This abstract sent to supervisor 5/4/12 Tyrosine hydroxylase is the enzyme which controls the ratelimiting step in the synthesis of noradrenaline. In order to discover whether the activity of tyrosine hydroxylase might serve as an indicator of noradrenergic function in the brain, two preparations for the direct measurement of TH activity in rat brain regions by a tritium-release method were employed: synaptosomal suspensions prepared from pinchedoff nerve terminals, and partially solubilised enzyme preparations prepared from frozen homogenates or synaptosomal suspensions and assayed at saturating concentrations of cofactor and tyrosine. There was evidence of an increase in tyrosine hydroxylase activity in hippocampal synaptosomes of rats killed immediately after a mild electrical footshock. Activation of synaptosomal enzyme activity was also found after single doses of clonidine and parachloroamphetamine, and after repeated handling; and single doses of morphine and of yohimbine appeared to lower tyrosine hydroxylase activity. Repeated administration of drugs such as clonidine, desipramine and 2-deoxyglucose, however, did not affect tyrosine hydroxylation rate. A preliminary finding , suggesting differences in synaptosomal tyrosine hydroxylase activity related to experience with different reinforcement schedules (continuous reward vs. partial reward) in a runway experiment, was not substantiated in later experiments; nor was there any difference between the synaptosomal tyrosine hydroxylase activity of naive controls and rats given repeated daily shocks for a week. The saturated TH assay performed on solubilised enzyme was, as predicted, unresponsive to the short term stimulation effects detected with the synaptosomal assay. However, other changes, such as a reduced maximal hydroxylation rate after repeated desipramine administration, and an increased rate several weeks after a course of electrical stimulation of the septal area, were established with the saturated assay. Although the changes in stimulated rats were associated with increased behavioural tolerance to stress, e.g. resistance to extinction of a running response in a runway, other experiments in which the behavioural stress-tolerance was induced by behavioural methods alone showed no accompanying changes in TH activity. Measures of synaptosomal and saturated soluble TH activity appear to constitute independent indicators of noradrenergic function. It seems that synaptosomal tyrosine hydroxylase activity is not, as anticipated, controlled by the firing rate of locus coeruleus neurones; but it may be subject to local regulation in noradrenergic terminals. The results are discussed in the context of theoretical aspects of the regulation of noradrenaline synthesis in the brain, and the mechanisms underlying physiological responses to stress and behavioural tolerance to stress.

Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

Because endorphin receptor activation alters the function of the central noradrenergic system, opiates may change the regenerative sprouting of neurons in response to adrenergic neurotoxins. To test this hypothesis, newborn rats were treated with several opioids and 6-hydroxydopa (6-OHDOPA) and the development of the noradrenergic system was evaluated. In combination with 6-OHDOPA morphine and naloxone potentiated the development of norepinephrine (NE) levels in the pons-medulla and cerebellum by four weeks of age. β-Endorphin, Leu- and Met-enkephalin and d-Ala2-enkephalinamide produced a similar effect in the pons-medulla. The effect of morphine was partially attenuated by naloxone. Increased cerebellar noradrenergic histofluorescent staining was observed with the morphine + 6-OHDOPA and naloxone + 6-OHDOPA treatments. Both naloxone and morphine decreased NE levels in the pons-medulla of adult rats treated with 6-OHDOPA. These results suggest that opiates and endorphins may enhance sprouting of noradrenergic neurons following neonatal treatment with 6-OHDOPA, by increasing the toxicity of this neurotoxin.

No description available.

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 β-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.

In order to demonstrate the influence of morphine on the developmental localization of regenerated noradrenergic fibers in rat cerebellum, a glyoxylic histofluorescent method and radiometric assay for norepinephrine (NE) were utilized. An initial reduction of NE in the cerebellum after 6-hydroxydopa [6-OHDOPA; 60 µg/g intraperitoneally (i.p.)] was followed by a return to control levels at 3 days, and an elevation above control levels at 7 days. The initial rates of recovery of NE in the cerebellum of the 6-OHDOPA group of rats and the group receiving morphine (20 µg/g i.p.) in combination with 6-OHDOPA were identical up to 7 days. However, by 14 days NE content was further elevated in the cerebellum of the morphine+6-OHDOPA group. Histofluorescent microscopic observations of the cerebellar cortex correlated with the biochemical findings. A reduction in cerebellar NE content at 3 days was associated with a reduction in the number of visible histofluorescent fibers in the cerebellar cortex. By 7 days the relative number of fibers in the 6-OHDOPA groups was similar to that seen in the control group, but by 9 days the relative number of fluorescent fibers in the cerebellar cortex was increased above control. By 13 days there was a further increase in the relative number of fluorescent fibers in the cerebellar cortex of the morphine+6-OHDOPA group, as compared to the group treated with 6-OHDOPA alone. These findings provide an anatomic correlate for recovery of noradrenergic fibers after 6-OHDOPA, and demonstrate an action of morphine in enhancing regenerative sprouting.

The amino acid, 6-hydroxydopa (6-OHDOPA), found at the active site of amine oxidases, exists as a keto-enol. Exogenously administered 6-OHDOPA is an excitotoxin like β-N-oxalylamino-L-alanine (BOAA) and β-N-methylamino-L-alanine (BMAA), acting at the non-N-methyl-D-aspartate (non-NMDA) α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor. BMAA and BOAA are causal factors of neurolathyrism in humans. Much exogenously administered 6-OHDOPA is biotransformed by aminoacid decarboxylase (AADC) to the highly potent and catecholamine-(CA) selective neurotoxin, 6-hydroxydopamine (6-OHDA). 6-OHDOPA destroys locus coeruleus noradrenergic perikarya and produces associated denervation of brain by norepinephrine-(NE) containing fibers. Opiopeptides and opioids enhance neurotoxic effects of 6-OHDOPA on noradrenergic nerves, by a naloxone-reversible process. An understanding of mechanisms underlying neurotoxic effects of 6-OHDOPA can be helpful in defining actions of known and newfound amino acids and for investigating their potential neurotoxic properties.

Degeneration of the noradrenergic neurons has been reported in the brain of patients suffering from neurodegenerative diseases. However, their pathological characteristics during the neurodegenerative course and underlying mechanisms remain to be elucidated. In the present study, we used the neurotoxin camptothecin (CPT) to induce the DNA damage response in neuroblastoma SH-SY5Y cells, normal fibroblast cells, and primarily cultured locus coeruleus (LC) and raphe neurons to examine cellular responses and repair capabilities after neurotoxin exposure. To our knowledge, the present study is the first to show that noradrenergic SH-SY5Y cells are more sensitive to CPT-induced DNA damage and deficient in DNA repair, as compared to fibroblast cells. Furthermore, similar to SH-SY5Y cells, primarily cultured LC neurons are more sensitive to CPT-induced DNA damage and show a deficiency in repairing this damage. Moreover, while N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) exposure also results in DNA damage in cultured LC neurons, neither CPT nor DSP4 induce DNA damage in neuronal cultures from the raphe nuclei. Taken together, noradrenergic SH-SY5Y cells and LC neurons are sensitive to CPT-induced DNA damage and exhibit a repair deficiency, providing a mechanistic explanation for the pathological characteristics of LC degeneration when facing endogenous and environmental DNA-damaging insults in vivo.

Distribution of prolactin-releasing peptide-like immunoreactivity (PrRP- LI) was investigated in the rat medulla with the use of a rabbit polyclonal antiserum against the human PrRP-31 peptide. PrRP-positive neurons were noted mainly in two areas of the caudal medulla: ventrolateral reticular formation and commissural nucleus of the nucleus of the solitary tract (NTS), corresponding to the A1 and A2 areas. PrRP-LI neurons were absent in the medulla rostral to the area postrema. Double-labeling the sections with PrRP antisera and tyrosine hydroxylase (TH) monoclonal antibodies revealed extensive colocalization of PrRP- and TH-like immunoreactivity (TH-LI) in neurons of the A1 and A2 areas. Our results show that PrRP-LI is expressed in a population of A1 and A2 noradrenergic neurons of the rat caudal medulla.

In order to attain a better understanding of the sprouting response of noradrenergic fibers in the central nervous system (CNS), noradrenergic innervation to the cerebellum was observed by the glyoxylic acid method after a variety of manipulations and in a genetic variant of mouse classified as "Purkinje cell degeneration" (pcd/pcd). It has been found that a midbrain lesion in rats at birth will result in a collateral sprouting response of noradrenergic fibers in the cerebellum at 8 weeks, as indicated by the increased number of histofluorescent fibers observed in the molecular layer of the cerebellar cortex. Another procedure, treatment of neonatal rats with nerve growth factor alone appears to produce a temporary stimulation of noradrenergic fiber growth in the cerebellum, as observed by the histofluorescent method, although the innervation at 6 weeks or later is ultimately unchanged from the control group. In contrast, NGF (500 units) given to rats in combination with 6-hydroxydopa (6-OHDOPA) (60 μg/g IP) at 3 days postbirth produces a hyperinnervation of the cerebellum by noradrenergic fibers by 2 weeks of age and until at least 8 weeks of age. A third procedure, locus coeruleus implantation, was generally unsuccessful using the procedures described, since the implant was usually non-viable after several days. In a few instances where histofluorescent nuclei were found within the implant, there was an abundance of histofluorescent fibers within and adjacent to the implant, with fibers appearing to grow into host cerebellum. In the final procedure, it was noted that the density of noradrenergic input to the molecular layer of the cerebellar cortex was markedly increased in a genetic mutant mouse, classified as "Purkinje cell degeneration" (pcd/pcd), which is characterized by the absence of Purkinje cells of the cerebellum in adulthood. However, because of the tissue shrinkage that occurs after loss of Purkinje cells during postnatal development, it is unclear as to whether this observation represents hyperinnervation or a normal complement of fibers in a smaller brain space. The above procedures demonstrate the plasticity of noradrenergic fibers in neonatal cerebellum, a brain region that undergoes considerable postnatal development. The cerebellum is thought to be a good site for studying development/ regeneration/sprouting of noradrenergic fibers in particular, and central axonal processes in general.