, but not M2-, muscarinic acetylcholine receptor binding site density in the somatosensory cortex and basal ganglia of only the female homozygous mutant mice. Such an increase can be regarded as a mechanism acting to compensate for the dramatically reduced cholinergic input, raising the possibility that the forebrain cholinergic system in female mice may be more plastic and responsive to situations of limited neurotransmitter availability. Finally, our study provides additional data for the sexual dimorphism of the forebrain cholinergic system, as female mice appear to have a lower density of M1-muscarinic acetylcholine receptors in the striatal areas of the basal ganglia and a higher density of M2-muscarinic acetylcholine receptors, in a number of cortical areas, as well as the striatal areas of the basal ganglia.

Addition of a signal peptide sequence to the α1D‐adrenoceptor gene increases the density of receptors, as determined by [3H]‐prazosin binding in the membranes

1. Both in mammalian tissues and in transfected cells, only low levels of alpha1D-adrenoceptors are detected in radioligand binding studies. It has been implicated that the comparatively long N-terminal tail of the alpha1D-adrenoceptor is responsible for the inefficient surface expression of the receptor. 2. In the present study, we created gene constructs for six N-terminally truncated variants of the human alpha1D-adrenoceptor. These constructs were used to transfect Neuro2A cells. We show that the density of alpha1D-adrenoceptors, observed by [3H]-prazosin binding, gradually increased with longer truncations of the N-terminus. This seems to indicate that the long N-terminal tail nonspecifically interferes with receptor translocation to the plasma membrane. 3. The addition of a 16 amino acids long signal peptide to the N-terminus of the wild-type alpha1D-adrenoceptor increased the density of receptor binding sites 10-fold in Neuro2A and COS-7 cells. This indicates that, after the addition of a signal peptide, the long N-terminal tail of the alpha1D-adrenoceptor does not interfere with proper translocation of the receptor to the plasma membrane. This, in turn, indicates that the N-terminal tail of the wild-type alpha1D-adrenoceptor, merely by its long length, hinders the first transmembrane helix of the receptor from being a signal anchor. 4. Neither the wild-type alpha1D-adrenoceptor (for which the expression level of [3H]-prazosin binding sites is low) nor the truncated alpha1D-adrenoceptor variant (for which the expression level of [3H]-prazosin binding sites is high) showed any constitutive activity in stimulating inositol phosphate accumulation. This indicates that the low expression level of [3H]-prazosin binding sites, after transfection with the wild-type alpha1D-adrenoceptor, is not caused by constitutive activity of the receptor and subsequent receptor downregulation.

Neurobiologie et pharmacothérapie de la phobie sociale

Resume Bien que Marks ait propose des 1970 une classification des phobies comprenant la phobie sociale, l’apparition de l’entite diagnostique phobie sociale est encore plus recente. Elle a lieu en 1980 avec le DSM III, 3e edition de la nomenclature de l’American Psychiatric Association, qui etablit des criteres operationnels pour son diagnostic. De ce fait, les recherches visant a identifier les facteurs genetiques, familiaux et temperamentaux, les correlats neurochimiques et neuroendocriniens ainsi que les structures cerebrales s’inscrivant dans la pathogenie de ce trouble en sont a leur debut. Neanmoins, elles ont permis d’apprecier l’intervention des influences genetiques, du milieu familial, et de traits temperamentaux, comme l’inhibition comportementale face a l’inconnu, dans le risque d’apparition d’une phobie sociale. Elles ont egalement contribue a mettre en evidence des perturbations du fonctionnement des systemes de neurotransmission monoaminergique dans la phobie sociale. Parallelement, les progres realises dans l’analyse des relations structure-fonction, et notamment l’apport recent de la neuro-imagerie, ont souligne l’importance des voies cortico-limbiques dans la physiopathologie de la phobie sociale, avec la participation de regions et structures cerebrales comme le cortex prefrontal, l’hippocampe, l’amygdale et le striatum. Ceci n’a rien de surprenant si l’on considere leur role essentiel dans la signification attribuee aux informations emanant de l’environnement, les processus emotionnels, mnesiques et de conditionnement contextuel, ainsi que dans la programmation des comportements dont on peut supposer l’alteration a la lumiere des symptomes caracteristiques de la maladie. La demarche pharmacotherapique aujourd’hui proposee repose sur l’efficacite demontree d’agents medicamenteux, au premier rang desquels figurent les inhibiteurs selectifs de la recapture de la serotonine dont l’emploi peut aujourd’hui prevaloir sur celui d’autres produits egalement actifs, comme les inhibiteurs de la monoamine oxydase non selectifs et irreversibles ou les benzodiazepines, de par la simplicite de leur utilisation et leur tres bonne tolerance.

Region specific changes in forebrain 5-hydroxytryptamine 1a and 5-hydroxytryptamine 2a receptors in isolation-reared rats: an in vitro autoradiography study

The neurochemical correlates of the behavioural consequences of isolation rearing of rats are complex and involve many neurotransmitters, including the serotonergic system. Impaired functioning of the ascending serotonergic system has been implicated in many neuropsychiatric syndromes, including attention deficit hyperactivity disorder and schizophrenia. In the present investigation serotonergic function was assessed using in vitro receptor autoradiography. The 5-hydroxytryptamine 2A (5-HT 2A) receptor antagonist [ 3H]ketanserin and the 5-HT 1A receptor antagonist, [ 3H]WAY100, 635 were used to compare 5-HT receptor subtype densities in the forebrains of socially and isolation-reared rats. Regions of highest receptor density were observed in the frontal cortex for 5-HT 2A receptors and in the frontal cortex, dorsal hippocampus and lateral septum for 5-HT 1A receptors. In isolation-reared rats, 5-HT 2A receptor binding site densities were significantly increased by between 36 and 67% in the prelimbic, motor and cingulate cortices compared with socially reared controls. By contrast, 5-HT 1A receptor binding site densities were significantly reduced by 22% in the prelimbic cortex, and significantly increased by between 10 and 50% in the motor cortex, somatosensory cortex, dentate gyrus and CA fields of the hippocampus. These data demonstrate that isolation-rearing produces significant effects on forebrain 5-HT 1A and 5-HT 2A receptor densities in the adult rat. It is hypothesised that altered serotonergic function, particularly in the hippocampus and prefrontal cortex, may underlie some of the behavioural abnormalities associated with isolation-rearing.

Subchronic phencyclidine administration alters central vasopressin receptor binding and social interaction in the rat

Arginine vasopressin (AVP) is a peptide involved in social behaviors in rodents. To investigate the mechanism underlying the deficits in social behavior induced by blockade of N-methyl- d-aspartate (NMDA) receptors, this study examined the effect of noncompetitive NMDA antagonists on AVP receptor binding and social interaction in the rat. Subchronic phencyclidine (PCP) administration (2 mg/kg/day, 14 days, i.p.) significantly reduced the density of V1a receptor binding sites, labeled by an [ 125I]-Linear AVP antagonist, in several brain regions. Subchronic treatment with PCP or MK-801 (0.13 mg/kg/day, 14 days, i.p.) impaired social interactions in rats, as has been previously reported. These results suggest that NMDA antagonists have modulatory effects on the central vasopressinergic system and social interaction.

Milking characteristics and their relation to adrenergic receptor mRNA expression and ligand binding in the mammary gland of dairy cows

Stimulation of α- and β-adrenergic receptors in the bovine mammary gland affects milking characteristics such as milk yield and peak flow rate. The aim of this study was to detect possible correlations between milkability, adrenergic receptor binding capacity and receptor expression at the mRNA level. In addition, dose–response relationships of α- and β-adrenergic receptor stimulation were evaluated after application of an α- and β-adrenergic receptor agonist, respectively in different dosages. Density and distribution of adrenergic receptor binding sites in the region around the large mammary ducts were investigated as well as adrenergic receptor mRNA expression. Milk flow of one-quarter was recorded in 10 cows without or with additional α- and β-adrenergic receptor stimulation in three dosages each. After slaughter, mammary tissue was taken from the region around the large mammary ducts in the previously investigated quarters. Protein and RNA were extracted for measuring α 1-, α 2-, and β 2-adrenergic receptor binding sites and mRNA expression levels by real-time reverse-transcription polymerase chain reaction (RT-PCR). Peak flow rate without additional adrenergic receptor stimulation was negatively correlated with α 2-adrenergic receptor binding (maximal binding capacity, B max) and positively correlated with α 2-adrenergic receptor expression at the mRNA level (crossing point (CP) of the real-time PCR). During α-adrenergic receptor stimulation, there was a positive correlation between milkability and α 2-adrenergic receptor mRNA expression, whereas during β-adrenergic receptor stimulation no correlations were detected. Dose–response relationships were existing during α-adrenergic receptor stimulations, but not during β-adrenergic receptor stimulations at four dosages each including control milking. Significant changes in milk yield and peak flow rate mainly occurred after application of an α-adrenergic receptor agonist. In conclusion, high mRNA expression levels or binding capacities of adrenergic receptors do not necessarily lead to according reactions in vivo, concerning milk yield and peak flow rate. To influence milking characteristics, individual reactions of the cow on adrenergic stimulation have to be considered.

Chronic nicotine administration does not increase nicotinic receptors labeled by [125I]epibatidine in adrenal gland, superior cervical ganglia, pineal or retina.

Neuronal nicotinic acetylcholine receptors (nAChRs) were measured in CNS and peripheral tissues following continuous exposure to saline or nicotine hydrogen tartrate (3.3 or 10 mg/kg/day) for 14 days via osmotic pumps. Initially, binding of [3H](-)nicotine, [3H]cytisine and [3H]epibatidine to nAChRs was compared to determine the suitability of each for these kinds of studies. The predominant nAChR labeled by agonists in the cerebral cortex is an alpha 4 beta 2 subtype, whereas the predominant nicotinic receptors in the adrenal gland, superior cervical ganglia and pineal gland contain an alpha 3 subunit, and they do not bind either [3H](-)nicotine or [3H]cytisine with high affinity. In retina some nAChRs bind all three ligands with high affinity, and others appear to bind only [3H]epibatidine. Thus, only [3H]epibatidine had high enough affinity to be useful for measuring the nAChRs in all of the tissues. The receptors from nicotine-treated rats were then measured using [125I]epibatidine, which has binding characteristics very similar to [3H]epibatidine. Treatment with the two doses of nicotine hydrogen tartrate increased binding sites in the cerebral cortex by 40% and 70%, respectively. In contrast, no significant changes in the density of receptor binding sites were found in the adrenal gland, superior cervical ganglia, pineal gland or retina. These data indicate that chronic administration of nicotine even at high doses does not increase all nicotinic receptor subtypes, and that receptors containing alpha 3 subunits may be particularly resistant to this nicotine-induced change.

N-terminal truncation of human α 1D-adrenoceptors increases expression of binding sites but not protein

The role of the N-terminus of human α 1D-adrenoceptors was examined by deleting the first 79 amino acids (Δ 1–79) and epitope-tagging to facilitate immunoprecipitation and detection. Following transfection into HEK293 cells, 6- to 13-fold increases in the density of specific [ 125I]BE 2254 binding sites were observed for both tagged and untagged Δ 1–79α 1D- compared to full-length α 1D-adrenoceptors, while agonist and antagonist affinities remained unchanged. In contrast, immunoprecipitation of tagged receptors showed that full-length α 1D-adrenoceptor protein was at least twice as abundant as Δ 1–79α 1D-adrenoceptor protein. Photoaffinity labelling with [ 125I]arylazidoprazosin showed much more intense labelling of tagged Δ 1–79α 1D- than of full-length α 1D-adrenoceptors. Substantial N-linked glycosylation of tagged Δ 1–79α 1D-adrenoceptors was observed, although full-length α 1D-adrenoceptors contain two consensus glycosylation sites but are not glycosylated. These results suggest that N-terminal truncation of α 1D-adrenoceptors enhances processing of a binding competent form in HEK293 cells; and show a clear dissociation between abundance of receptor protein and density of receptor binding sites.