Developmental changes of nerve growth factor levels in sympathetic ganglia and their target organs

The predominant source of nerve growth factor (NGF) used by mature sympathetic neurons originates in their target organs (Heumann, R., Korsching, S., Scott, J., and Thoenen, H. (1984), EMBO J. 3, 3183-3189; Korsching, S., and Thoenen, H. (1985), J. Neurosci. 5, 1058-1061). We have determined the NGF content of two sympathetically innervated mouse organs, submandibular gland and heart ventricle, and of sympathetic ganglia from mouse and rat between embryonic Day 12 (E12) and adulthood. NGF levels were measured by a two-site enzyme immunassay (Korsching, S., and Thoenen, H. (1983), Proc. Natl. Acad. Sci. USA 80, 3513-3516). In heart ventricle and submandibular gland, NGF first became detectable around the time of initial innervation by sympathetic neurons (E12 and E13, respectively) and increased respectively 14- and 7-fold in the following 2 days, to reach adult levels already at E14 for heart ventricle (1.4 +/- 0.2 ng NGF/g wet wt). NGF in the superior cervical ganglion (SCG) was first detected at the same time as in its target organ, the submandibular gland. NGF content in the SCG then increased 6-fold during the next 2 days and continued to increase until the end of the third postnatal week, when adult levels were reached. Although the levels of NGF in the adult mouse submandibular gland are sexually dimorphic and six orders of magnitude higher than those in other sympathetic target organs, no sex difference in the NGF content was found in either developing submandibular gland or SCG until the end of the third postnatal week. Moreover, the steep NGF increase observed in the male submandibular gland after postnatal Day 18 (250-fold within the following 3 days and up to the 55,000-fold in the next 7 days) was not reflected in a corresponding increase in the NGF content of the male SCG. These data indicate that, in accordance with earlier findings (see Levi-Montalcini, R., and Angeletti, P. U. (1968), Physiol. Rev. 48, 534-569), SCG neurons do not have access to the large amounts of NGF synthesized during and after adolescence in the mouse submandibular gland. Our results support the concept that initial fiber outgrowth of sympathetic neurons is neither dependent on NGF nor mediated by it. The time course of NGF levels in the SCG is consistent with the concept that sympathetic neurons are provided with NGF by means of retrograde axonal transport from the innervated organs already early in development.     

Inhibition of parasympathetic axonal sprouting in the cat submandibular gland by sympathetic axons

Summary The sprouting of parasympathetic axons into the submandibular sympathetic nerve trunk following sympathetic denervation has been investigated. It was found that a permanent sympathetic denervation was necessary in order for the sprouting to develop and be maintained: if reinnervation by adrenergic nerves was delayed, the sprouting developed but was reduced at longer survival times when the original innervation was reestablished. The evidence for suppression of the cholinergic sprouting by the adrenergic axons is discussed, as is the evidence that these sprouts arise from the submandibular gland.     

Developmental status of sympathetic innervation in relation to calcium accumulation by submandibular gland following reserpine, surgical sympathectomy or cyclocytidine

Sympathectomy (Sx) of the submandibular gland was induced at various postnatal ages by ip administration of a single dose of reserpine or by unilateral excision of a superior cervical ganglion. If animals were 12 days old or less at the time of drug administration, [Ca] of the submandibular gland was not measurably increased 24 hr later; if rats were 14 days of age or older, [Ca] of the gland 24 hr after reserpine injection was nearly double that of untreated controls. Two days after surgical Sx, [Ca] of the denervated submandibular gland was unchanged from that of the innervated member of a pair if animals were less than 14 days of age at the time of denervation; [Ca] was twice that of glands of control rats if animals were older than 14 days of age when the denervation was performed. The anti-tumor agent, cyclocytidine (CC), given daily for 3 days in an ip dose of 500 mg/kg, also caused a two- to threefold increase in [Ca] of the submandibular gland when rats were more than 12 days of age at the time of the initial injection of the drug, but in rats younger than this age, CC caused no change in the [Ca] of the submandibular gland. Present data show that there are age-related differences in the ability of the submandibular gland to accumulate calcium following sympathetic denervation or treatment with a norepinephrine-releasing drug. These differences may be attributed either to incomplete development of calcium transport mechanisms, or incomplete development of the sympathetic innervation before 14 days of age.     

Macrophage populations and cardiac sympathetic denervation during L-NAME-induced hypertension in rats

The rat model of hypertension induced by prolonged treatment with Nomega-nitro-L-arginine methyl ester (L-NAME) has been extensively used. However, the effects on cardiac autonomic innervation are unknown. Here, the cardiac sympathetic innervation is analyzed in parallel with myocardial lesions and leukocyte infiltration during L-NAME (40 mg/Kg body weight/day, orally) treatment. The occurrence of cardiomyocyte hypertrophy, a controversial matter, is also addressed. Degenerating cardiomyocytes and focal inflammation occurred one day after treatment. Inflammatory lesions became gradually more frequent until day 7. At day 14 fibroblast-like cells were outstanding. Interstitial and perivascular connective tissue increased from day 28 on. In the left ventricle, cardiomyocyte hypertrophy occurred only around the damaged area during the first 14 days. After 28 days, it became more widespread. In the right ventricle, the hypertrophic cardiomyocytes were restricted to damaged areas. Significant reduction of the noradrenergic nerve terminals occurred from day 3 to 28. The area occupied by ED1+ (hematogenous) macrophages increased until day 7, and dropped to control levels by day 10. ED2+ (resident) macrophages increased from day 3 to 7 and remained higher than control values up to day 77. Animals receiving both L- NAME and aminoguanidine (AG), an inducible nitric oxide synthase (iNOS) inhibitor (65 mg/Kg body weight/day, orally), showed significant decrease in the nitrite serum levels, sympathetic denervation and macrophage infiltration at day 7. No denervation was detectable at day 14 of double treatment, using subcutaneous AG. Our findings favor a role for ED1+ macrophages and iNOS in the hypertension-induced denervation process.     

Immunocytochemical demonstration of nerve growth factor and histofluorescence of catecholaminergic nerves in the salivary glands of diabetic mice

Summary Nerve growth factor (NGF) was localized in the submandibular, sublingual, and parotid salivary glands of male and female diabetic mice and their normal littermates by immunoperoxidase staining usingp-phenylenediamine-pyrocatechol as a chromogen for the cytochemical demonstration of peroxidase activity. In the normal male submandibular gland, immunoreactive NGF was localized in the apical regions of granular, intercalated and collecting duct cells, while in the normal female submandibular gland, NGF was present throughout the cytoplasm of granular duct cells. The localization of NGF in the diabetic male and female submandibular glands was similar and resembled that of the normal female. NGF immunoreactivity was also observed in the striated duct cells in the sublingual and parotid glands of all four types of mice.The sympathetic innervation of the submandibular glands of normal and diabetic mice was demonstrated using glyoxylic acid-induced histofluorescence. The pattern of sympathetic innervation and the intensity of catecholamine fluorescence was consistently different in the four types of mice. In the normal male submandibular gland the fluorescence was very intense, particularly in nerves adjacent to the granular ducts. In the normal female submandibular gland, the fluorescence was weak, while in the diabetic male and female the fluorescence was moderate.The correlation between the intensity of the immunocytochemical staining for NGF and the catecholamine fluorescence adjacent to the granular ducts suggests a trophic influence of the NGF-containing granular ducts on their sympathetic innervation.     

Nitric oxide synthase immunoreactive nerves in rat and ferret salivary glands, and effects of denervation

Nitric oxide has been implicated in mechanisms mediating nerve-evoked vasodilatory and secretory responses in salivary glands. In the present study, the occurrence and distribution of nitric oxide synthase (NOS)-immunoreactive nerves in ferret and rat salivary glands were investigated using immunocytochemistry with rabbit and sheep NOS antisera, and using NADPH-diaphorase enzyme histochemistry. In the parotid, submandibular and sublingual glands of the rat and the ferret, NOS-immunoreactive varicose terminals encircled acini and arteries of various sizes. In the ferret, collecting ducts were also supplied with NOS-immunoreactive fibres. In the rat, only the granular ducts of the submandibular gland were supplied with such fibres. The NOS-immunoreactive innervation of acinar cells was more abundant in the rat than in the ferret, whereas the opposite was true for the innervation of blood vessels. No NOS immunoreactivity was observed in the vascular endothelium. In both species, NOS-positive ganglionic cell bodies were found in the hilar regions of the submandibular and sublingual glands, whereas none could be detected in the parotid glands. NADPH-diaphorase reactivity had the same neuronal distribution as NOS immunoreactivity and, in addition, NADPH-diaphorase reactivity was expressed in ductal epithelium. Neither sympathetic denervation (by removal of the superior cervical ganglion) nor treatment with the sensory neurotoxin capsaicin reduced the NOS-immunoreactive innervation of the parotid gland. However, parasympathetic denervation (by cutting the auriculo-temporal nerve) caused an almost total disappearance of the NOS-immunoreactive innervation. The present findings provide a morphological background to the suggested role of nitric oxide in parasympathetic secretory and vascular responses of salivary glands. This revised version was published online in November 2006 with corrections to the Cover Date.     

Nitric oxide is involved in the lesions of the peripheral autonomic neurons observed in the acute phase of experimental Trypanosoma cruzi infection

Our aim was to investigate the possible involvement of nitric oxide (NO) in peripheral denervation during the acute phase of murine experimental Trypanosoma cruzi infection. Wistar male rats were infected with the Y strain of T. cruzi. One group of animals was also treated with the NO synthase inhibitor N-nitro-l-arginine. A group of uninfected animals was the control. At the 18th day of infection the animals were sacrificed. Quantification of neurons in the colon and heart and tissue parasitism in the heart was performed. Serum concentration of nitrate was measured and a histochemical technique for assessing NADPH-diaphorase activity in the colon was also performed. The infected animals presented a statistically significant decrease in the number of peripheral neurons in the colon and heart and a 2-fold increase in serum NO(3) concentration compared with controls. The animals treated with N-nitro-l-arginine showed almost an absence of NO(3) concentration in the serum and did not show loss of neurons compared with controls. These treated animals displayed a 15-fold increase in tissue parasitism compared with nontreated infected animals. The NADPH-diaphorase activity was much more intense in the muscle layers of the colon of the infected animals than in those of the controls. Taken together, these data suggest that NO is involved in the peripheral denervation observed in the acute phase of experimental T. cruzi infection.     

Infarction alters both the distribution and noradrenergic properties of cardiac sympathetic neurons

Regional changes occur in the sympathetic innervation of the heart after myocardial infarction (MI), including loss of norepinephrine (NE) uptake and depletion of neuronal NE. This apparent denervation is accompanied by increased cardiac NE spillover. One potential explanation for these apparently contradictory findings is that the sympathetic neurons innervating the heart are exposed to environmental stimuli that alter neuronal function. To understand the changes that occur in the innervation of the heart after MI, immunohistochemical, biochemical, and molecular analyses were carried out in the heart and stellate ganglia of control and MI rats. Immunohistochemistry with panneuronal markers revealed extensive denervation in the left ventricle (LV) below the infarct, but sympathetic nerve fibers were retained in the base of the heart. Western blot analysis revealed that tyrosine hydroxylase (TH) expression (normalized to a panneuronal marker) was increased significantly in the base of the heart and in the stellate ganglia but decreased in the LV below the MI. NE transporter (NET) binding sites, normalized to total protein, were unchanged, except in the LV, where [3H]nisoxetine binding was decreased. TH mRNA was increased significantly in the left and right stellate ganglia after MI, while NET mRNA was not. In the base of the heart, increased TH coupled with no change in NET may explain the increase in extracellular NE observed after MI. Coupled with substantial denervation in the LV, these changes likely contribute to the onset of cardiac arrhythmias.     

Functional and anatomical variability of canine cardiac sympathetic efferent pathways: implications for regional denervation of the left ventricle

To further elucidate the functional anatomy of canine cardiac innervation as well as to assess the feasibility of producing regional left ventricular sympathetic denervation, the chronotropic and (or) regional left ventricular inotropic responses produced by stellate or middle cervical ganglion stimulation were investigated in 22 dogs before and after sectioning of individual major cardiopulmonary or cardiac nerves. Sectioning the right or left subclavian ansae abolished all cardiac responses produced by ipsilateral stellate ganglion stimulation. Sectioning a major sympathetic cardiopulmonary nerve, other than the right interganglionic nerve, usually reduced, but seldom abolished, regional inotropic responses elicited by ipsilateral middle cervical ganglion stimulation. Sectioning the dorsal mediastinal cardiac nerves consistently abolished the left ventricular inotropic responses elicited by right middle cervical ganglion stimulation but minimally affected those elicited by left middle cervical ganglion stimulation. In contrast, cutting the left lateral cardiac nerve decreased the inotropic responses in lateral and posterior left ventricular segments elicited by left middle cervical ganglion stimulation but had little effect on the inotropic responses produced by right middle cervical ganglion stimulation. In addition, the ventral mediastinal cardiac nerve was found to be a significant sympathetic efferent pathway from the left-sided ganglia to the left ventricle. These results indicate that the stellate ganglia project axons to the heart via the subclavian ansae and thus effective sympathetic decentralization can be produced by cutting the subclavian ansae; the right-sided cardiac sympathetic efferent innervation of the left ventricle converges intrapericardially in the dorsal mediastinal cardiac nerves; and the left-sided cardiac sympathetic efferent innervation of the left ventricle diverges to innervate the left ventricle by a number of nerves including the dorsal mediastinal, ventral mediastinal, and left lateral cardiac nerves. Thus consistent denervation of a region of the left ventricle can not be accomplished by sectioning an individual cardiopulmonary or cardiac nerve because of the functional and anatomical variability of the neural components in each nerve, as well as the fact that overlapping regions of the left ventricle are innervated by these different nerves.     

Interleukin-1 increases norepinephrine turnover in the spleen and lung in rats

To clarify effects of interleukin-1 on sympathetic nerve activity, norepinephrine turnover in various organs was assessed in rats after intraperitoneal injection of recombinant human interleukin-1 beta. Interleukin-1 administration increased norepinephrine turnover in the spleen, lung and hypothalamus without appreciable effect in the heart, liver, submandibular gland, thymus, pancreas, brown adipose tissue and medulla oblongata. Similar changes in norepinephrine turnover were also found after the administration of bacterial endotoxin. It was concluded that interleukin-1 activates the sympathetic nerves specifically in the spleen and lung.     

Trypanosoma cruzi: peripheral blood monocytes and heart macrophages in the resistance to acute experimental infection in rats

The contribution of mononuclear phagocytes to host resistance against acute Chagas disease has been studied in vivo in a rat model inoculated with Trypanosoma cruzi, Y strain. Acute T. cruzi infection triggered a dramatic increase (937%) in peripheral blood monocyte number at day 12 of infection. At this point, histological analysis of the heart showed high parasitism and diffuse and a moderate to intense mononuclear inflammatory process. Ultrastructural study revealed a large number of macrophages, in addition to lymphocytes and undiffer entiated cells. Clusters of macrophages exhibited different morphologi cal phenotypes, with evident signs of activation (increase in size, surface rufflings, and amount of cytoplasmic organelles). Cell-to-cell contacts involving macrophages and lymphocytes or macrophages and mono cytes were observed. Depletion of macrophages by treatment with silica, a selective cytotoxic agent for these cells, caused a significant increase in the number of amastigote nests in cardiomyocytes. The present findings indicate that the early phase of infection with T. cruzi induces rapid production, maturation, and activation of the monocyte/macrophage system so as to control T. cruzi replication, emphasizing the crucial role for macrophages in the rat resistance to Chagas disease.     

Enteric Neuronal Damage,Intramuscular Denervation and Smooth Muscle Phenotype Changes as Mechanisms of Chagasic Megacolon: Evidence from a Long-Term Murine Model of Tripanosoma cruzi Infection

We developed a novel murine model of long-term infection with Trypanosoma cruzi with the aim to elucidate the pathogenesis of megacolon and the associated adaptive and neuromuscular intestinal disorders. Our intent was to produce a chronic stage of the disease since the early treatment should avoid 100% mortality of untreated animals at acute phase. Treatment allowed animals to be kept infected and alive in order to develop the chronic phase of infection with low parasitism as in human disease. A group of Swiss mice was infected with the Y strain of T. cruzi. At the 11^th day after infection, a sub-group was euthanized (acute-phase group) and another sub-group was treated with benznidazole and euthanized 15 months after infection (chronic-phase group). Whole colon samples were harvested and used for studying the histopathology of the intestinal smooth muscle and the plasticity of the enteric nerves. In the acute phase, all animals presented inflammatory lesions associated with intense and diffuse parasitism of the muscular and submucosa layers, which were enlarged when compared with the controls. The occurrence of intense degenerative inflammatory changes and increased reticular fibers suggests inflammatory-induced necrosis of muscle cells. In the chronic phase, parasitism was insignificant; however, the architecture of Aüerbach plexuses was focally affected in the inflamed areas, and a significant decrease in the number of neurons and in the density of intramuscular nerve bundles was detected. Other changes observed included increased thickness of the colon wall, diffuse muscle cell hypertrophy, and increased collagen deposition, indicating early fibrosis in the damaged areas. Mast cell count significantly increased in the muscular layers. We propose a model for studying the long-term (15 months) pathogenesis of Chagasic megacolon in mice that mimics the human disease, which persists for several years and has not been fully elucidated. We hypothesize that the long-term inflammatory process mediates neuronal damage and intramuscular and intramural denervation, leading to phenotypic changes in smooth muscle cells associated with fibrosis. These long-term structural changes may represent the basic mechanism for the formation of the Chagasic megacolon.     

Infection of Calomys callosus (Rodentia Cricetidae) with strains of different Trypanosoma cruzi biodemes: pathogenicity, histotropism, and fibrosis induction

The influence of different Trypanosoma cruzi biodemes on the evolution of the infection and on the histopathological lesions of the heart and skeletal muscles, during the experimental infection of Calomys callosus, was investigated. Three groups of C. callosus were infected, respectively, with parasite strains representative of three different Biodemes: Type I (Y strain), Type II (21 SF strain), and Type III (Colombian strain). For each group, normal C. callosus were also used as controls. Marked differences have been detected in the responses of C. callosus to the infection with the three strains in this model. The strains Types I and II (Y and 21 SF) determined moderate lesions, mostly in the myocardium, with low parasitism, a rapid course, and total regression of the lesions by the 60th day of infection. Differently, Type III strain (Colombian), was more pathogenic for C. callosus and induced necrotic-inflammatory lesions in skeletal muscles and myocardium, in correspondence to intracellular parasitism. Proliferation of fibroblasts and amorphous matrix deposits, followed by interstitial fibrosis were present. Progressive regression of the inflammatory changes and collagen deposits occurred spontaneously. The progression and regression of both inflammation and fibrosis induced by the Colombian strain were further submitted to quantitative evaluation by morphometry. Results of the morphometric studies presented good correlation with the histopathological findings. The results confirm the importance of the different biodemes in the determination of tissue lesions and the peculiarities of response of C. callosus to infection with T. cruzi.     

Met5-enkephalin-Arg6-Gly7-Leu8-immunoreactive nerve fibers in the major salivary glands of the rat: evidence for both sympathetic and parasympathetic origin

Summary The localization of the proenkephalin A-derived octapeptide, Met⁵-enkephalin-Arg⁶-Gly⁷-Leu⁸ (MEAGL), was studied in the major salivary glands of Sprague-Dawley and Wistar rats with the indirect immunofluorescence method. MEAGL-immunoreactive nerve fibers were found around the acini, along intra-and interlobular salivary ducts and in close contact with blood vessels. In the parotid and submandibular glands tyrosine hydroxylase (TH) immunoreactivity was observed in nerve fibers around the acini, in association with intra- and interlobular salivary ducts and around blood vessels, while in the sublingual gland TH-immunoreactive nerve fibers were only seen around blood vessels. Parasympathetic neurons in submandibular ganglia contained MEAGL immunoreactivity. Moderate TH immunoreactivity was seen in some neurons of the submandibular ganglia. A subpopulation of sympathetic principal neurons in the superior cervical ganglion were immunoreactive for both MEAGL and TH. In the trigeminal ganglion, no MEAGL-immunoreactive sensory neurons or nerve fibers were observed. Superior cervical ganglionectomies resulted in a complete disappearance of TH-immunoreactive nerve fibers, while MEAGL-immunoreative nerve fibers were still present in the glands. The presence of MEAGL immunoreactivity in neurons of both sympathetic superior cervical ganglia and parasympathetic submandibular ganglia and the results of superior cervical ganglionectomies suggest, that MEAGL-immunoreactive nerve fibers in the major salivary glands of the rat have both sympathetic and parasympathetic origin.     

Blockade of endothelin ET(A)/ET(B) receptors favors a role for endothelin during acute Trypanosoma cruzi infection in rats

Endothelin has been implicated in the pathogenesis of experimental and human Chagas' disease (American trypanosomiasis). In the present study, we tested the effect of bosentan, an antagonist of both ET(A) and ET(B) endothelin receptors, on parasitemia, histopathology (heart and diaphragm), heart levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-10, interferon (IFN)-gamma, CCL2, CCL3 and CCL5, and the serum levels of nitrate/nitrite (NOx). Bosentan treatment was accompanied by a significant increase in parasitemia and tissue parasitism or inflammation. In vehicle-treated rats, Trypanosoma cruzi infection increased the cardiac levels of TNF-alpha, IFN-gamma and IL-10, at day 9 post inoculation, and the TNF-alpha remained elevated until day 13. The infection also caused a significant increase in the cardiac levels of the chemokines CCL2 (9, 13 and 18 days) and CCL3 (13 and 18 days). Bosentan-treatment had no significant effect on the infection-associated increase in IFN-gamma and chemokine concentrations. There was a lower increase in IL-10 at day 9 and this was mirrored by a greater increase of TNF-alpha at day 13, in comparison with vehicle-treated rats. These latter findings correlated well with the enhanced inflammatory process in hearts of bosentan-treated infected rats. Bosentan treatment reduced the infection-associated increase in NOx serum concentration. Altogether, our data suggest that ET action on ET(A) and ET(B) receptors may play a role in the initial control of T. cruzi infection in rats probably by interfering in NO production.     

Effects of heterologous cross-suture between the postganglionic sympathetic and the preganglionic parasympathetic nerve trunks of submandibular glands in cats

Summary After sectioning the postganglionic adrenergic sympathetic nerve trunk for the submandibular gland, as close to the submandibular artery as practicable, its central end was sutured to the peripheral end of the preganglionic cholinergic parasympathetic nerve trunk for the gland, the chorda, which had been sectioned where it left the lingual nerve. The effects of this heterologous cross-sature were studied at different times, up to 1 year afterwards, by assessing the physiological and pharmacological responses of the glands and the neuro-histochemical changes in the nerve trunks and in the nerves within the glands.In all cases adrenergic sympathetic nerves grew across the site of suture and down the erstwhile cholinergic parasympathetic trunk, eventually to develop connections in the gland. In some cases the functional adrenergic reinnervation of the submandibular gland appeared to result exclusively or predominantly from the direct downgrowth of adrenergic axons to the gland, via the crossed nerves. In other cases however, in addition to a direct glandular reinnervation, there was some physiological and morphological evidence which suggested that possible heterogenous synaptic contacts may have been created between postganglionic sympathetic axons and cholinergic ganglion cells in the chorda nerve.This work was supported by a grant from the Joint Research Committee, King's College Hospital.     

Ontogenesis of Monoamine-Synthesizing Enzyme Activities and Biopterin Levels in Rat Brain or Salivary Glands, and the Effect of Thyroxine Administration

Neonatal changes in the activities of tyrosine hydroxylase (TH) and tryptophan hydroxylase (TrpH) and in the content of the co-factor, biopterin, were studied in rat midbrain for the first 20 days after birth. Changes in TH activity in the parotid and submandibular glands were also examined. Changes in TH activity per unit weight in the developing rat brain were briefly similar to those in the salivary glands; the activity increased from day 2 or 4 to day 9 after birth, and remained constant or slightly decreased at day 12, then rapidly increased on day 16. TrpH activity in the midbrain increased about twofold up to day 16. The biopterin concentration in the brain increased, reached a maximum level on day 12 after birth, and thereafter decreased. The effect of hyperthyroidism in rats given 0.2 mg/kg i.p. of thyroxine every 2 days postnatally was studied on the activity of TH in rat salivary glands at 12-day-old rats. In parotid or submandibular gland of hyperthyroid rats, TH activity increased at day 12 postnatally. In comparison with the effect on TH activity in the salivary glands, TH activity in the midbrain on day 20 postnatally was not induced by hyperthyroidism. Furthermore, increase of the TrpH activity and biopterin and catecholamine levels in the midbrain of hyperthyroid rats was not found on day 20 after birth in comparison with the corresponding controls. From these data, we suppose that postnatal hyperthyroidism may cause precocious induction of TH in rat salivary gland, but may not increase the activity of TH or TrpH, and the level of their co-factor, biopterin, in rat midbrain.