Agonist Binding to α2-Adrenoceptors Is Elevated in the Locus Coeruleus from Victims of Suicide

Abstract: The binding of an agonist, p-[¹²⁵I]iodoclonidine, and an antagonist, [³H]yohimbine, to α₂-adrenoceptors was measured autoradiographically in the locus coeruleus from 10 pairs of antidepressant-free victims of suicide and age-matched controls. Agonist binding to α₂-adrenoceptors was significantly greater in the locus coeruleus from victims of suicide compared with control subjects. In contrast, antagonist binding to α₂-adrenoceptors in the locus coeruleus did not differ significantly between control and suicide subjects. HPLC analysis of norepinephrine in tissue sections of the locus coeruleus did not reveal any differences between control subjects and suicide victims, suggesting that differences in agonist binding are not a result of differences in retention of the endogenous agonist norepinephrine in tissue sections. The increase in agonist binding to α₂-adrenoceptors in the locus coeruleus of victims of suicide links an altered expression of the high-affinity state of autoinhibitory α₂-adrenoceptors with suicide.     

Effects of Chronic and Subchronic Nicotine on Tyrosine Hydroxylase Activity in Noradrenergic and Dopaminergic Neurones in the Rat Brain

Chronic nicotine (0.8 mg/kg by daily subcutaneous injection) over a 7 to 28-day period was found to increase the activity of tyrosine hydroxylase in predominantly noradrenergically innervated regions but not in dopaminergic projection areas. Increases in tyrosine hydroxylase activity were observed in dopaminergic cell body regions only after nicotine treatment for 3 to 5 days. The increase in tyrosine hydroxylase activity in noradrenergic neurones was evident first in the cell bodies in the locus coeruleus from 3 to 7 days, reaching 223% of control activities, and was followed by increases of up to 205% in the terminals up to 3 weeks later. It was then established that nicotine for 7 days was sufficient to increase the activity of the enzyme to the same extent in the terminals at 21 days even without further nicotine administration. This is consistent with axonal transport preceded by induction of the enzyme in noradrenergic cell bodies, whereas "delayed activation" might account for the transient effect seen in dopaminergic cell body regions. The response in the locus coeruleus to nicotine for 7 days was completely blocked by daily preinjection with mecamylamine but not with hexamethonium, which is consistent with the effect of nicotine on tyrosine hydroxylase being mediated by central nicotinic receptors.     

Neuropeptide Y in Frontal Cortex Is Not Altered in Major Depression

Abstract: Previously, we reported a modest but significant reduction in the concentration of neuropeptide Y in frontal cortices from victims of suicide relative to age-matched natural or accidental death control subjects. The reduction in neuropeptide Y appeared to be greatest in a subgroup of victims of suicide for which there was indirect evidence of histories of depression. We pursued these initial findings in the present study by measuring neuropeptide Y concentrations in frontal cortices from natural or accidental death control subjects and from suicide victims in whom a firm diagnosis of major depression was established by psychiatric autopsy. Because several subjects with major depression had a comorbid diagnosis of alcoholism, a group of victims of suicide that had an Axis I diagnosis of alcohol dependence was also studied. No significant differences in neuropeptide Y concentrations were observed between control subjects and victims of suicide with major depression or victims of suicide with alcohol dependence. These findings do not support a role for neuropeptide Y in major depression.     

Direct Transfer into Nitrocellulose and Quantitative Radioautographic Anatomical Determination of Brain Tyrosine Hydroxylase Protein Concentration

An improved quantitative immunochemical determination of brain tyrosine hydroxylase (TH) concentrations was designed using direct transfer into nitrocellulose from 20-microns thick brain sections, followed by immunodetection and quantitative radioautography in three reference brain structures (locus ceruleus, substantia nigra, and ventral tegmental area). Results obtained by this methodology were similar to those obtained after extraction and Western blotting of the TH protein in control and reserpine-treated animals. Moreover, this methodology allows the combination of high sensitivity and high anatomical resolution in the study of the distribution of pharmacological effects. The locus ceruleus exhibited a significant posteroanterior distribution of TH protein concentration in control and reserpine-treated animals.     

Quantitation of tyrosine hydroxylase, protein levels: spot immunolabeling with an affinity-purified antibody

Tyrosine hydroxylase was purified from bovine adrenal chromaffin cells and rat pheochromocytoma using a rapid (less than 2 days) procedure performed at room temperature. Rabbits were immunized with purified enzyme that was denatured with sodium dodecylsulfate, and antibodies to tyrosine hydroxylase were affinity-purified from immune sera. A Western blot procedure using the affinity-purified antibodies and 125I-protein A demonstrated a selective labeling of a single Mr approximately 62,000 band in samples from a number of different tissues. The relative lack of background 125I-protein A binding permitted the development of a quantitative spot immunolabeling procedure for tyrosine hydroxylase protein. The sensitivity of the assay is 1-2 ng of enzyme. Essentially identical standard curves were obtained with tyrosine hydroxylase purified from rat pheochromocytoma, rat corpus striatum, and bovine adrenal medulla. An extract of PC 12 cells (clonal rat pheochromocytoma cells) was calibrated against purified rat pheochromocytoma tyrosine hydroxylase and used as an external standard against which levels of tyrosine hydroxylase in PC12 cells and other tissue were quantified. With this procedure, qualitative assessment of tyrosine hydroxylase protein levels can be obtained in a few hours and quantitative assessment can be obtained in less than a day.     

TYROSINE HYDROXYLASE ACTIVITY IN NORADRENERGIC NEURONS OF THE LOCUS COERULEUS AFTER RESERPINE ADMINISTRATION: SEQUENTIAL INCREASE IN CELL BODIES AND NERVE TERMINALS

The effect of a single systemic injection of reserpine on tyrosine hydroxylase activity in the locus coeruleus, cerebellum, hypothalamus, and hippocampus was examined. Increases in enzyme activity were seen in all four brain areas; the time-course of the changes, however, was different in each case. In the locus coeruleus the maximum change in enzyme activity was seen 3 days after drug administration; in the cerebellum, 7-11 days; in the hypothalamus, 8-11 days; and in the hippocampus, 21 days. Since tyrosine hydroxylase in the cerebellum and hippocampus is present in terminals of neurons whose cell bodies are located in the locus coeruleus, the delayed increase in enzyme activity in cerebellum and hippocampus probably depends upon the slow rate of transport of TH molecules in these neurons.     

Normal levels of tryptophan hydroxylase immunoreactivity in the dorsal raphe of depressed suicide victims

A variety of evidence suggests that serotonin neurotransmission is altered in the brain of suicide victims and depressed patients. While numerous post-mortem studies have investigated serotonin transporters and receptors, few studies have examined the biosynthetic integrity of the rate-limiting enzyme, tryptophan hydroxylase (TPH), in post-mortem specimens of depressed suicide subjects. Therefore, the aim of the present study was to test the hypothesis that the levels of TPH immunoreactivity (IR) are altered in specific subnuclei of the dorsal raphe (DR) in depressed suicide victims. Suicide victims with a confirmed diagnosis of major depression were matched with non-psychiatric controls based on age, gender and post-mortem interval. Frozen tissue sections containing the DR were selected from two anatomical levels and processed for TPH radioimmunocytochemistry. The optical density corresponding to the regional levels of TPH-IR was quantified in specific subnuclei of the DR from the film autoradiographic images. No significant differences in the levels of TPH-IR were found in any DR subnuclei between depressed suicide victims and control subjects. The lack of change in TPH-IR levels does not necessarily imply that serotonin synthesis or neurotransmission is not altered in the brain of depressed subjects. Many factors influence and regulate serotonin synthesis, and it is conceivable that alterations exist at other levels of regulation of serotonin biosynthesis in depression. Our findings indicate that TPH biosynthesis, at least at the protein level, is not significantly altered in the DR of depressed suicide victims.     

Short-Term Regulation of Hydroxylase Cofactor Activity in Rat Brain

Differential drug effects on hydroxylase cofactor activities were observed in the corpus striatum and the locus coeruleus when conditions of sacrifice were controlled. A conformational stability-dependent variable degree of stoichiometric coupling between quinonoid dihydropteridine reductase and tyrosine hydroxylase is proposed as a short-latency influence on hydroxylase cofactor levels.     

Tyrosine Hydroxylase Content of Residual Striatal Dopamine Nerve Terminals Following 6-Hydroxydopamine Administration: A Flow Cytometric Study

Fluorescence-activated cell sorting based on immunolabeling with a monoclonal antibody to tyrosine hydroxylase and a fluorescein-conjugated secondary antibody was used to identify striatal synaptosomes derived from nigrostriatal dopamine nerve terminals. The amount of tyrosine hydroxylase immunoreactivity in dopaminergic striatal synaptosomes prepared from control rats was compared to the amount in dopaminergic synaptosomes prepared from rats that had received intraventricular injections of 6-hydroxydopamine. Although the absolute number of dopaminergic synaptosomes was decreased in lesioned animals, those residual dopamine terminals present contained more tyrosine hydroxylase than did dopamine terminals from control rats. Both the decrease in the absolute number of dopamine terminals and the increase in tyrosine hydroxylase immunoreactivity in residual terminals were proportional to the extent of the lesion, as determined by measurement of striatal dopamine levels. These results suggest that an increase in the amount of tyrosine hydroxylase protein in residual terminals may represent one compensatory mechanism by which residual dopamine neurons maintain normal striatal function after partial destruction of the nigrostriatal dopamine projection.     

Bee venom acupoint stimulation increases Fos expression in catecholaminergic neurons in the rat brain

Fos immunocytochemistry was combined with tyrosine hydroxylase (TH) or dopamine-beta-hydroxylase (DBH) immunolabeling to examine brainstem catecholaminergic neuronal activation resulting from bee venom (BV) stimulation of the Zusanli acupoint (ST36) in Sprague-Dawley rats. BV injection into the Zusanli acupoint caused increased Fos expression in catecholaminergic neurons located in the hypothalamic arcuate nucleus (Arc), the dorsal raphe (DR), the A5 cell group (A5) and the locus coeruleus (LC). BV acupoint stimulation significantly increased Fos-TH double-labeled neurons in the Arc, LC and DR. Fos-DBH positive neurons were also increased by BV acupoint stimulation in the LC and A5. In contrast BV stimulation of a non-acupoint only increased Fos expression and Fos-TH double-labeled neurons in the Arc. These data indicate that BV acupoint stimulation activates brainstem catecholaminergic neurons and that this activation underlies BV acupoint-induced antinociception.     

BMPs, FGF8 and Wnts regulate the differentiation of locus coeruleus noradrenergic neuronal precursors

In the present study, we investigated the involvement of rhombomere 1 patterning proteins in the regulation of the major noradrenergic centre of the brain, the locus coeruleus. Primary cultures of rat embryonic day 13.5 locus coeruleus were treated with fibroblast growth factor-8, noggin and members of the bone morphogenetic and Wnt protein families. We show that bone morphogenetic proteins 2, 5 and 7 increase and noggin decreases the number of tyrosine hydroxylase-positive locus coeruleus neurons. Interestingly, from all Wnts expressed in the first rhombomere by embryonic day 12.5 in the mice, we only found expression of wnt5a mRNA in the vicinity of the locus coeruleus. In agreement with this finding, from all Wnts studied in vitro, only Wnt5a increased the number of tyrosine hydroxylase-positive neurons in locus coeruleus cultures. Finally, we also found that fibroblast growth factor-8 increased the number of tyrosine hydroxylase-positive cells in locus coeruleus cultures. Neither of the identified factors affected the survival of tyrosine hydroxylase-positive locus coeruleus noradrenergic neurons or the proliferation of their progenitors or neurogenesis. Instead, our results suggest that these patterning signals of rhombomere 1 may work to promote the differentiation of noradrenergic progenitors at later stages of development.     

Differential and independent manifestation within co-containing neurones of neuropeptide Y and tyrosine hydroxylase during ontogeny of the rat central nervous system

The development of tyrosine hydroxylase- and neuropeptide Y-immunoreactive cell bodies in the foetal rat brain was analyzed immunohistochemically using antibodies raised against tyrosine hydroxylase and neuropeptide Y. Possible co-existence of these two substance within the same neurones was investigated by comparison of adjacent sections.

In the ventral medulla oblongata, neurones containing both neuropeptide Y- and tyrosine hydroxylase-like immunoreactivity were demonstrable in and around the lateral reticular nucleus as early as the 17th day of gestation. The total number and the proportion of cells exhibiting this co-existence increased from this stage up to birth. In the nucleus of the solitary tract in the dorsal medulla oblongata, NPY-immunoreactive cells bodies were first visualized at day 13 of gestation. However, although tyrosine hydroxylase-immunoreactive cells could also be seen within the nucleus at this and later ages, they occupied a different, more caudal and medial part. Consequently, no neurones containing both neuropeptide Y and tyrosine hydroxylase were apparent up to the day of birth. Finally, in the locus coeruleus, tyrosine hydroxylase-immunoreactive neurones were also demonstrable at day 13 of gestation. In this case, however, no neuropeptide Y-immunoreactive somata could be seen in the nucleus until day 21.

The present study indicates that the existence of neuropeptide Y and tyrosine hydroxylase in co-containing neurones is not inextricably linked, and suggests that the factors controlling the synthesis of these two substances are not identical.     

Deficits in the Activation and Phosphorylation of Hippocampal Tyrosine Hydroxylase in the Aged Fischer 344 Rat Following Intraventricular Administration of 6-Hydroxydopamine

Abstract: Tyrosine hydroxylase activity was measured under optimal and suboptimal assay conditions in hippocampal extracts from young (2 month), mature (12 month), and old (24 month) Fischer 344 male rats 72 h after the infusion of 200 µg of the neurotoxin 6-hydroxydopamine or vehicle into the lateral ventricle. The lesion resulted in a 45–55% decrease of tyrosine hydroxylase activity measured under optimal conditions (pH 6.1, 3.0 m M 6-methyl-5,6,7,8-tetrahydropterin) and an ∼35% decrease in the relative concentration of immunoreactive tyrosine hydroxylase. When measured under suboptimal conditions (pH 6.6, 0.7 m M 6-methyl-5,6,7,8-tetrahydropterin), tyrosine hydroxylase activity in 2- and 12-month-old lesioned animals was twice that measured in vehicle-treated animals. However, in the old lesioned animals, tyrosine hydroxylase activity measured under suboptimal conditions was not different from that measured in age-matched vehicle-treated animals. Isoforms of tyrosine hydroxylase were identified on immunoblots after two-dimensional gel electrophoresis using enhanced chemiluminescence. The relative proportion of lower pl isoforms of tyrosine hydroxylase in the 2-month-old lesioned animals was greater than that observed in vehicle-treated controls. In contrast, no difference was seen in the relative proportion of tyrosine hydroxylase isoforms in the 24-month-old lesioned versus control animals. These data indicate that the ability of locus ceruleus neurons to rapidly respond to and compensate for insult is attenuated in 24-month-old Fischer 344 rats due to a deficit in stimulus-evoked enzyme phosphorylation.     

Four Forms of Tyrosine Hydroxylase Are Present in Human Adrenal Medulla

Alternative splicing can result in up to four forms of human tyrosine hydroxylase (HTH) mRNA (mRNAHTH). In the adrenal gland, a major site of catecholamine biosynthesis, the presence of all four mRNAHTH forms is controversial. In the present study, postmortem human adrenal medullary tissue was analyzed for the presence of multiple forms of HTH protein by blot immunolabeling. Electrophoretic transfers were developed with affinity-purified rabbit anti-HTH antibodies raised against peptides that reproduced the unique amino acid sequences predicted by the four mRNAHTH forms. All four of the predicted HTH protein forms were present in the adrenal glands from a diverse sample population.     

Comparative studies on the distribution of protein-o-carboxylmethyltransferase and tyrosine hydroxylase in rat brain by immunocytochemistry

The distribution of protein-O-carboxylmethyltransferase and tyrosine hydroxylase immunoreactivity in brain was compared with the use of highly specific polyclonal antibodies prepared against the native form of each enzyme. Protein-O-carboxylmethyltransferase was found in brain areas rich in catecholamine neurons as identified by tyrosine hydroxylase immunoreactivity. Rabbit anti-protein-O-carboxylmethyltransferase labeled cell bodies in the locus coeruleus, substantia nigra, and paraventricular nucleus whereas rabbit anti-tyrosine hydroxylase prepared against highly purified, native tyrosine hydroxylase from cultured PC12 cells labelled cell bodies in the same brain regions. In addition, the antibody to tyrosine hydroxylase made possible the visualization of very fine cortical processes containing tyrosine hydroxylase and very dense neuronal networks throughout the nigrostriatal pathway. The coincidence of protein-O-carboxylmethyltransferase and tyrosine hydroxylase in catecholamine rich brain areas provide an anatomical basis for the possibility that protein-O-carboxylmethyltransferase could modulate catecholaminergic neurotransmission.     

Dopamine transporter expression distinguishes dopaminergic neurons from other catecholaminergic neurons in the developing zebrafish embryo

To characterize the formation of the dopaminergic system in the developing zebrafish CNS, we cloned cDNAs encoding tyrosine hydroxylase (th), an enzyme in dopamine synthesis, and the dopamine transporter (dat), a membrane transport protein which terminates dopamine action by re-uptake. Dopaminergic neurons are first detected between 18 and 19 h post-fertilization in a cluster of cells in the ventral diencephalon. Subsequently, th and dat detection identifies dopaminergic neurons in the olfactory bulb, the pretectum, the retina and the locus coeruleus. Neurons expressing th but not dat are adrenergic or noradrenergic, and are found in the locus coeruleus, the medulla, the likely analog of the carotid body, and precursors of the enteric and sympathetic nervous system.     

Crucial role of TrkB ligands in the survival and phenotypic differentiation of developing locus coeruleus noradrenergic neurons

The role of glial cell-line derived neurotrophic factor (GDNF) and neurotrophins in the development of locus coeruleus noradrenergic neurons was evaluated. We found that two neurotrophic factors previously reported to prevent the degeneration of lesioned adult central noradrenergic neurons, GDNF and neurotrophin 3 (NT3), do not play significant roles in the prenatal development of locus coeruleus noradrenergic neurons, as demonstrated by: (1) the lack of alterations in double Gdnf/Nt3 null mutant mice; and (2) the lack of survival-promoting effects of GDNF and/or NT3 in rat E13.5 primary cultures. In contrast, null mutant mice for TrkB, the tyrosine kinase receptor for brain-derived neurotrophic factor and neurotrophin 4, displayed a clear loss of locus coeruleus noradrenergic neurons. In accordance with this, treatment of rat E13.5 primary cultures with TrkB ligands prevented the early loss of noradrenergic neurons and maintained their survival for up to 6 days in vitro. Moreover, an additional 5-10-fold increase in the number of tyrosine hydroxylase positive noradrenergic neurons was detected after 12 hours in culture. This second effect of TrkB ligands involved neither proliferation nor survival, because the number of BrdU- or TUNEL-positive noradrenergic neurons did not change and the effect was elicited by delayed administration of either factor. Because TrkB ligands increased the number of tyrosine hydroxylase-positive cells expressing Phox2a, a paired homeodomain protein required for the development of locus coeruleus noradrenergic neurons, but did not affect the number of Phox2a-positive tyrosine hydroxylase-negative cells, our results suggest that the second effect of TrkB ligands may involve promoting or inducing a noradrenergic phenotype. In summary, our findings suggest that, unlike NT3 and GDNF, TrkB ligands are required and sufficient to promote the development of central noradrenergic neurons.     

Reduced Neuropeptide Y Concentrations in Suicide Brain

Neuropeptide Y (NPY) was measured in postmortem brain tissue from victims of suicide and from individuals dying a sudden natural or accidental death (controls). Concentrations of NPY-immunoreactivity were measured by radioimmunoassay in frontal cortex (BA 10), temporal cortex (BA 22), caudate nucleus, and cerebellum. Concentrations of NPY-immunoreactivity were significantly lower in postmortem frontal cortex (-14%) and caudate nucleus (-27%) from suicide victims compared with age-matched controls. A subgroup of suicides with evidence of a history of depression revealed more robust reductions in concentrations of NPY-immunoreactivity in frontal cortex and caudate nucleus, as did four individuals who died from natural causes and also were described as having a possible history of depression. Concentrations of NPY-immunoreactivity in temporal cortex and cerebellum from victims of suicide or from the subgroup of subjects with a possible history of depression were not significantly different from those of age-matched controls. We suggest there is a deficit in the brain NPY system leading to region-specific reductions in peptide concentrations in subjects who have a history of depression.     

Chronic Morphine Administration Increases β-Adrenergic Receptor Kinase (βARK) Levels in the Rat Locus Coeruleus

Abstract: Based on the established role of β-adrenergic receptor kinase (βARK) and β-arrestin in the desensitization of several G protein-coupled receptors, we investigated the effect of chronic morphine administration on βARK and β-arrestin levels in selected brain areas. Levels of βARK were measured by blot immunolabeling analysis using antibodies specific for two known forms of βARK, i.e., βARK1 and βARK2. It was found that chronic morphine treatment produced an ∼35% increase in levels of βARK1 immunoreactivity in the locus coeruleus, but not in several other brain regions studied. In contrast, chronic morphine treatment failed to alter levels of βARK2 immunoreactivity in any of the brain regions studied. Levels of β-arrestin immunoreactivity, measured using an antiserum that recognizes two major forms of this protein in brain, were also found to increase (by ∼20%) in the locus coeruleus. It is proposed that chronic morphine regulation of βARK1 and β-arrestin levels may contribute to opioid-receptor tolerance that is known to occur in this brain region.