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Biomedical subjects

Gregory A Ordway

Publications and source records attributed to Gregory A Ordway.

18 recordsLinked to original sources
PubMed2006

Effects of cocaine on monoamine uptake as measured ex vivo.

The increase in extracellular dopamine (DA) following cocaine administration plays a major role in cocaine abuse. In vitro, cocaine binds to DA transporters (DAT) and blocks DA uptake. Moreover, cocaine can increase extracellular DA concentration as measured by in vivo neurochemical methods. The present study examined the effects of cocaine and other drugs on DA, NE and 5-HT uptake using an ex vivo assay. Rats were injected i.v. with saline or drug and sacrificed at various time points after injections. Brains were dissected for regional monoamine uptake studies ex vivo. In most brain regions, cocaine given in vivo blocked monoamine uptake as expected. [3H]DA uptake in nucleus accumbens was inhibited with an ED50=22.3 micromol/kg. Cocaine fully inhibited [3H]NE uptake (ED50=4.58 micromol/kg) in the occipital cortex and partially inhibited [3H]5-HT uptake (33% at 30 micromol/kg) in the midbrain. However, under the same conditions [3H]DA uptake in the striatum was not inhibited after injections of cocaine up to 56 micromol/kg. Although the mechanism for this discrepancy is unclear, DA binding and uptake sites may be distinct and/or there may be regional differences in DA transporters.

Analysis of Variance↗
PubMed2006

Normal [3H]flunitrazepam binding to GABAA receptors in the locus coeruleus in major depression and suicide.

Major depression and suicide are associated with altered concentrations of specific noradrenergic proteins in the human locus coeruleus (LC). Based on experimental studies that can reproduce these LC abnormalities in laboratory animals, we hypothesized that noradrenergic pathobiology in depression is a result of overactivity of the LC. LC activity is under the control of both excitatory and inhibitory inputs. A major inhibitory input to the LC is GABAergic, arising from the nucleus prepositus hypoglossi. Numerous studies demonstrating low levels of GABA in the CSF and plasma of subjects with major depressive disorder (MDD) raise the possibility that LC overactivity in depression may be secondary to reduced GABAergic input to the LC. Here, GABAergic input to the LC in depression was evaluated by studying the binding of [(3)H]flunitrazepam to GABA(A) receptors at three anatomically defined levels of the human postmortem LC. LC tissues were collected from subjects with MDD, subjects with depressive disorders including MDD that died as a result of suicide, and psychiatrically normal control subjects. A modest rostral-caudal gradient of GABA(A) receptor binding density was observed among all subjects. No significant differences in the amount of binding to GABA(A) receptors were observed between control subjects (n=21) and MDD subjects (n=9) or depressed suicide victims (n=17). These results demonstrate that GABA(A) receptor binding in the LC measured with [(3)H]flunitrazepam is not altered in subjects with depressive illnesses.

Analysis of Variance↗
PubMed2006

KCl stimulation increases norepinephrine transporter function in PC12 cells.

The norepinephrine transporter (NET) plays a pivotal role in terminating noradrenergic signaling and conserving norepinephrine (NE) through the process of re-uptake. Recent evidence suggests a close association between NE release and regulation of NET function. The present study evaluated the relationship between release and uptake, and the cellular mechanisms that govern these processes. KCl stimulation of PC12 cells robustly increased [3H]NE uptake via the NET and simultaneously increased [3H]NE release. KCl-stimulated increases in uptake and release were dependent on Ca2+. Treatment of cells with phorbol-12-myristate-13-acetate (PMA) or okadaic acid decreased [3H]NE uptake but did not block KCl-stimulated increases in [3H]NE uptake. In contrast, PMA increased [3H]NE release and augmented KCl-stimulated release, while okadaic acid had no effects on release. Inhibition of Ca2+-activated signaling cascades with KN93 (a Ca2+ calmodulin-dependent kinase inhibitor), or ML7 and ML9 (myosin light chain kinase inhibitors), reduced [3H]NE uptake and blocked KCl-stimulated increases in uptake. In contrast, KN93, ML7 and ML9 had no effect on KCl-stimulated [3H]NE release. KCl-stimulated increases in [3H]NE uptake were independent of transporter trafficking to the plasma membrane. While increases in both NE release and uptake mediated by KCl stimulation require Ca2+, different intracellular mechanisms mediate these two events.

Analysis of Variance↗
PubMed2006

The norepinephrine transporter and its regulation.

For many years, the norepinephrine transporter (NET) was considered a 'static' protein that contributed to the termination of the action of norepinephrine in the synapse of noradrenergic neurons. The concept that the NET is dynamically regulated, adjusting noradrenergic transmission by changing its function and/or expression, was considered initially in the mid 1980s. Since that time, a plethora of studies demonstrate that the NET is regulated by several intracellular and extracellular signaling molecules, and that phosphorylation of the NET is a major pathway regulating its cell surface expression and thereby its function. The NET is a target of action of a number of drugs that are used long-term therapeutically or abused chronically. This has driven numerous investigations of how the NET and its function are regulated by long-term exposure to drugs. While repeated exposure to many drugs has been shown to affect NET function and expression, the intracellular mechanisms for these effects remains elusive.

Animals↗
PubMed2006

Expression of arginine decarboxylase in brain regions and neuronal cells.

After our initial report of a mammalian gene for arginine decarboxylase, an enzyme for the synthesis of agmatine from arginine, we have determined the regional expression of ADC in rat. We have analyzed the expression of ADC in rat brain regions by activity, protein and mRNA levels, and the regulation of expression in neuronal cells by RNA interference. In rat brain, ADC was widely expressed in major brain regions, with a substantial amount in hypothalamus, followed by cortex, and with least amounts in locus coeruleus and medulla. ADC mRNA was detected in primary astrocytes and C6 glioma cells. While no ADC message was detected in fresh neurons (3 days old), significant message appeared in differentiated neurons (3 weeks old). PC12 cells, treated with nerve growth factor, had higher ADC mRNA compared with naive cells. The siRNA mixture directed towards the N-terminal regions of ADC cDNA down-regulated the levels of mRNA and protein in cultured neurons/C6 glioma cells and these cells produced lower agmatine. Thus, this study demonstrates that ADC message is expressed in rat brain regions, that it is regulated in neuronal cells and that the down-regulation of ADC activity by specific siRNA leads to lower agmatine production.

Amino Acid Sequence↗
PubMed2005

Effects of depression, cigarette smoking, and age on monoamine oxidase B in amygdaloid nuclei.

Altered concentrations of dopamine transporter and D2/D3 receptors have been observed in the amygdaloid complex of subjects with major depression. These findings are suggestive of neurochemical abnormalities in the limbic dopamine system in depression. Monoamine oxidase-B (MAO-B) is a key enzyme in the catabolism of biogenic amines, including dopamine, and alterations in this enzyme may underlie dopaminergic abnormalities associated with depression. The specific binding of [(3)H]lazabemide to MAO-B was measured in the right amygdaloid complex of 15 major depressive subjects and 16 psychiatrically normal controls. Subjects of the two study groups were matched as close as possible for age, sex, and postmortem interval. Examination of the regional distribution of MAO-B revealed lower [(3)H]lazabemide binding to MAO-B in the lateral and basal nuclei of the amygdala and higher binding in the medial nucleus. A modest elevation in binding to MAO-B observed in all amygdaloid nuclei in major depressive subjects as compared to control subjects failed to reach statistical significance. A significant decrease in binding to MAO-B was observed when cigarette smokers were compared to nonsmoking subjects. The amount of MAO-B binding positively correlated with the age of subjects in all nuclei investigated. A decreased amount of MAO-B in smokers further validates the pharmacological effect of tobacco smoke on this enzyme.

Adult↗
PubMed2005

Elevated levels of the NR2C subunit of the NMDA receptor in the locus coeruleus in depression.

Low levels of the intracellular mediator of glutamate receptor activation, neuronal nitric oxide synthase (nNOS) were previously observed in locus coeruleus (LC) from subjects diagnosed with major depression. This finding implicates abnormalities in glutamate signaling in depression. Receptors responding to glutamate in the LC include ionotropic N-methyl-D-aspartate receptors (NMDARs). The functional NMDAR is a hetero-oligomeric structure composed of NR1 and NR2 (A-D) subunits. Tissue containing the LC and a nonlimbic LC projection area (cerebellum) was obtained from 13 and 9 matched pairs, respectively, of depressed subjects and control subjects lacking major psychiatric diagnoses. NMDAR subunit composition in the LC was evaluated in a psychiatrically normal subject. NR1 and NR2C subunit immunoreactivities in LC homogenates showed prominent bands at 120 and 135 kDa, respectively. In contrast to NRI and NR2C, very weak immunoreactivity of NR2A and NR2B subunits was observed in the LC. Possible changes in concentrations of NR1 and NR2C that might occur in depression were assessed in the LC and cerebellum. The overall amount of NR1 immunoreactivity was normal in the LC and cerebellum in depressed subjects. Amounts of NR2C protein were significantly higher (+ 61%, p = 0.003) in the LC and modestly, but not significantly, elevated in the cerebellum (+ 35%) of depressives as compared to matched controls. Higher levels of NR2C subunit implicate altered glutamatergic input to the LC in depressive disorders.

Adult↗
PubMed2005

Age-associated changes in mRNA levels of Phox2, norepinephrine transporter and dopamine beta-hydroxylase in the locus coeruleus and adrenal glands of rats.

Age-related changes in the gene expression of the transcription factors, Phox2a and 2b, and two marker proteins, norepinephrine transporter (NET) and dopamine beta-hydroxylase (DBH), of noradrenergic neurons were characterized in the locus coeruleus (LC) and adrenal glands using in situ hybridization. Analysis of changes was performed in rats that were 1-23 months of age. Compared to 1-month-old rats, there was a 62% increase of Phox2a messenger RNA (mRNA) in the LC of 3-month-old rats, and a decline of 37% in 23-month-old rats. In contrast, levels of Phox2b mRNA in the LC remained unchanged in 3-month-old rats, but declined to a 30% reduction in 23-month-old rats. Interestingly, mRNA levels of NET in the LC decreased with increasing age to a reduction of 29%, 30% and 43% in 3-, 8- and 23-month-old rats, respectively. Similarly, DBH mRNA in the LC declined with increasing age to a 56% reduction in 23-month-old rats. mRNA levels of Phox2a, Phox2b, NET and DBH in the adrenal medulla of 23-month-old rats were significantly lower than those of 1-month-old rats. Semi-quantitative reverse transcription assays of the same genes yielded data similar to in situ hybridization experiments, with beta-actin mRNA levels being unchanged across the ages. Taken together, these data reveal that reduced Phox2 mRNAs in the LC and adrenal medulla of aging rats are accompanied by a coincidental decline in mRNA levels of NET and DBH and suggest a possible relationship between Phox2 genes and the marker genes in noradrenergic neurons after birth.

Adrenal Glands↗
PubMed2004

Norepinephrine transporter function and desipramine: residual drug effects versus short-term regulation.

Previous research has shown that exposure of norepinephrine transporter (NET)-expressing cells to desipramine (DMI) downregulates the norepinephrine transporter, although changes in the several transporter parameters do not demonstrate the same time course. Exposures to desipramine for <1 day reduces only radioligand binding and uptake capacity while transporter-immunoreactivity is unaffected. Recent demonstration of persistent drug retention in cells following desipramine exposures raises the possibility that previous reported changes in the norepinephrine transporter may be partly accountable by residual drug. In this study, potential effects of residual desipramine on norepinephrine transporter binding and uptake were re-evaluated following exposures of PC12 cells to desipramine using different methods to remove residual drug. Using a method that minimizes residual drug, exposure of intact PC12 cells to desipramine for 4h had no effect on uptake capacity or [(3)H]nisoxetine binding to the norepinephrine transporter, while exposures for > or =16 h reduced uptake capacity. Desipramine-induced reductions in binding to the transporter required >24 h or greater periods of desipramine exposure. This study confirms that uptake capacity of the norepinephrine transporter is reduced earlier than changes in radioligand binding, but with a different time course than originally shown. Special pre-incubation procedures are required to abolish effects of residual transporter inhibitor when studying inhibitor-induced transporter regulation.

Adaptation, Physiological↗
PubMed2004

Effect of repeated treatment with olanzapine or olanzapine plus fluoxetine on tyrosine hydroxylase in the rat locus coeruleus.

Repeated treatment of rats with antidepressant drugs down-regulates tyrosine hydroxylase (TH) in the locus coeruleus (LC). Using this effect as a model system, this study evaluated the antidepressant potential of the atypical antipsychotic drug, olanzapine. In an initial study, rats were treated i.p. with saline, olanzapine (3 mg/kg), or imipramine (15 mg/kg) once daily for 18 d. Three additional groups of rats received the same treatments that were overlapped with reserpine administration (0.5 mg/kg.d for 21 d). In a second study, groups of rats were treated twice daily with saline, olanzapine (5 mg/kg.d), fluoxetine (15 mg/kg.d), or fluoxetine (15 mg/kg.d) plus olanzapine (5 mg/kg.d) for 1, 6, 12 and 18 d. In the initial study, imipramine produced a 45% reduction in LC TH levels, while olanzapine had no effect. In reserpinized rats, olanzapine exhibited an action that was opposite to that of imipramine, although this effect did not reach statistical significance. In the second study, olanzapine treatment alone or in combination with fluoxetine up-regulated TH in the LC, while fluoxetine alone had no effect. When fluoxetine was co-administered, olanzapine-induced increases in LC TH were more robust and occurred earlier in the time-course of treatment. Based on this preclinical model alone, olanzapine did not exhibit typical antidepressant properties. The unique effect of olanzapine to elevate LC TH may result from olanzapine-induced increases in LC activity. Such an action may contribute to novel behavioural effects of this atypical antipsychotic drug, including enhanced attention.

Actins↗
PubMed2004

Reinforcing effect of pseudoephedrine isomers and the mechanism of action.

It has been proposed that ephedrine and its isomers may have abuse potential. When made available to rhesus monkeys (n = 4) for self-administration, +-pseudoephedrine functioned as a positive reinforcer in all monkeys, as did (-)-pseudoephedrine in two of three monkeys. Pseudoephedrine isomers were 10- to 33-fold less potent than cocaine. In in vitro binding in monkey brain tissue, both isomers had low affinity for dopamine and serotonin transporters by at least 200-fold relative to cocaine, but comparable affinity for norepinephrine transporters. +-Pseudoephedrine also blocked dopamine uptake in 293 hDAT cells with low potency relative to cocaine. When given in vivo +-pseudoephedrine significantly displaced radioligand binding to dopamine transporters with a potency comparable to that in self-administration. Therefore, pseudoephedrine isomers can function as reinforcers and the mechanism at dopamine transporters may underlie this effect. However, pseudoephedrine appears to be a weak reinforcer and may have relatively low abuse potential.

Animals↗
PubMed2004

The persistent membrane retention of desipramine causes lasting inhibition of norepinephrine transporter function.

The present study examined the potential membrane retention of desipramine (DMI) following exposures of 293-hNET cells to DMI, and its effect on [3H]NE uptake. Incubation of cells with 500 nM DMI for 1 h or 1 day persistently inhibited the uptake of [3H]NE up to 7 days, despite daily repeated washing of cells with drug-free medium. Uptake inhibition was paralleled by persistent retention of DMI associated with cells, as determined by HPLC and by radiotracer experiments using [3H]DMI. [3H]DMI trapped in membranes was displaceable by the structurally unrelated NET inhibitor, nisoxetine, in a concentration-dependent manner, implying interaction of retained [3H]DMI with the NET. A similar cellular retention was observed following incubation of cells with nisoxetine. The results demonstrate that DMI and nisoxetine are persistently retained in cell membranes, at least partly in association with the NET. The retention and slow diffusion of DMI and nisoxetine from membranes may contribute to their pharmacological and modulatory action on NET.

Adrenergic Uptake Inhibitors↗
PubMed2003

The alpha2C-adrenoceptor modulates GABA release in mouse striatum.

The alpha(2C)-adrenoceptor occurs in high density in the striatum relative to other brain regions, but its biological role in striatal physiology is perplexing because of the paucity of noradrenergic terminals in this region. In this study, mice with a targeted inactivation of the alpha(2C)-adrenoceptor gene (alpha(2C)-KO mice), and genetically related mice (WT mice), were used to study the potential role of the striatal alpha(2C)-adrenoceptor in modulating GABA release. Perfused brain slices were pre-loaded with [(3)H]GABA and were stimulated electrically. In WT mice, the alpha(2)-adrenoceptor agonist, UK14304 (brimonidine), significantly enhanced [(3)H]GABA release from striatal slices, while the alpha(2)-adrenoceptor antagonist, RX821002, alone evoked a significant decrease in [(3)H]GABA release. In alpha(2C)-KO mice, the effect of RX821002 was absent, while UK14304 retained its ability to enhance [(3)H]GABA release. Pharmacological depletion of monoamines in WT mice also abolished the effect of RX821002 on [(3)H]GABA release. In hippocampal slices, RX821002-induced reduction in [(3)H]GABA release was present in WT and alpha(2C)-KO mice. In the presence of tetrodotoxin, RX821002 increased [(3)H]GABA release in striatal slices from both WT and alpha(2C)-KO mice. Together, these data imply that alpha(2A)- and alpha(2C)-adrenoceptors are located on different neurons in the striatum, that alpha(2C)-adrenoceptor-mediated effects on striatal GABA release are mediated by an endogenous catecholamine that could be dopamine, and that the alpha(2C)-adrenoceptor effect of RX821002 does not occur at the GABAergic terminal.

Adrenergic alpha-Agonists↗
PubMed2003

Elevated agonist binding to alpha2-adrenoceptors in the locus coeruleus in major depression.

BACKGROUND: Recent postmortem studies demonstrate disrupted neurochemistry of the noradrenergic locus coeruleus (LC) in major depression (MD). Increased levels of tyrosine hydroxylase and decreased levels of norepinephrine transporter implicate a norepinephrine deficiency in the LC in MD. Here we describe a study of alpha2-adrenoceptors in the LC and raphe nuclei of subjects with MD compared with psychiatrically normal control subjects. METHODS: The specific binding of p-[125I]iodoclonidine to alpha2-adrenoceptors was measured at multiple levels along the rostrocaudal extent of the LC in postmortem tissue from 14 control and 14 MD subjects. In addition, p-[125I]iodoclonidine binding was measured in the dorsal and median raphe nuclei in the same tissue sections. RESULTS: The specific binding of p-[125I]iodoclonidine to alpha2-adrenoceptors was significantly elevated throughout the LC from MD compared with matched control subjects. No significant differences were observed in p-[125I]iodoclonidine binding to alpha2-adrenoceptors in the raphe nuclei comparing MD and control subjects. CONCLUSIONS: Given that alpha2-adrenoceptors are upregulated in laboratory animals by treatment with drugs that deplete norepinephrine, our findings implicate a premortem deficiency of brain norepinephrine in the region of the locus coeruleus in subjects with MD.

Adrenergic alpha-2 Receptor Agonists↗
PubMed2002

Dopaminergic abnormalities in amygdaloid nuclei in major depression: a postmortem study.

BACKGROUND: A deficiency of mesolimbic dopamine (DA) is a leading candidate for the etiology of certain symptoms of depression (e.g., anhedonia and loss of motivation). Here we show amounts of dopaminergic proteins in the amygdala, a key brain structure involved in the integration of emotions and stress, in subjects with major depression and in psychiatrically normal control subjects. METHODS: The specific binding of [(125)I]RTI 55 to the DA transporter, [(3)H]SCH 23390 to the D1 receptor and [(125)I]epidepride to D2/D3 receptors were measured in the right amygdaloid complex in postmortem brains from 11 subjects with major depression and 11 matched control subjects. RESULTS: The binding of [(125)I]RTI 55 to DA transporter was significantly lower in the basal and central amygdaloid nuclei, whereas the binding of [(125)I]epidepride to D2/D3 receptors was significantly higher in the basal, central, and lateral amygdaloid nuclei in major depression compared with control subjects. No difference in the binding of [(3)H]SCH 23390 to D1 receptors was observed. CONCLUSIONS: Given that DA depletion in rats can induce a reduction in the DA transporter and an upregulation of D2/D3 receptors, our data are consistent with the hypothesis that major depression is associated with a deficiency of mesolimbic DA.

Adult↗
PubMedDate not supplied

Serotonin transporter and MAO-B levels in monoamine nuclei of the human brainstem are normal in major depression.

Neurochemical imbalance between noradrenergic and serotonergic systems has been postulated to underlie the pathophysiology of psychiatric illnesses involving mood disorders. The present study was designed to examined the possibility that serotonergic innervation of the locus coeruleus (LC) is abnormal in major depression, by measuring two proteins expressed by serotonergic neurons, but not by noradrenergic neurons, in the region of the LC. The specific binding of [(3)H]paroxetine to serotonin transporter (SERT) and of [(3)H]lazabemide to monoamine oxidase (MAO-B) were measured autoradiographically in tissue sections cut transversely at multiple levels along the rostro-caudal extent of the LC, as well as in the caudal portion of the dorsal raphe nucleus, from psychiatrically normal subjects and age-matched subjects with major depression. Under the conditions of the assays, [(3)H]paroxetine binding in the LC was specific for the SERT, based on the rank order of affinity of compounds for inhibiting [(3)H]paroxetine binding in the LC, i.e. citalopram > imipramine > desipramine > mazindol. The binding of [(3)H]paroxetine to SERT and [(3)H]lazabemide to MAO-B were higher in the raphe nuclei than in the LC. Comparison of control subjects to major depressive subjects revealed no differences in the amount of [(3)H]paroxetine binding to SERT and [(3)H]lazabemide to MAO-B in the LC, as well as in the raphe nuclei. These findings imply that serotonergic innervation of the LC is intact in major depression.

Adult↗
PubMedDate not supplied

Differential expression of alpha2-adrenoceptor vs. imidazoline binding sites in postmortem orbitofrontal cortex and amygdala of depressed subjects.

Clonidine is a well established antihypertensive agent that is also used effectively to treat a variety of psychiatric disorders. Clonidine is a prototypic imidazoline compound that acts as an alpha(2)-adrenergic agonist but possesses nearly equivalent affinity for non-adrenergic imidazoline binding sites (I-sites). Receptor autoradiography of [(3)H]-clonidine binding presented herein compares densities of alpha(2)-adrenoceptors and I-sites (under a noradrenergic-mask) in Brodmann's area 47 of the left orbitofrontal cortex (OFC) and in six amygdaloid nuclei of subjects with major depression (n=12) vs. controls with no psychiatric history (n=11). Postmortem diagnoses were made from psychiatric interviews with next-of-kin. [(3)H]-Clonidine binding to alpha(2)-adrenoceptors in each of six OFC layers was lower, although not reaching statistical significance in any one layer by multivariate analysis, in depressives vs. control subjects. Binding to I-sites was conversely higher in depressives compared to control OFC layers, but did not reach statistical significance alone. However, the ratios of alpha(2)-adrenoceptor : I-sites in all six layers of OFC of depressed subjects were nearly half that of control subjects (P<0.008). In amygdalas from a different group of depressed patients there were no changes in alpha(2)-adrenoceptors or I-sites, or their ratios, compared with controls. The results support previous western blot data indicating a cortex-selective shift away from alpha(2)AR towards I-site preponderance in depressed patients.

Adrenergic alpha-Agonists↗