Transcription factor Phox2 upregulates expression of norepinephrine transporter and dopamine ß-hydroxylase in adult rat brains.

Degeneration of the noradrenergic locus coeruleus (LC) in aging and neurodegenerative diseases is well documented. Slowing or reversing this effect may have therapeutic implications. Phox2a and Phox2b are homeodomain transcriptional factors that function as determinants of the noradrenergic phenotype during embryogenesis. In the present study, recombinant lentiviral eGFP-Phox2a and -Phox2b (vPhox2a and vPhox2b) were constructed to study the effects of Phox2a/2b over-expression on dopamine ß-hydroxylase (DBH) and norepinephrine transporter (NET) levels in central noradrenergic neurons. Microinjection of vPhox2 into the LC of adult rats significantly increased Phox2 mRNA levels in the LC region. Over-expression of either Phox2a or Phox2b in the LC was paralleled by significant increases in mRNA and protein levels of DBH and NET in the LC. Similar increases in DBH and NET protein levels were observed in the hippocampus following vPhox2 microinjection. In the frontal cortex, only NET protein levels were significantly increased by vPhox2 microinjection. Over-expression of Phox2 genes resulted in a significant increase in BrdU-positive cells in the hippocampal dentate gyrus. The present study demonstrates an upregulatory effect of Phox2a and Phox2b on the expression of DBH and NET in noradrenergic neurons of rat brains, an effect not previously shown in adult animals. Phox2 genes may play an important role in maintaining the function of the noradrenergic neurons after birth, and regulation of Phox2 gene expression may have therapeutic utility in aging or disorders involving degeneration of noradrenergic neurons.

Low nNOS protein in the locus coeruleus in major depression.

Disruptions of glutamatergic and noradrenergic signaling have been postulated to occur in depressive disorders. Glutamate provides excitatory input to the noradrenergic locus coeruleus (LC). In this study, the location of immunoreactivity against neuronal nitric oxide synthase (nNOS), an intracellular mediator of glutamate receptor activation, was examined in the normal human LC, and potential changes in nNOS immunoreactivity that might occur in major depression were evaluated. Tissue containing LC, and a non-limbic, LC projection area (cerebellum) was obtained from 11 to 12 matched pairs of subjects with major depression and control subjects lacking major psychiatric diagnoses. In the LC region, nNOS immunoreactivity was found in large neuromelanin-containing neurons, small neurons lacking neuromelanin, and glial cells. Levels of nNOS immunoreactivity were significantly lower in the LC (- 44%, p < 0.05), but not in the cerebellum, when comparing depressed with control subjects. nNOS levels were positively correlated with brain pH values in depressed, but not control, subjects in both brain regions. Low levels of nNOS in the LC may reflect altered excitatory input to this nucleus in major depression. However, pH appears to effect preservation of nNOS immunoreactivity in subjects with depression. This factor may contribute, in part, to low levels of nNOS in depression.

Effect of dietary protein on lean body wasting in dogs: correlation between loss of lean mass and markers of proteasome-dependent proteolysis.

To determine the effect of dietary protein intake on lean body wasting in adult canines a study was undertaken to investigate the Ubiquitin Proteasome (UP) pathway and concurrent changes in lean and fat body mass of canines fed variable sources and concentrations of dietary protein. Purpose-bred, intact female canines (56) between the ages of 2 and 3 years were fed either 12 or 28% protein diet for 10 weeks. Each diet contained variable amounts of corn gluten meal and chicken protein sources in ratios of 100 : 0, 67 : 33, 33 : 67 and 0 : 100 per cent (w/w), respectively. All diets were isocaloric with calories coming from protein : fat : carbohydrate at the respective ratios of 12 : 40 : 48% for the 12% diets, and 28 : 40 : 32% for the 28% diets. Standard dual energy X-ray absorptiometry was performed to assess total body lean and fat mass at weeks 0 and 10 of the dietary trial. Muscle biopsies were also taken and processed for protein determination and standard gel electrophoresis with subsequent Western blotting for 20S proteasome and PA700 regulatory cap subunit p31. Statistical analysis revealed a moderate degree of correlation between increasing quantities of corn gluten, which is low in essential amino acids (i.e. lysine, tryptophan), and increasing loss of lean body mass over the 10-week study (R = 0.56). Furthermore, a moderate degree of correlation was observed between increasing concentrations of corn gluten protein and decreased expression of the p31 subunit of the 26S proteasome (R = 0.49). Additionally, the dogs consuming the 12% protein diets had a significant increase in fat mass regardless of the protein source. These findings suggest that lean body wasting in adult canines can be associated with the consumption of low protein diets consisting of predominantly corn gluten, which is likely due to imbalances or subclinical deficiencies of specific essential amino acids, and that low protein diets may augment accumulation of adipose tissue. Although the mechanisms remain unclear, alteration of molecular targets of skeletal muscle proteolysis, specifically involving the UP pathway occur.

Reinforcing strength of a novel dopamine transporter ligand: pharmacodynamic and pharmacokinetic mechanisms.

Drugs that block dopamine uptake often function as positive reinforcers but can differ along the dimension of strength or effectiveness as a positive reinforcer. The present study was designed to examine pharmacological mechanisms that might contribute to differences in reinforcing strength between the piperidine-based cocaine analog (+)-methyl 4beta-(4-chlorophenyl)-1-methylpiperidine-3-alpha-carboxylate [(+)-CPCA] and cocaine. Drugs were made available to rhesus monkeys (n = 5) for i.v. self-administration under a progressive ratio schedule. Both compounds maintained responding with sigmoidal or biphasic dose-response functions (0.1-1.0 mg/kg/injection). (+)-CPCA was one-fourth as potent as cocaine and maintained fewer injections per session, at maximum. For in vitro binding in monkey brain tissue, (+)-CPCA was about one-half as potent as cocaine at the dopamine transporter (DAT), and the two compounds had similar affinities at the norepinephrine transporter. (+)-CPCA was less than 1/10 as potent as cocaine at the serotonin transporter. In ex vivo binding in rat striatum, occupancy of the DAT increased directly with dose to a maximum of approximately 80% for both compounds, and (+)-CPCA was about one-fourth as potent as cocaine. Ex vivo DAT occupancy was significantly higher for cocaine than (+)-CPCA at 2 min after injection but similar at other times. Thus, the primary differences between these compounds were in serotonin transporter affinity and the kinetics of DAT binding. These results suggest that (+)-CPCA is a weaker positive reinforcer than cocaine because it has a slower onset of action over the first few minutes after i.v. injection.

Role of the dopamine transporter and the sodium channel in the cocaine-like discriminative stimulus effects of local anesthetics in rats.

RATIONALE: Local anesthetics bind to the dopamine transporter (DAT), inhibit dopamine (DA) uptake and have been reported to have cocaine-like discriminative stimulus effects. The hypothesis of the present study was that affinity at the DAT and potency as a DA uptake blocker determines potency as a cocaine-like discriminative stimulus among local anesthetics, and maximum DA uptake inhibition determines maximum cocaine-like discriminative stimulus effects. OBJECTIVES: Cocaine-like discriminative potency was compared to DAT affinity and DA uptake inhibition potency, and maximum cocaine-like discriminative stimulus effects were compared to maximum DA uptake inhibition for procaine, chloroprocaine, dimethocaine, tetracaine and lidocaine. METHODS: Discriminative stimulus effects were determined in two groups of rats using 10 mg/kg and 3.0 mg/kg cocaine training doses. DAT affinity and DA uptake inhibition effects were determined in vitro in rat caudate nucleus tissue. Additionally, sodium channel affinity was determined in rat frontal cortex tissue. RESULTS: In the 10 mg/kg group, none of the local anesthetics fully substituted for cocaine and all decreased response rate. Rate decreasing potencies were positively correlated with sodium channel affinities. In the low training dose group, all the local anesthetics except tetracaine substituted fully for cocaine. Discriminative potencies were positively correlated with sodium channel affinities. Maximum DA uptake inhibition did not adequately predict maximum discriminative stimulus effects. CONCLUSIONS: Cocaine-like discriminative stimulus effects of local anesthetics were more prominent at a low than at a high training dose of cocaine. Sodium channels seem to have a direct influence on discriminative effects at low cocaine doses, whereas they have an indirect influence on discriminative effects at high cocaine doses by decreasing response rates.

Effects of long-term cigarette smoking on the human locus coeruleus.

BACKGROUND: It has been hypothesized that cigarette smoking among subjects with major depression is a form of self-medication. To explore a possible biological basis for this hypothesis, noradrenergic proteins in the locus coeruleus (LC) were measured in long-term cigarette smokers and in nonsmokers. The LC was studied because elevated amounts of alpha2-adrenoceptors and tyrosine hydroxylase have been observed postmortem in the LCs of subjects with major depression or who commit suicide, and because long-term administration of antidepressant drugs to rats down-regulates these proteins in the LC. METHODS: Postmortem LCs were obtained from long-term cigarette smokers (n=7) and from nonsmokers (n = 9), all of whom lacked diagnoses of major depression. Amounts of tyrosine hydroxylase immunoreactivity and radioligand binding to the norepinephrine transporter, monoamine oxidase A, and alpha2-adrenoceptors were measured. RESULTS: Amounts of tyrosine hydroxylase immunoreactivity and radioligand binding to alpha2-adrenoceptors were significantly lower (approximately 60% and 40%, respectively) along the axis of the LCs of long-term smokers compared with nonsmokers. Smoking had no statistically significant effects on binding to monoamine oxidase A or to the norepinephrine transporter. CONCLUSION: This is the first demonstration that cigarette smoking affects noradrenergic proteins in the LC. The direction of these changes is opposite to that observed when comparing subjects who have major depression with normal controls and the same as that produced by long-term antidepressant treatment in animals. If the present observations reflect long-term effects of smoking on premortem noradrenergic biochemistry, smoking-induced changes in LC biochemistry may strengthen the smoking habit among subjects with major depression.

Noradrenergic pathology in psychiatric disorders: postmortem studies.

In this paper, we review research utilizing postmortem brain tissue in order to investigate the potential neuropathology of the noradrenergic system in psychiatric disorders. The postmortem tissue approach to the study of the noradrenergic system has been used primarily in investigations of the biology of suicide and depression. Findings from postmortem studies provide data generally consistent with the hypothesis that a norepinephrine deficiency exists in depression, and possibly in the victims of suicide. However, postmortem studies do not presently provide irrefutable evidence of noradrenergic neuropathology. Technical shortcomings, issues of reproducibility, and the strengths of postmortem research are reviewed. More rigorously performed postmortem research is needed to aid researchers in pinpointing specific neuropathologies associated with psychiatric disease.

Imidazoline receptor proteins are decreased in the hippocampus of individuals with major depression.

BACKGROUND: A downregulation of I(2)-imidazoline binding sites has been reported in frontal cortices of depressed suicide victims, according to I(2)-radioligand binding and confirmed by Western blotting. We now report Western blots of imidazoline receptor proteins in hippocampi of subjects with and without depression at the time of death. METHODS: Postmortem diagnoses were obtained from 17 cases of Axis I major depressive disorder and 17 cases without Axis I psychopathology. No psychotropic compounds were found in body fluids. Hippocampi were removed, sectioned, and assessed histologically. Throughout the analysis, each major depressive disorder sample was paired with a sample from a psychiatrically healthy subject based on equivalent life spans and postmortem delays. The antiserum was identical to that used in previous studies that reported a downregulation of cortical 29/30-kd imidazoline receptor-binding proteins in depression. RESULTS: A triad of imidazoline receptor-binding protein bands (40-50 kd) was detected in the human hippocampus. Subjects with major depressive disorder had significantly less intensity in each imidazoline receptor-binding proteins band compared with control subjects (p =. 01 for overall bands). CONCLUSIONS: The present results can be aligned with previous reports of downregulation of I(2)-radioligand binding sites in both cortices and platelets of depressed patients.

Autoradiographic comparison of [3H]-clonidine binding to non-adrenergic sites and alpha(2)-adrenergic receptors in human brain.

UNLABELLED: Clonidine is a partial agonist at brain alpha(2)-adrenoceptors (alpha(2)AR), but also has high affinity (K(D) = 51 nM) in homogenate binding assays for non-adrenergic imidazoline-binding sites (I-sites; imidazoline receptors). Herein, an autoradiographic comparison of [3H]-clonidine binding to I-sites and alpha(2)AR in sections of human brain is reported. For I-sites, the adrenergic component of 50 nM [3H]-clonidine binding was masked with either 60 microM norepinephrine (NE; alpha(2)AR agonist) or 12.5 microM methoxy-idazoxan (MIDX; selective alpha(2)AR antagonist), whereas the remaining non-adrenergic sites were studied by displacement with 20 microM cirazoline. Levels of [3H]-clonidine binding to alpha(2)AR and I-sites, determined in adjacent tissue sections, were positively correlated across 27 brain regions (p = 0.0003; r(2) = 0.385). The principal olivary nucleus and the rostral portion of the ventrolateral medulla had highest ratios of I-sites: alpha(2)AR (>4:1). Quantitative transepts drawn across hippocampal images revealed alpha(2)AR enrichments in the CA-1 and inner molecular layers of the dentate gyrus-areas not enriched in I-sites. Competition curves were generated for I-sites in caudate sections using 10 ligands known to distinguish between I(1) and I(2) subtypes. The rank-order of affinities were cirazoline > harmane > BDF6143 > idazoxan = tizanidine (affinities of agmatine, efaroxan, moxonidine, NE, and oxymetazoline were too low to be reliable). Only the endogenous I-site ligand, harmane, had a monophasic displacement curve at the non-adrenergic sites (Ki = 521 +/- 12 nM). IN CONCLUSION: 1) the distribution of non-adrenergic [3H]-clonidine binding sites in human brain sections was correlated with, but distinct from alpha(2)AR; and 2) the affinities of these sites was distinct from alpha(1)AR, alpha(2)AR, I(1) or I(2) sites as previously defined in membrane binding assays. The properties of this non-adrenergic [3H]-clonidine binding site are consistent with I-sites previously labeled by [3H]-cirazoline in rat brain.

Regulation of the human norepinephrine transporter by cocaine and amphetamine.

Certain antidepressant and psychostimulant drugs block the uptake of norepinephrine from the synaptic cleft by inhibiting norepinephrine transporter (NET) function. The effects of chronic occupation of the NET by these drugs on NET expression are poorly understood. We previously described down-regulation of the NET in cultured cells after continuous exposure to the tricyclic antidepressant desipramine. Here, the effects of structurally unrelated NET ligands, cocaine and amphetamine, on levels of NET and on NET function in HEK-293 cells transfected with human NET cDNA were investigated. All drug exposures were followed by incubation in drug-free media before harvesting and assays. Exposure of intact cells to cocaine for 3 days did not significantly affect the B(max) or K(D) of [(3)H]nisoxetine binding to NET in membrane homogenates, and did not alter levels of NET immunoreactivity or NET mRNA. In contrast, incubation of cells with amphetamine significantly reduced [(3)H]nisoxetine binding to NET and levels of NET immunoreactivity in a time-dependent manner, although levels of NET mRNA appeared to be unaffected. Exposures to cocaine or amphetamine resulted in significant reductions of [(3)H]norepinephrine uptake, although the magnitude of the reduction produced by amphetamine was much greater than cocaine. [(3)H]Nisoxetine binding to NET and NET protein levels were also reduced by exposure of cells to high concentrations of norepinephrine, although norepinephrine exposures were accompanied by changes indicative of cellular toxicity. Cocaine and amphetamine have distinctly different effects on NET expression after continuous exposure. The ability of only certain drugs to down-regulate the NET may provide clues to the unique therapeutic effects of antidepressants that are NET ligands.

Transcriptional regulation of gene expression in human skeletal muscle during recovery from exercise.

Exercise training elicits a number of adaptive changes in skeletal muscle that result in an improved metabolic efficiency. The molecular mechanisms mediating the cellular adaptations to exercise training in human skeletal muscle are unknown. To test the hypothesis that recovery from exercise is associated with transcriptional activation of specific genes, six untrained male subjects completed 60-90 min of exhaustive one-legged knee extensor exercise for five consecutive days. On day 5, nuclei were isolated from biopsies of the vastus lateralis muscle of the untrained and the trained leg before exercise and from the trained leg immediately after exercise and after 15 min, 1 h, 2 h, and 4 h of recovery. Transcriptional activity of the uncoupling protein 3 (UCP3), pyruvate dehydrogenase kinase 4 (PDK4), and heme oxygenase-1 (HO-1) genes (relative to beta-actin) increased by three- to sevenfold in response to exercise, peaking after 1-2 h of recovery. Increases in mRNA levels followed changes in transcription, peaking between 2 and 4 h after exercise. Lipoprotein lipase and carnitine pamitoyltransferase I gene transcription and mRNA levels showed similar but less dramatic induction patterns, with increases ranging from two- to threefold. In a separate study, a single 4-h bout of cycling exercise (n = 4) elicited from 5 to >20-fold increases in UCP3, PDK4, and HO-1 transcription, suggesting that activation of these genes may be related to the duration or intensity of exercise. These data demonstrate that exercise induces transient increases in transcription of metabolic genes in human skeletal muscle. Moreover, the findings suggest that the cumulative effects of transient increases in transcription during recovery from consecutive bouts of exercise may represent the underlying kinetic basis for the cellular adaptations associated with exercise training.

Quantitation of tyrosine hydroxylase protein in the locus coeruleus from postmortem human brain.

In this study, we developed an immuno-autoradiographic method to obtain quantitative estimates of tyrosine hydroxylase (TH) protein in tissue sections from post-mortem human brain. Protein from tissue sections containing the locus coeruleus (LC) was directly transferred to a polyvinylidene fluoride (PVDF) membrane. Immunoreactive TH on PVDF membranes was identified with optimized concentrations of TH antibody followed by application of [125I]labeled secondary antibody. Quantities of TH on autoradiograms were estimated by comparing optical densities of transferred immunoblots to a calibrated standard curve produced with purified recombinant TH dotted onto the same PVDF membranes. Amounts of TH-immunoreactivity in the LC were proportional to the thickness of tissue sections up to 15 micrometer. However, the amounts of total protein, as measured by Ponceau S staining, were linearly related to section thicknesses up to 30 micrometer. Comparisons of quantities of immunoreactive TH in the LC using this method to amounts determined using traditional Western blotting, in which LC tissue was punched from adjacent sections from the same subject, showed a positive correlation (r(2)=0.99, P<0.01). Using the transfer immunoblot method, an uneven distribution of TH protein was observed along the rostrocaudal axis of the human LC (P<0.01). This method may provide a sensitive and useful tool for the study of the role of human TH expression in the pathophysiology of psychiatric disease.

Deciphering the mysteries of myoglobin in striated muscle.

Myoglobin (Mb) is a large protein that reversibly binds oxygen in the muscle cell and is thought to be critical for O2 supply to the mitochondria during exercise. The role of Mb in aerobic function is evaluated based on the physical properties of Mb as an O2 carrier and experimental evidence of Mb function in vivo. This role depends on the reversible binding of O2 by Mb depending on PO2, which results in: (1) storage of O2; (2) buffering of PO2 in the cell to prevent mitochondrial anoxia; and (3) parallel diffusion of O2 (so-called, 'facilitated diffusion'). The storage role is well established in diving mammals and buffering of cell PO2 above anoxic levels is shown here by in vivo magnetic resonance spectroscopy (MRS). However, the quantitative role of Mb in 'facilitated' or parallel diffusion of O2 is controversial. Evidence in support of this role is from MRS analyses, which reveal rapid Mb desaturation with exercise, and from the proportionality of Mb content of a muscle to the O2 diffusion limitation. Recent experiments with myoglobin knockout mice demonstrating high levels of aerobic function in normal and myoglobin-free mice argue against a link between Mb and oxidative phosphorylation. Thus, the current evidence supports the role of Mb in the physical diffusion of O2; however, the unimpaired aerobic function of Mb knockout mice indicates that this role may not be critical to O2 supply in active muscle.

Chronic contractile activity upregulates the proteasome system in rabbit skeletal muscle.

Remodeling of skeletal muscle in response to altered patterns of contractile activity is achieved, in part, by the regulated degradation of cellular proteins. The ubiquitin-proteasome system is a dominant pathway for protein degradation in eukaryotic cells. To test the role of this pathway in contraction-induced remodeling of skeletal muscle, we used a well-established model of continuous motor nerve stimulation to activate tibialis anterior (TA) muscles of New Zealand White rabbits for periods up to 28 days. Western blot analysis revealed marked and coordinated increases in protein levels of the 20S proteasome and two of its regulatory proteins, PA700 and PA28. mRNA of a representative proteasome subunit also increased coordinately in contracting muscles. Chronic contractile activity of TA also increased total proteasome activity in extracts, as measured by the hydrolysis of a proteasome-specific peptide substrate, and the total capacity of the ubiquitin-proteasome pathway, as measured by the ATP-dependent hydrolysis of an exogenous protein substrate. These results support the potential role of the ubiquitin-proteasome pathway of protein degradation in the contraction-induced remodeling of skeletal muscle.

On the relationship between the dopamine transporter and the reinforcing effects of local anesthetics in rhesus monkeys: practical and theoretical concerns.

RATIONALE: Drugs that are self-administered appear to vary in their potency and effectiveness as positive reinforcers. Understanding mechanisms that determine relative effectiveness of drugs as reinforcers will enhance our understanding of drug abuse. OBJECTIVES: The hypothesis of the present study was that differences among dopamine transporter (DAT) ligands in potency and effectiveness as a positive reinforcers were related to potency and effectiveness as DA uptake inhibitors. Accordingly, self-administration of a group of local anesthetics that are DAT ligands was compared to their effects as DA uptake blockers in vitro in brain tissue. METHODS: Rhesus monkeys were allowed to self-administer cocaine and other local anesthetics i.v. under a progressive-ratio schedule. The same compounds were compared in standard in vitro DA uptake assays using monkey caudate tissue. RESULTS: The rank order of both potency and effectiveness as reinforcers was cocaine > dimethocaine > procaine > chloroprocaine. Tetracaine did not maintain self-administration. For inhibiting DA uptake, the potency order was cocaine > dimethocaine > tetracaine > procaine > chloro-procaine. At maximum, these compounds were equally effective in blocking DA uptake. Lidocaine did not inhibit DA uptake. CONCLUSIONS: The potency of local anesthetics as positive reinforcers is likely related to their potency as DA uptake inhibitors. Variation in their effectiveness as positive reinforcers was not a function of differences in effectiveness as DA uptake blockers, but may be related to relative potency over the concentrations that are achieved in vivo. Effects at sodium channels may limit the reinforcing effects of local anesthetics.

Elevated levels of tyrosine hydroxylase in the locus coeruleus in major depression.

BACKGROUND: Levels of tyrosine hydroxylase (TH) are regulated in the noradrenergic locus coeruleus (LC) in response to changes in the activity of LC neurons and in response to changes in brain levels of norepinephrine. To study the potential role of central noradrenergic neurons in the pathobiology of major depression, TH protein was measured in the LC from postmortem brains of 13 subjects with a diagnosis of major depression and 13 age-matched control subjects having no Axis I psychiatric diagnosis. Most of the major depressive subjects died as a result of suicide. METHODS: Protein from sections cut through multiple rostro-caudal levels of LC was transferred to Immobilon-P membrane, immunoblotted for TH, and quantified autoradiographically. RESULTS: The distribution of TH-immunoreactivity (TH-ir) along the rostro-caudal axis of the LC was uneven and was paralleled by a similar uneven distribution of neuromelanin-containing cells in both major depressive and psychiatrically normal control subjects. Amounts of TH-ir in the rostral, middle and caudal levels of the LC from major depressive subjects were significantly higher than that of matched control subjects. There were no significant differences in the number of noradrenergic cells at any particular level of the LC comparing major depressive subjects to control subjects. CONCLUSIONS: Elevated expression of TH in the LC in major depression implies a premortem overactivity of these neurons, or a deficiency of the cognate transmitter, norepinephrine.

Quantitative distribution of monoamine oxidase A in brainstem monoamine nuclei is normal in major depression.

An abnormal expression of noradrenergic proteins (e.g., tyrosine hydroxylase, norepinephrine transporters) in the locus coeruleus has recently been demonstrated in subjects with major depression and/or victims of suicide. Monoamine oxidase A (MAO-A) is a key enzyme in the catabolism of biogenic amines and is expressed in brain noradrenergic neurons. In this study, the binding of [3H]Ro41-1049 to MAO-A was measured by quantitative autoradiography at multiple levels along the rostral-caudal axis of the noradrenergic locus coeruleus from subjects with major depression and age- and postmortem interval-matched control subjects who were psychiatrically normal. [3H]Ro41-1049 binding to MAO-A was unevenly distributed along the axis of the locus coeruleus, paralleling an uneven number of neuromelanin-containing (noradrenergic) neurons throughout the nucleus. Accordingly, there was a significant correlation between the number of neuromelanin-containing neurons per section and the specific binding of [3H]Ro41-1049 at any particular level of the locus coeruleus in control subjects (r(2)=0.25; p<0.001) and in subjects with major depression (r(2)=0.14; p<0. 001). Moderate levels of [3H]Ro41-1049 binding were observed in regions surrounding the locus coeruleus, including the central gray and the dorsal and median raphe nuclei. No significant differences in [3H]Ro41-1049 binding to MAO-A were observed at any level of the locus coeruleus, or raphe nuclei, comparing subjects with major depression to psychiatrically normal control subjects. These findings demonstrate that the pathophysiology of major depression is not likely to involve abnormalities in MAO-A.

Brain noradrenergic receptors in major depression and schizophrenia.

The binding of [125I]p-iodoclonidine to alpha-2, and/or [125I]iodopindolol to beta-1 and beta-2 adrenoceptors was measured in right prefrontal cortex (Brodmann's area 10) and right hippocampus from subjects with DSM-III-R diagnoses of major depression (n = 15) or schizophrenia (n = 8) as well as from control subjects (n = 20). No significant differences between study groups were observed in binding to alpha-2 adrenoceptors in any of the six layers of prefrontal cortex or in any of the hippocampal fields. Likewise, there were no significant differences in beta-1 or beta-2 adrenoceptor binding in any of the hippocampal fields between control and major depressive subjects. In contrast, binding to beta-1 adrenoceptors, but not beta-2 adrenoceptors, was significantly lower (-13 to -27%) in most hippocampal fields of schizophrenic subjects as compared to control subjects or to major depressives. Alterations in beta-1 adrenoceptor binding in the hippocampus of schizophrenics provide further evidence for a role of central noradrenergic neurons in the neurochemical pathology of schizophrenia.

alpha2C adrenoceptors inhibit adenylyl cyclase in mouse striatum: potential activation by dopamine.

alpha2C adrenoceptors occur in high density in the striatum, but the functional role of these receptors is uncertain. Mice with targeted inactivation of the alpha2C adrenoceptor gene (Adra2c-/-) and genetically related control mice expressing the wild-type alpha2C adrenoceptor (Adra2c+/+) were used to determine whether striatal alpha2C adrenoceptors modulate adenylyl cyclase activation. In striatal slices from Adra2c+/+ mice, the alpha2 adrenoceptor antagonist RX821002 facilitated forskolin-stimulated cyclic AMP accumulation in a concentration-dependent manner. In contrast, RX821002 had no effect on forskolin-stimulated cAMP accumulation in striatal slices from Adra2c-/- mice or in striatal slices from Adra2c+/+ mice treated with reserpine and alpha-methyl-rho-tyrosine to deplete monoamine neurotransmitters. Given the sparse innervation of the striatum by noradrenergic neurons, the possibility that dopamine can activate the mouse alpha2C adrenoceptor at physiologically relevant concentrations was investigated using normal rat kidney (NRK) cells transfected with the mouse alpha2A or alpha2C adrenoceptor cDNA (NRK-alpha2A or NRK-alpha2C cells). Inhibition of [3H]RX821002 binding by agonists in homogenates of transfected cells revealed an affinity of dopamine for alpha2C adrenoceptors that was higher than the affinity of norepinephrine for its cognate receptor, the alpha2A adrenoceptor. Both norepinephrine and dopamine inhibited forskolin-stimulated cAMP accumulation in intact NRK-alpha2C cells. In NRK-alpha2A cells, norepinephrine facilitated forskolin-stimulated cAMP accumulation, an effect not observed for dopamine. Together, these data demonstrate that the alpha2C adrenoceptor is negatively coupled to adenylyl cyclase and is tonically activated in mouse striatal slices. The endogenous activator of the striatal alpha2C adrenoceptor may be dopamine, as well as norepinephrine.