Dong H. Park

Chemical and structural analysis of the relation between cortical inputs and tyrosine hydroxylase-containing terminals in rat neostriatum

Levels of tyrosine hydroxylase (TH) and the ultrastructural relation between axons from cerebral cortex and TH containing, predominantly dopaminergic terminals were examined in the adult rat neostriatum at 2 and 12 days following unilateral decortication. The caudate nuclei from the unlesioned and lesioned hemispheres were biochemically assayed for TH processed for light or electron microscopic localization of the enzyme. At both time intervals examined, there was no statistically significant alteration in TH activity or apparent change in the intensity of reactive labeling visualized by light microscopy. However, electron microscopic examination of the caudate nucleus homolateral to the decortication at two days following surgery revealed the presence of numerous small, osmiophilic boutons which were much less frequently seen on the contralateral side. Further ultrastructural examination showed that the osmiophilic boutons formed predominantly asymmetric, axodendritic synapses. In sections containing both degenerating and TH labeled terminals, two patterns of connectivity could be discovered. First and most commonly, the degenerating and TH-labeled terminals formed synapses with the same dendrite or dendritic spine. Less frequently, the two types of terminals were in direct contact with each other. In this axo-axonic relation, the outer membranes between the terminals were in apposition but usually failed to exhibit pre- or postsynaptic specializations. These findings indicate that the cortical and dopaminergic nigral efferents have actions on common recipient neurons in the rat caudate nucleus and provide support for a possible direct axonal interrelationship between these two primary inputs.

Aromaticl-amino acid decar☐ylase in the rat brain: Immunocytochemical localization in neurons of the brain stem

Neurons containing the enzyme aromatic L-amino acid decarboxylase were immunocytochemically localized in the brain stem of the rat. The enzyme occurred as expected in previously well characterized monoaminergic cell groups, and in addition in some nuclei with unknown neurotransmitters. Major aggregates of neurons that were immunoreactive for aromatic L-amino acid decarboxylase but contained neither tyrosine hydroxylase nor serotonin, were found in the pretectal nuclei, the lateral parabrachial nucleus, and the dorsolateral subdivision of the nucleus tractus solitarius. Aromatic L-amino acid decarboxylase was also present in serotonin neurons and the majority of catecholamine cell groups. Dopamine, noradrenaline, and adrenaline cells exhibited characteristic staining intensities to anti-aromatic L-amino acid decarboxylase reflective of relative enzyme levels in the different groups. Some cells in the dorsal motor nucleus of the vagus that were previously classified as dopaminergic lacked immunoreactivity to aromatic L-amino acid decarboxylase.

Adrenaline synthesizing neurons in the rostral ventrolateral medulla: a possible role in tonic vasomotor control

Adrenaline-containing neurons of the rostral ventrolateral medulla (the C1 group) project selectively to autonomic spinal neurons in rats. Stimulation of these neurons electrically or chemically elevates arterial pressure, while neuronal blockade by microinjection of tetrodotoxin bilaterally drops arterial pressure to levels comparable to those produced by spinal cord transection. Adrenaline neurons of the ventral medulla appear necessary for maintaining normal levels of blood pressure, and thus may constitute a tonic vasomotor center.

Phenylethanolamine N-methyltransferase-containing neurons in the rostral ventrolateral medulla of the rat. I. Normal ultrastructure

The electron microscopic localization of the adrenaline-synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT) was examined in the rostral ventrolateral medulla (RVL) of adult rats. The brains were fixed by perfusion with 3.75% acrolein and 2.0% paraformaldehyde in phosphate buffer. Coronal Vibratome sections through the RVL were immunocytochemically labeled using a rabbit polyclonal antiserum to PNMT and the peroxidase-antiperoxidase method. A semi-quantitative ultrastructure analysis revealed that the perikarya constituted 9% of the total immunoreactive profiles observed in the RVL. The labeled somata were large (18-24 microns) and were characterized by an indented nucleus and abundant cytoplasm with numerous mitochondria. An average of 136.8 +/- 11.6 mitochondria were present per 100 microns2 cytoplasm, which is 38% greater than the numbers found for PNMT-immunoreactive neurons in the nucleus of the solitary tract. Moreover, the labeled somata were often found in direct apposition to the basal lamina of small capillaries and neighboring astrocytic processes. The remaining labeled profiles were neuronal processes of which 72% were dendrites. Both the PNMT-labeled somata and dendrites received primarily symmetric contacts from unlabeled axon terminals. Only a few axons and terminals containing immunoreactivity for PNMT were observed. The axons were both unmyelinated and myelinated. The PNMT-immunoreactive terminals were characterized by a mixed population of vesicles and by the formation of synaptic junctions with both unlabeled dendrites and PNMT-labeled perikarya and dendrites. The ultrastructural morphology and proximity to blood vessels and glia suggest a high metabolic activity and possibly a chemosensory function of PNMT neurons in the RVL. The existence of myelinated and unmyelinated axons could imply that PNMT-containing neurons have different conduction velocities in efferent pathways to the spinal cord or other brain regions. Furthermore, the multiple types of synaptic interactions between labeled and unlabeled axons and dendrites support the concept that adrenergic neurons modulate and are modulated by neurons containing the same or other putative transmitters in the RVL.

A new group of neurons in hypothalamus containing phenylethanolamine N-methyltransferase (PNMT) but not tyrosine hydroxylase

Intraventricular injection of colchicine in rat results in the appearance within hypothalamus of numerous neurons containing the adrenaline-synthesizing enzyme, phenylethanolamine N-methyltransferase, but not the other catecholamine biosynthetic enzymes. Increased PNMT staining in hypothalamus was paralleled by an increase in PNMT activity measured in micropunch preparations. Immunotitration demonstrated that this increase was due to accumulation of specific enzyme protein. The finding that hypothalamic neurons express PNMT without tyrosine hydroxylase suggests that such neurons may produce methylated amines other than adrenaline.

Molecular cloning and characterization of cDNA encoding tryptophan hydroxylase from rat central serotonergic neurons

Tryptophan hydroxylase (TPH) from central serotonergic neurons in the dorsal raphe nucleus (DRN) and that from the endocrine pineal gland (PG) have been shown to exhibit difference biochemical characteristics. We further report here that the isoelectric point determined by chromatofocusing differs between TPH from the rat brainstem and PG. In addition, the levels of TPH mRNA are much greater in the PG than the DRN despite a higher enzymatic activity in the DRN. These data raise the question as to whether different forms of TPH may exist in the DRN and the PG. To address this question, we amplified TPH cDNAs by the polymerase chain reaction (PCR) using poly(A)+ RNA purified from both tissues. Several combinations of oligonucleotide primers encompassing different regions of the published coding sequence of rat pineal TPH were employed for this purpose. Subsequent analysis by gel electrophoresis and Southern blotting of PCR products indicated that DNA fragments of identical length were amplified from both sources. Furthermore, the nucleotide sequences of three independent subclones containing the putative full-length coding region of DRN TPH were determined and found to be identical to that of PG. In situ hybridization using the amplified cDNA as a probe demonstrated specific labeling within the DRN of the rat brain. These data support the hypothesis that tissue-specific differences in TPH characteristics result from differential post-translational events and clearly indicate that a TPH mRNA transcript identical in coding sequence to the PG form is expressed in the DRN.

Phenylethanolamine N-methyltransferase-containing neurons in the rostral ventrolateral medulla. II: Synaptic relationships with GABAergic terminals

The ultrastructural morphology of terminals synthesizing gamma-aminobutyric acid (GABA), as indicated by peroxidase immunoreactivity for its synthetic enzyme L-glutamate decarboxylase (GAD), was examined in the rostral ventrolateral medulla (RVL) of the adult rat brain. The objective of the study was to determine the types of synaptic associations between the GABAergic terminals and other neurons in the RVL, particularly the C1-adrenergic neurons containing phenylethanolamine N-methyltransferase (PNMT). The brains were fixed by perfusion with 3.75% acrolein and 2.0% paraformaldehyde in phosphate buffer. Coronal Vibratome sections through the RVL were singly labeled with a sheep antiserum to GAD using the peroxidase-antiperoxidase (PAP) method. Additional sections were dually labeled using the PAP technique for the GAD antiserum and immunogold labeling for a rabbit antiserum against PNMT. Ultrastructural analysis revealed that peroxidase labeling for GAD was localized primarily to axons and axon terminals in both single and dual labeled material. The axons were small and unmyelinated. The GAD-labeled terminals were 0.5-2.0 microns in diameter and contained a large population of small clear vesicles usually associated with a few mitochondria. These terminals formed synapses with many dendrites, a few nerve cell bodies and axon terminals. The junctions were all symmetric and the postsynaptic structures failed to exhibit immunoreactivity when processed only for GAD labeling. In sections incubated with both GAD and PNMT antisera, the peroxidase-labeled GABAergic terminals formed symmetric synapses with nerve cell bodies and dendrites showing immunogold labeling for PNMT. In addition, the GAD-labeled terminals were presynaptic to other dendrites which appeared to have equal access to the antisera and gold markers, but failed to exhibit detectable immunoreactivity for PNMT. Both the PNMT-labeled and unlabeled somata and dendrites also received symmetric and asymmetric contacts from terminals containing neither GAD nor PNMT-immunoreactivity. We conclude that GABA is at least one of the inhibitory transmitters regulating adrenergic as well as non-adrenergic outflow from the RVL.

Cholinergic neurons of the chick ciliary ganglia express adrenergic traits in vivo and in vitro

In this study, we sought to determine whether neurons of the chick embryo ciliary ganglia (CG), a parasympathetic cholinergic ganglia, can express catecholaminergic (CA) traits. To accomplish this, we used immunocytochemical techniques to examine the presence of the CA enzymes tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) in CGs removed from chick embryo at day 8 of development (E8). Few neurons containing TH but not PNMT were found in the E8 CG. To examine whether CG neurons express CA enzymes in vitro, CGs removed from E8 chick embryo were dissociated and kept in culture for 3 to 12 days. In 50% of the culture dishes, some neurons contain TH or PNMT after 5 days in vitro. In an equal proportion of culture plates, CG neurons did not express the enzymes. To determine whether the proportion of CG neurons expressing TH or PNMT is increased by tissue influences, ganglion cells were co-cultured with notochord. In 90% of the co-culture experiments, most neurons present in the culture dishes stained with TH or PNMT after 5 days in vitro. To test for the presence of aromatic L-amino acid decarboxylase (AADC), another CA enzyme, cultures of CGs and CGs plus notochord were incubated with levodopa and processed for the detection of CA histofluorescence. Dopamine histofluorescence was present in all neurons after 3 days in vitro irrespective of the presence of notochord, suggesting that the expressions of TH and PNMT and that of AADC are differentially regulated. This study, therefore, demonstrates that cholinergic neurons of the CG contain CA enzymes in vivo and in vitro and that the proportion of neurons expressing CA traits during development in vitro can be increased by environmental cues such as those released by the notochord.

Parallel Up‐Regulation of Catecholamine Biosynthetic Enzymes by Dexamethasone in PC12 Cells

Abstract: We sought to investigate whether dexamethasone produces a coordinated, time‐dependent effect on all enzymes in the catecholamine biosynthetic pathway in PC12 cells. The levels of mRNAs of tyrosine hydroxylase (TH), aromatic L‐amino acid decarboxylase (AADC), and dopamine γ‐hydroxylase (DBH) were examined at 0, 6, 12, 24, and 48 h after dexamethasone (5 μM) treatment to PC12 cells. The levels of all enzyme mRNAs steadily increased for 24 h, although the increase of AADC mRNA content was slow. The increased mRNA levels of TH and AADC were maintained at 48 h, whereas the level of DBH mRNA was sharply decreased at 48 h. The maximally induced mRNA levels were ∼5.0‐, 2.4‐, and 7.0‐fold higher than the control levels of TH, AADC, and DBH, respectively. The elevation of enzyme activities was detected later than the increase in levels of mRNAs. The maximal activities of TH, AADC, and DBH were reached between 48 and 72 h with 3.6‐, 1.8‐, and 8.0‐fold increases, respectively. Low, but detectable, phenylethanolamine N‐methyltransferase (PNMT) activity was observed in PC12 cells, and dexamethasone increased its activity 5.6‐fold at 72 h. The PNMT mRNA was easily detected by northern blot analysis after exposure for 24 h to dexamethasone. The data suggest that, in PC12 cells, dexamethasone up‐regulates all catecholamine biosynthetic enzyme genes in a parallel fashion.

Different Forms of Adrenal Phenylethanolamine N-Methyltransferase: Species-Specific Posttranslational Modification

Abstract: Phenylethanolamine N‐methyltransferase was purified from rat and cow adrenal glands. The enzymes from the two species have the same molecular weight of 31,000, but differ in electrophoretic mobility. During polyacrylamide gel electrophoresis, the rat form migrates faster than the bovine form. Antibodies to bovine enzyme precipitated equally well the rat and cow form of the enzyme, but antibodies against rat enzyme precipitated poorly the bovine form. In contrast, both antibodies recognized a similar protein in the in vitro translation products of poly(A+)mRNA isolated from cow adrenal glands. The results suggest that the primary protein structure of rat and bovine enzyme is similar and that differences in electrophoretic mobility are due to posttranslational modification of the enzyme molecule.