Effects of imidazoline I2 receptor agonists on reserpine-induced hyperalgesia and depressive-like behavior in rats.

Pharmacotherapies for fibromyalgia treatment are lacking. This study examined the antinociceptive and antidepressant-like effects of imidazoline I2 receptor (I2R) agonists in a reserpine-induced model of fibromyalgia in rats. Rats were treated for 3 days with vehicle or reserpine. The von Frey filament test was used to assess the antinociceptive effects of I2 receptor agonists, and the forced swim test was used to assess the antidepressant-like effects of these drugs. 2-BFI (3.2-10 mg/kg, intraperitoneally), phenyzoline (17.8-56 mg/kg, intraperitoneally), and CR4056 (3.2-10 mg/kg, intraperitoneally) all dose-dependently produced significant antinociceptive effects, which were attenuated by the I2R antagonist idazoxan. Only CR4056 significantly reduced the immobility time in the forced swim test in both vehicle-treated and reserpine-treated rats. These data suggest that I2R agonists may be useful to treat fibromyalgia-related pain and comorbid depression.

A new, simple and robust radioligand binding method used to determine kinetic off-rate constants for unlabeled ligands. Application at α2A- and α2C-adrenoceptors.

Kinetic on and off rate constants for many receptor ligands are difficult to determine with regular radioligand binding technique since only few of the ligands are available in labeled form. Here we developed a new and simple radioligand binding method for determining the kinetic off-rate constant for unlabeled ligands, using whole cells expressing α2A- and α2C-adrenoceptors. The new method involves pre-incubation with unlabeled ligand, centrifugation of microtiter plates in order to adhere the cells to the bottom surface, and then upside-down centrifugation of the plates for few seconds to wash away the non-bound fraction of the pre-incubated ligand. The final on-reaction assay for the radioligand is then started by quick addition of a relatively fast-associating radioligand to the cells. The curve obtained is defined by a fairly simple mathematical formula that reflects the simultaneous dissociation of pre-incubated ligand and association of the radioligand. The method proved to produce highly reproducible results in determining the koff constants for various unlabeled ligands. The results show that the α2C-selectivity of MK912 depends mainly on a very slow off-rate at the α2C-adrenoceptor subtype. Regarding the markedly α2C- over α2A-selective compound spiroxatrine, its much faster on-rate at α2C- than α2A-adrenoceptors explains much of its exceptional α2C-selectivity. Several new techniques for determining the kinetic component of ligand-receptor interactions at molecular level are currently developing. As a reference, based on standard radioligand binding techniques, the present study describes a simple and robust experimental and mathematical procedure for determining koff constants of unlabeled drugs.

The predicted N-terminal signal sequence of the human α2C-adrenoceptor does not act as a functional cleavable signal peptide.

The N-terminal region of the human α(2C)-adrenoceptor has a 22 amino acid sequence MASPALAAALAVAAAAGPNASG. This stretch is predicted to be a cleavable signal peptide. Signal peptides facilitate the translocation of membrane proteins from ribosomes into the endoplasmatic reticulum (ER) for further transport to the plasma membrane. However, recently it has been suggested that the hydrophobic stretch ALAAALAAAAA in the N-tail of the rat α(2C)-adrenoceptor, rather than being part of a signal peptide, is an ER retention signal (Angelotti, 2010). Here, we have investigated the functionality of the N-terminal region of the human α(2C)-adrenoceptor further. The predicted signal peptide was found to be non-cleavable, as shown for a modified α(2C)-adrenoceptor construct equipped with a FLAG epitope. The influence of the N-terminal region on receptor translocation to the plasma membrane was investigated by rebuilding the N-tail and then by analyzing the expression level of binding-competent receptors in transfected COS-7 cell membranes. Truncated α(2C)-adrenoceptor constructs showed decreased expression levels as compared to the wild type α(2C)-adrenoceptor. Addition of, or exchange for, the influenza virus hemagglutinin signal peptide to the α(2C)-adrenoceptor had no effect, respectively decreased, the expression level of binding-competent receptor in the membranes. Our analysis supports the conclusions that the predicted signal peptide in the N-terminal tail of the α(2C)-adrenoceptor does not act as a cleavable signal peptide. In addition, the results indicate that the presence of an intact N-tail is augmenting the amount of binding-competent α(2C)-adrenoceptors at the cell surface.

Locus coeruleus stimulation and noradrenergic modulation of hippocampo-prefrontal cortex long-term potentiation.

Stimulation of the subiculum/CA1 of the hippocampal formation evokes monosynaptic field potentials in the prefrontal cortex (PFC). High-frequency stimulation of the hippocampus (HPC) can induce long-term potentiation (LTP) in this hippocampo-prefrontal cortical (hippo-PFC) pathway. Previous studies have shown that dopamine and serotonin modulate hippo-PFC LTP. Here, we investigated whether the locus coeruleus (LC) and noradrenaline (NA) can modulate LTP in the rat hippo-PFC pathway. Stimulation of the LC in combination with stimulation of the HPC increased hippo-PFC LTP. Infusion of lidocaine into the LC reduced hippo-PFC LTP. Administration of the noradrenaline reuptake inhibitor, nisoxetine or the alpha2 adrenoceptor antagonist, idazoxan prior to high-frequency stimulation of the HPC enhanced hippo-LTP. In contrast, administration of clonidine, an alpha2 adrenoceptor agonist, impaired hippo-PFC LTP. Partial noradrenergic (NAergic) lesioning with DSP-4 also impaired hippo-PFC LTP. In conclusion, the LC and NAergic mechanisms modulate hippo-PFC LTP.

Adjunctive alpha2-adrenoceptor blockade enhances the antipsychotic-like effect of risperidone and facilitates cortical dopaminergic and glutamatergic, NMDA receptor-mediated transmission.

Compared to both first- and second-generation antipsychotic drugs (APDs), clozapine shows superior efficacy in treatment-resistant schizophrenia. In contrast to most APDs clozapine possesses high affinity for alpha2-adrenoceptors, and clinical and preclinical studies provide evidence that the alpha2-adrenoceptor antagonist idazoxan enhances the antipsychotic efficacy of typical D2 receptor antagonists as well as olanzapine. Risperidone has lower affinity for alpha2-adrenoceptors than clozapine but higher than most other APDs. Here we examined, in rats, the effects of adding idazoxan to risperidone on antipsychotic effect using the conditioned avoidance response (CAR) test, extrapyramidal side-effect (EPS) liability using the catalepsy test, brain dopamine efflux using in-vivo microdialysis in freely moving animals, cortical N-methyl-D-aspartate (NMDA) receptor-mediated transmission using intracellular electrophysiological recording in vitro, and ex-vivo autoradiography to assess the in-vivo alpha2A- and alpha2C-adrenoceptor occupancies by risperidone. The dose of risperidone needed for antipsychotic effect in the CAR test was approximately 0.4 mg/kg, which produced 11% and 17% in-vivo receptor occupancy at alpha2A- and alpha2C-adrenoceptors, respectively. Addition of idazoxan (1.5 mg/kg) to a low dose of risperidone (0.25 mg/kg) enhanced the suppression of CAR, but did not enhance catalepsy. Both cortical dopamine release and NMDA receptor-mediated responses were enhanced. These data propose that the therapeutic effect of risperidone in schizophrenia can be enhanced and its EPS liability reduced by adjunctive treatment with an alpha2-adrenoceptor antagonist, and generally support the notion that the potent alpha2-adrenoceptor antagonistic action of clozapine may be highly important for its unique efficacy in schizophrenia.

Attenuating effect of melatonin on pyridoxal-stimulated release of adrenomedullary catecholamines in the rat.

AIMS: To investigate the ability of melatonin (MEL) to suppress adrenomedullary catecholamine (CAT) release in the rat, with pyridoxal (PL) being used as an adrenomedullary stimulus and liver and gastrocnemius muscle glycogenolysis acting as indices of CAT release. MAIN METHODS: MEL (1-4 mg/kg, i.p.) and PL (300 mg/kg, i.p.) were administered separately or together to male Sprague-Dawley rats (275-300 g), and blood samples for the assay of plasma glucose and CATs were periodically collected for up to 3 h after PL. Immediately thereafter, the liver and gastrocnemius muscle were surgically removed and used for the assay of glycogen. The role of adrenoceptors in PL-induced glycogenolysis was examined by parallel experiments in which idazoxan (IDX, 1 mg/kg), propranolol (PRO, 2 mg/kg) or metoprolol (MET, 2 mg/kg) were administered alongside MEL. In addition, MEL (4 mg/kg) was co-administered with taurine (TAU, 2.4 mmol/kg), a known adrenomedullary membrane stabilizer. KEY FINDINGS: MEL attenuated the release of adrenomedullary CATs and accompanying liver and gastrocnemius muscle glycogenolysis due to PL in a dose-dependent manner. A co-treatment with MEL and an adrenoceptor blocker had a greater attenuating effect on PL-induced glycogenolysis and hyperglycemia than MEL but without impinging on the CAT levels seen with MEL alone. Evidence of maximal inhibitory action by MEL on PL-induced plasma CAT elevation was suggested by the about equal levels of plasma CATs after treatments with MEL and with MEL plus TAU. SIGNIFICANCE: The present study demonstrates the modulatory effect of MEL of exogenous origin on adrenomedullary CAT secretion when present in supraphysiological concentrations.

Interplay among catecholamine systems: dopamine binds to alpha2-adrenergic receptors in birds and mammals.

Dopaminergic and adrenergic receptors are G-protein-coupled receptors considered to be different based on their pharmacology and signaling pathways. Some receptor subtypes that are members of one family are actually closer in phylogenetic terms to some subtypes belonging to the other family, suggesting that the pharmacological specificity among these receptors from different families is not perfect. Indeed, evidence is accumulating that one amine can cross-talk with receptors belonging to the other system. However, most of these observations were collected in vitro using artificial cell models transfected with cloned receptors, so that the occurrence of this phenomenon in vivo as well as its distribution in the central nervous system is not known. In this study the pharmacological basis of possible in vivo interactions between dopamine and alpha(2)-adrenergic receptors was investigated in quail, zebra finches, and rats. Binding competitions showed that dopamine displaces the binding of the selective alpha(2)-adrenergic ligand, [(3)H]RX821002, in the brain of the three species with an affinity approximately 10-28-fold lower than that of norepinephrine. Dopamine also displaces with an affinity 3-fold lower than norepinephrine the binding of [(3)H]RX821002 to human alpha(h2A)-adrenergic receptors expressed in Sf9 cells. The anatomical distribution of this interaction was assessed in brain slices of quail and rat based on autoradiographic methods. Both norepinephrine and dopamine significantly displace [(3)H]RX821002 binding in all brain nuclei considered. Together, these data provide evidence for an interaction between the dopaminergic and noradrenergic systems in the vertebrate brain, albeit with species variations.

Dopamine binds to alpha(2)-adrenergic receptors in the song control system of zebra finches (Taeniopygia guttata).

A commonly held view is that dopamine exerts its effects via binding to D1- and D2-dopaminergic receptors. However, recent data have emerged supporting the existence of a direct interaction of dopamine with adrenergic but this interaction has been poorly investigated. In this study, the pharmacological basis of possible in vivo interactions between dopamine and alpha(2)-adrenergic receptors was investigated in zebra finches. A binding competition study showed that dopamine displaces the binding of the alpha(2)-adrenergic ligand, [(3)H]RX821002, in the brain. The affinity of dopamine for the adrenergic sites does not differ between the sexes and is 10- to 28-fold lower than that for norepinephrine. To assess the anatomical distribution of this interaction, binding competitions were performed on brain slices incubated in 5nM [(3)H]RX821002 in the absence of any competitor or in the presence of norepinephrine [0.1microM] or dopamine [1microM]. Both norepinephrine and dopamine displaced the binding of the radioligand though to a different extent in most of the regions studied (e.g., area X, the lateral part of the magnocellular nucleus of anterior nidopallium, HVC, arcopallium dorsale, ventral tegmental area and substantia grisea centralis) but not in the robust nucleus of the arcopallium. Together these data provide evidence for a direct interaction between dopamine and adrenergic receptors in songbird brains albeit with regional variation.

Characterization of an antiproliferative effect of imidazoline receptor ligands on PC12 cells.

The present study aimed at investigating the influence of imidazoline receptor ligands on the proliferation of PC12 cells and the involvement of the sphingosine-1-phosphate (S1P) signaling system in this effect. In cultured PC12 cells, S1P (0.3-100 nM) and the I(1) imidazoline receptor ligands moxonidine (0.3 and 1 mM), agmatine (1 mM), idazoxan (10-100 microM) and efaroxan (1-100 microM) concentration-dependently reduced protein contents which were used as estimates for cell number. The antiproliferative effects elicited by the compounds were abolished after knock-down of S1P(1), S1P(2) and S1P(3) receptors by RNA interference indicating an involvement of S1P receptors. In conclusion, the present data add further evidence to the recent finding that effects of imidazoline receptor ligands in PC12 cells are mediated by homo- and heterodimers of members of the S1P receptor family.

Analysis of brain adrenergic receptors in dopamine-beta-hydroxylase knockout mice.

The biosynthesis of norepinephrine occurs through a multi-enzymatic pathway that includes the enzyme dopamine-beta-hydroxylase (DBH). Mice with a homozygous deletion of DBH (Dbh-/-) have a selective and complete absence of norepinephrine. The purpose of this study was to assess the expression of alpha-1, alpha-2 and beta adrenergic receptors (alpha1-AR, alpha2-AR and beta-AR) in the postnatal absence of norepinephrine by comparing noradrenergic receptors in Dbh-/- mice with those in Dbh heterozygotes (Dbh+/-), which have normal levels of norepinephrine throughout life. The densities of alpha1-AR, alpha2-AR and beta-AR were assayed with [3H]prazosin, [3H]RX21002 and [125I]-iodo-pindolol autoradiography, respectively. The alpha2-AR agonist high affinity state was examined with [125I]-para-iodoclonidine autoradiography and alpha2-AR functionality by alpha2-AR agonist-stimulated [35S]GTPgammaS autoradiography. The density of alpha1-AR in Dbh-/- mice was similar to Dbh+/- mice in most brain regions, with an up-regulation in the hippocampus. Modest decreases in alpha2-AR were found in septum, hippocampus and amygdala, but these were not reflected in alpha2-AR functionality. The density of beta-AR was up-regulated to varying degrees in many brain regions of Dbh-/- mice compared to the heterozygotes. These findings indicate that regulation of noradrenergic receptors by endogenous norepinephrine depends on receptor type and neuroanatomical region.

Prenatal cocaine exposure alters alpha2 receptor expression in adolescent rats.

BACKGROUND: Prenatal cocaine exposure produces attentional deficits which to persist through early childhood. Given the role of norepinephrine (NE) in attentional processes, we examined the forebrain NE systems from prenatal cocaine exposed rats. Cocaine was administered during pregnancy via the clinically relevant intravenous route of administration. Specifically, we measured alpha2-adrenergic receptor (alpha2-AR) density in adolescent (35-days-old) rats, using [3H]RX821002 (5 nM). RESULTS: Sex-specific alterations of alpha2-AR were found in the hippocampus and amygdala of the cocaine-exposed animals, as well as an upregulation of alpha2-AR in parietal cortex. CONCLUSION: These data suggest that prenatal cocaine exposure results in a persistent alteration in forebrain NE systems as indicated by alterations in receptor density. These neurochemical changes may underlie behavioral abnormalities observed in offspring attentional processes following prenatal exposure to cocaine.

Autoradiographical distribution of imidazoline binding sites in monoamine oxidase A deficient mice.

This study has used receptor autoradiography to characterize imidazoline binding sites (I-BS) in monoamine oxidase (MAO) A knockout and wild-type mice. A comparison between MAO-A and MAO-B, binding of the endogenous beta-carboline [(3)H]harmane, and I-BS, has been made using sections from brain and kidney. The loss of binding to MAO-A in the knockout animals was confirmed using the selective radioligand [(3)H]Ro41-1049, with labelling reduced to background levels. The binding of [(3)H]Ro19-6327 to MAO-B was unaffected, indicating no change in this isoform in response to the loss of MAO-A. A reduction in binding to the I(2)-BS, as labelled by both [(3)H]idazoxan and [(3)H]2-BFI (2-(2-benzofuranyl)-2-imidazoline), was seen in the MAO-A knockout animals in both brain and kidney sections, whereas binding to the I(1)-BS in kidney sections remained unchanged. The loss of I(2) binding was found to be regionally dependent and was positively correlated with the relative expression of MAO-A in specific regions in the wild-type animals. Using the MAO-A knockout mice it was also possible to demonstrate a non-MAO-A population of binding sites labelled by the putative I-BS endogenous ligand, harmane.

Compensatory changes in the noradrenergic nervous system in the locus ceruleus and hippocampus of postmortem subjects with Alzheimer's disease and dementia with Lewy bodies.

In Alzheimer's disease (AD), there is a significant loss of locus ceruleus (LC) noradrenergic neurons. However, functional and anatomical evidence indicates that the remaining noradrenergic neurons may be compensating for the loss. Because the noradrenergic system plays an important role in learning and memory, it is important to determine whether compensation occurs in noradrenergic neurons in the LC and hippocampus of subjects with AD or a related dementing disorder, dementia with Lewy bodies (DLB). We observed profound neuronal loss in the LC in AD and DLB subjects with three major changes in the noradrenergic system consistent with compensation: (1) an increase in tyrosine hydroxylase (TH) mRNA expression in the remaining neurons; (2) sprouting of dendrites into peri-LC dendritic zone, as determined by alpha2-adrenoreceptors (ARs) and norepinephrine transporter binding sites; and (3) sprouting of axonal projections to the hippocampus as determined by alpha2-ARs. In AD and DLB subjects, the postsynaptic alpha1-ARs were normal to elevated. Expression of alpha1A- and alpha2A-AR mRNA in the hippocampus of AD and DLB subjects were not altered, but expression of alpha1D- and alpha2C-AR mRNA was significantly reduced in the hippocampus of AD and DLB subjects. Therefore, in AD and DLB subjects, there is compensation occurring in the remaining noradrenergic neurons, but there does appear to be a loss of specific AR in the hippocampus. Because changes in these noradrenergic markers in AD versus DLB subjects were similar (except neuronal loss and the increase in TH mRNA were somewhat greater in DLB subjects), the presence of Lewy bodies in addition to plaques and tangles in DLB subjects does not appear to further affect the noradrenergic compensatory changes.

Long-lasting effects of yohimbine on the ejaculatory function in male dogs.

Previous studies have demonstrated that systemic administration of a low dose of the alpha2-adrenoceptor antagonists stimulates the ejaculatory response of male dogs, when this function is analyzed using the amount of ejaculated semen in response to genital stimulation. The present study was designed to further examine the features of the stimulatory effects of the alpha2-adrenoceptor antagonists on ejaculation, especially the duration of action. Treatment with yohimbine (0.1 mg/ kg, i.p.) to male dogs, at 0.5, 1, 3, or 5 h before the testing, produced a significant stimulatory effects on the ejaculatory response elicited by manual penile stimulation; the amount of ejaculated semen was increased and the onset of ejaculation was shortened following each treatment. However, such effects were not observed in the treatment with yohimbine at 8 and 24 h before the testing, indicating that the ejaculatory stimulation induced by yohimbine lasted for a relative long period. By contrast, the stimulatory effects of RX821002 (0.1 mg/kg, i.p.), a selective alpha2-adrenoceptor antagonist, on ejaculation were observed only for 1 h after administration. To determine the contribution of the alpha2-adrenoceptor blockade for the long-lasting effect of yohimbine, we tested whether yohimbine can prevent the ejaculatory inhibition induced by clonidine, an alpha2-adrenoceptor agonist. The ejaculatory inhibition (a decrease in the amount of ejaculated semen and a delay onset of ejaculation) elicited by clonidine (0.05 mg/kg, i.p.; 1 h before testing) was completely blocked by pretreatment with yohimbine at 1 or 5 h before the testing, whereas the pretreatment with the drug at 24 h before the testing did not affect the clonidine-induced ejaculatory inhibition. These results indicate that yohimbine-induced ejaculatory stimulation is continued for a relative long period (at least 5 h after administration), and this long-lasting effects may be related to the alpha2-adrenoceptor blocking property of the drug.

Characterisation of imidazoline I2 binding sites in pig brain.

The imidazoline I2 binding sites in the central nervous system have previously been described in several different species including rat, mouse, rabbit and frog. The present study has investigated the imidazoline I2 binding site, and its relationship to the monoamine oxidase isoforms, in pig whole brain and compared the results obtained with data from other species. Results from saturation binding studies revealed that the imidazoline I2-selective ligand, [3H]2BFI (2-(2-benzofuranyl)-2-imidazoline) labelled a single saturable population of sites with a KD=6.6 nM and Bmax=771.7 fmol/mg protein. The pharmacological characterisation of the sites was similar to that previously reported with a rank order of potency for the imidazoline I2 ligands of 2BFI>BU224>Idazoxan>BU226. Displacement by the imidazoline I1 ligands was low affinity and the monoamine oxidase inhibitors displaced with micromolar affinity. The majority of compounds displaced the binding in a monophasic manner, however, displacement by the putative endogenous ligand, harmane was biphasic. The relative populations of the two monoamine oxidase isoforms revealed a 10 fold greater expression of monoamine oxidase B relative to monoamine oxidase A. These data confirm the presence of imidazoline I2 binding sites in pig brain and show that their pharmacology is characteristic of that seen in other species. The proportion of monoamine oxidase A and B expressed in the pig brain is similar to that seen in the human brain therefore, given the association between imidazoline I2 binding sites and monoamine oxidase, the pig may provide a more useful model for human imidazoline I2 binding sites than other species such as the rat.

Autoradiographic localisation of [3H]2-BFI imidazoline I2 binding sites in mouse brain.

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.

Clonidine increases caspase-3 mRNA level and DNA fragmentation in the developing rat brainstem.

The densities of alpha2-adrenergic receptors, labeled by 3H-clonidine or 3H-RX821002, reach a peak in the rat brainstem during the first week of its life. This enables the agonist of alpha2-adrenergic receptor clonidine, which is used as a component of anaesthetic solution in infants and children, to have specific effects in this structure of the developing brain. Clonidine was injected into the fetal brain (5 microg in 5 microl of saline) or subcutaneously to the pups (1, 10 microg in 50 microl of saline) 3 days before investigation. Clonidine increased the level of apoptotic enzyme caspase-3 mRNA expression, as measured by RT-PCR and enhanced the DNA fragmentation, as determined by gel electrophoresis, in the brainstem of the 21-day-old fetuses and 8-day-old rats. In the cortex of 8-day-old rat, the alpha2-adrenergic receptors are at a much lower level than the brainstem. Clonidine treatment had no evident effects on caspase-3 mRNA level and DNA fragmentation in the cortex of an 8-day-old rat. The data suggest that clonidine facilitates cell death in the developing brainstem. This drug effect provides a potential mechanism whereby clonidine during early life could induce long-lasting alterations in brain neurochemistry, autonomic functions and behavior.

Imidazoline I(2) receptor density increases with the malignancy of human gliomas.

BACKGROUND: Current glioma grading schemes are limited by subjective histological criteria. Imidazoline I(2) receptors are principally expressed on glial cells. OBJECTIVE: To investigate the feasibility of using the measurement of imidazoline I(2) receptor expression to differentiate glial tumours from other types of brain tumours and for grading the different gliomas. METHODS: The specific binding of [(3)H]idazoxan to imidazoline I(2) receptors was measured in homogenates from human gliomas of different grades. RESULTS: The density of imidazoline I(2) receptors was significantly greater in the three types of malignant glial tumours than in postmortem control brain or non-glial tumours. The increase in density correlated with the malignancy grade of the gliomas. No significant differences in affinity values were observed. CONCLUSION: These results suggest that the density of imidazoline I(2) receptors may be a useful radioligand parameter for the differentiation of glial tumours from other types of brain tumours and for grading the different gliomas.

Alpha-2 adrenergic receptor development in rat CNS: an autoradiographic study.

During development norepinephrine plays a role in determining the morphologic organization of the CNS and the density and future responsiveness of adrenergic receptors. alpha-2 Adrenergic receptors, one of three adrenergic receptor types, regulate important adult CNS functions and may have a distinct role during development. We examined alpha-2 receptor distribution and density in the rat brain at postnatal days 1, 5, 10, 15, 21, 28 and in adults using the antagonist [(3)H]RX821002 for autoradiography. Binding kinetics and pharmacology for alpha-2 adrenergic receptors were the same in adults and neonates. There was an overall increase in alpha-2 receptor levels during postnatal development with great variability in pattern and timing of receptor density changes among brain regions. Three major patterns were apparent. First, in many regions receptor density increased during postnatal development, generally reaching adult levels around postnatal day 15. Within this group there was variability in timing between regions and there were several regions with receptor densities higher than adult levels during the postnatal period. Second, there were regions with very high levels of receptors at birth and little or no change in density during the postnatal period. Third, some regions demonstrated decreasing or transient expression of alpha-2 adrenergic receptors in the course of postnatal development, including white matter regions, cerebellum and many brainstem nuclei, suggesting specific roles for alpha-2 receptors during development. This study investigates the development of alpha-2 adrenergic receptors in the rat CNS. It demonstrates there is region-specific regulation of alpha-2 receptor development and identifies brain regions where these receptors may play a specific and critical role in the regulation normal development.

The alpha-2B adrenoceptor in the paraventricular thalamic nucleus is persistently upregulated by chronic psychosocial stress.

Stress has been reported to regulate adrenergic receptors but it is not known whether it has an impact on the alpha-2 adrenoceptor subtype B that is strongly expressed in distinct nuclei of the thalamus. So far little is known about effects of stress on the thalamus. Using the chronic psychosocial stress paradigm in male tree shrews, we analyzed alpha-2B adrenoceptor expression in the paraventricular and the anteroventral nucleus of the thalamus after a six-week period of daily social stress and after a 10-day post-stress recovery period. In situ hybridization with a specific alpha-2B adrenoceptor probe was performed to quantify receptor gene expression in single neurons, and receptor binding was determined by in vitro receptor autoradiography using the radioligand [3H]RX821002. To determine the stress level in the animals, we measured urinary cortisol excretion and body weight. In the neurons of the paraventricular thalamic nucleus, expression of the alpha-2B adrenoceptor transcript was increased after both the six-week chronic-stress period and the post-stress recovery period. Combination of in situ hybridization and immunocytochemistry revealed expression of alpha-2B adrenoceptor transcript in neurons that were stained with an antibody against glutamate but not in neurons immunoreactive for GABA. Alpha-2 adrenoceptor radioligand binding was also increased after both time periods in the paraventricular thalamic nucleus. No significant effects of stress and recovery were observed in the anteroventral thalamic nucleus. Urinary cortisol excretion was increased during the stress period but normalized thereafter. Body weight was reduced during weeks 1 to 3 of stress and then normalized. These data show that long-term chronic psychosocial stress has an impact on alpha-2B adrenoceptor expression in the thalamus and that the effect persists throughout a post-stress recovery period though activity of the hypothalamic pituitary adrenal axis normalizes after stress. Upregulation of the receptor probably alters neurotransmission in the paraventricular thalamic nucleus and may thus influence information transfer to limbic and cortical brain areas.