Catecholamine Histofluorescence Research Articles

Splenic 6‐hydroxydopamine Worsens Renal Inflammation and Injury in a Murine Model of Systemic Lupus Erythematosus

Renal inflammation is prevalent in the chronic autoimmune disease systemic lupus erythematosus (SLE) and drives the progression of kidney injury, which contributes to the morbidity and mortality. Inflammation in SLE results from a loss of self‐tolerance and the production of autoantibodies against nuclear antigens (e.g., anti‐double‐stranded (ds) DNA autoantibodies); however, other immunoregulatory mechanisms such as the cholinergic anti‐inflammatory pathway may also be dysregulated and contribute to aberrant systemic and renal inflammation in the disease. The cholinergic anti‐inflammatory pathway is a protective, neuroimmune mechanism thought to involve neurotransmission between the parasympathetic vagus and the sympathetic splenic nerves. Although a direct anatomical connection between the vagus and the splenic nerve is controversial, stimulation of these nerves results in a decrease in the production and release of pro‐inflammatory cytokines from splenic immune cells. In order to confirm the importance of the splenic nerve in this pathway, others have used 6‐hydroxydopamine (6OHDA), a neurotoxin that depletes catecholamines, to chemically denervate the spleen and thereby dampen the sympathetic component of the cholinergic anti‐inflammatory pathway. We hypothesized that splenic injection of 6OHDA would further disrupt the cholinergic anti‐inflammatory pathway in a mouse model of SLE and result in increased renal inflammation and worsened disease severity, which would highlight the importance of the splenic nerve in quelling renal inflammation. In the current study, female SLE and control mice were injected with 6OHDA (120 μg in 60 μl saline) or vehicle directly into the spleen at 33 weeks of age (n = 6/group). To confirm splenic denervation with 6OHDA, we utilized the glyoxylic acid condensation reaction on 12 μm spleen sections of representative animals and verified that catecholamine histofluorescence was diminished in 6OHDA‐treated mice (205 vs. 111 histofluorescent foci). Renal cortical TNF‐α (normalized to total protein) was increased in SLE mice compared to controls (3.1e6 ± 1.2e5 vs. 7.8e5 ± 1.6e4 intensity units; p = 0.029), and 6OHDA exacerbated this pro‐inflammatory cytokine in SLE mice (7.6e6 ± 2.4 intensity units; p = 0.048) with no effect in controls (1.8e6 ± 7.0e5 intensity units; p = 0.697). Anti‐dsDNA autoantibodies were elevated in SLE mice compared to controls (2.1e4 ± 5.7e3 vs. 1.0e4 ± 4.7e3 activity units; p = 0.013), but 6OHDA did not alter this measure of disease severity in SLE mice (1.8e4 ± 5.2e3 activity units; p = 0.692). Albumin excretion rate (AER) was elevated in SLE mice compared to controls (160.7 ± 28.0 vs. 2.7 ± 2.7 mg/dL; p < 0.001). 6OHDA accentuated the increase in AER in SLE mice (454.2 ± 150.2 mg/dL; p = 0.010) but had no effect in controls (27.5 ± 17.0 mg/dL; p = 0.754). In summary, 6OHDA aggravated renal inflammation and injury in SLE mice indicated by heightened renal cortical TNF‐α and AER. This suggests that chemical splenic denervation may disrupt endogenous, sympathetically‐mediated anti‐inflammatory mechanisms. Further studies are needed to confirm the role of the splenic nerve in regulating renal inflammation.Support or Funding InformationFunded by AHA grants 14SDG18320033 (KWM), 16PRE29910012 (GSP), and UNTHSC Intramural FundingThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Development of the nervous system in Platynereis dumerilii (Nereididae, Annelida)

BackgroundThe structure and development of the nervous system in Lophotrochozoa has long been recognized as one of the most important subjects for phylogenetic and evolutionary discussion. Many recent papers have presented comprehensive data on the structure and development of catecholaminergic, serotonergic and FMRFamidergic parts of the nervous system. However, relatively few papers contain detailed descriptions of the nervous system in Annelida, one of the largest taxa of Lophotrochozoa. The polychaete species Platynereis dumerilii has recently become one of the more popular model animals in evolutionary and developmental biology. The goal of the present study was to provide a detailed description of its neuronal development. The data obtained will contribute to a better understanding of the basic features of neuronal development in polychaetes.ResultsWe have studied the development of the nervous system in P. dumerilii utilizing histo- and immunochemical labelling of catecholamines, serotonin, FMRFamide related peptides, and acetylated tubulin. The first neuron differentiates at the posterior extremity of the protrochophore, reacts to the antibodies against both serotonin and FMRFamide. Then its fibres run forwards along the ventral side. Soon, more neurons appear at the apical extreme, and their basal neurites form the basel structure of the developing brain (cerebral neuropil and circumesophageal connectives). Initial development of the nervous system starts in two rudiments: anterior and posterior. At the nectochaete stage, segmental ganglia start to differentiate in the anterior-to-posterior direction, and the first structures of the stomatogastric and peripheral nervous system appear. All connectives including the unpaired ventral cord develop from initially paired nerves.ConclusionsWe present a detailed description of Platynereis dumerilii neuronal development based on anti-acetylated tubulin, serotonin, and FMRFamide-like immunostaining as well as catecholamine histofluorescence. The development of the nervous system starts from peripheral pioneer neurons at both the posterior and anterior poles of the larva, and their neurites form a scaffold upon which the adult central nervous system develops. The anterior-to-posterior mode of the ventral ganglia development challenges the primary heteronomy concept. Comparison with the development of Mollusca reveals substantial similarities with early neuronal development in larval Solenogastres.

Early Postnatal Administration of Growth Hormone Increases Tuberoinfundibular Dopaminergic Neuron Numbers in Ames Dwarf Mice

Hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons secrete dopamine, which inhibits pituitary prolactin (PRL) secretion. PRL has demonstrated neurotrophic effects on TIDA neuron development in PRL-, GH-, and TSH-deficient Ames (df/df) and Snell (dw/dw) dwarf mice. However, both PRL and PRL receptor knockout mice exhibit normal-sized TIDA neuron numbers, implying GH and/or TSH influence TIDA neuron development. The current study investigated the effect of porcine (p) GH on TIDA neuron development in Ames dwarf hypothalamus. Normal (DF/df) and dwarf mice were treated daily with pGH or saline beginning at 3 d of age for a period of 42 d. After treatment, brains were analyzed using catecholamine histofluorescence, tyrosine hydroxylase immunocytochemistry, and bromodeoxyuridine (BrdU) immunocytochemistry to detect BrdU incorporation. DF/df males and df/df treated with pGH experienced increased (P </= 0.01) weight gain compared with those treated with saline. DF/df had greater (P </= 0.01) TIDA neuron numbers than df/df, regardless of treatment. TIDA neuron number in pGH-treated df/df was greater (P </= 0.01) than in saline-treated df/df. Zona incerta and periventricular dopamine neurons were not affected by treatment or genotype. There was no effect of genotype or treatment on BrdU incorporation in the arcuate nucleus, median eminence, or periventricular region surrounding the third ventricle. Saline-treated df/df experienced decreased (P </= 0.05) dentate gyrus BrdU incorporation compared with saline-treated DF/df. In the lateral ventricle, pGH-treated males had greater BrdU immunoreactivity than pGH-treated females. The results show an effect of pGH on TIDA neuron development, although this effect is less potent than that of PRL, and likely GH-induced preservation of TIDA neurons rather than generation of new TIDA neurons via neurogenesis.

Prolactin Induces Tuberoinfundibular Dopaminergic Neurone Differentiation in Snell Dwarf Mice if Administered Beginning at 3 Days of Age

The hypothalamic tuberoinfundibular dopaminergic (TIDA) neurones secrete dopamine, which inhibits prolactin secretion. TIDA neurone numbers are deficient in Ames (df/df) and Snell (dw/dw) dwarf mice, which lack prolactin, growth hormone and thyroid-stimulating hormone. Prolactin therapy initiated before 21 days maintains normal-sized TIDA neurone numbers in df/df mice and, when initiated as early as 7 days, maintains the maximum TIDA neurone numbers observed in dw/dw development, which are decreased compared to those in normal mice. The present study investigated the effect of prolactin dose and species on TIDA neurone development. Snell dwarf and normal mice were treated with saline, 5 μg of ovine prolactin (oPRL), 50 μg of oPRL, or 50 μg of recombinant mouse prolactin (rmPRL) beginning at 3 days of age. Brains were analysed at 45 days using catecholamine histofluorescence, and immunohistochemistry for tyrosine hydroxylase or bromodeoxyuridine. Normal mice had greater (P ≤ 0.01) TIDA neurones than dw/dw, regardless of treatment. TIDA neurones in 50 μg oPRL-treated dw/dw mice were greater (P ≤ 0.05) than those in 5 μg oPRL- and rmPRL-treated dw/dw mice, which were greater (P ≤ 0.01) than those in saline-treated dw/dw mice. Fifty microgram oPRL-treated dw/dw mice also had greater (P < 0.01) TIDA neurone numbers than the maximum numbers observed in untreated dw/dw mice development. Among saline, 5 μg oPRL and 50 μg oPRL treatments, but not rmPRL, A14 neurone numbers were higher (P ≤ 0.01) in normal compared to in dw/dw mice. The mechanism of TIDA neurone recruitment was investigated using bromodeoxyuridine (BrdU) treatment at intervals after 21 days. Mice treated with rmPRL, but not oPRL, had increased BrdU incorporation in the periventricular area surrounding the third ventricle and median eminence and in the arcuate nucleus. The data obtained in the present study indicate that oPRL, but not rmPRL, when given at a high enough dose, induces TIDA neurone differentiation in dw/dw mice. This supports neurotrophic effects of prolactin on TIDA neurones in early postnatal development that extends beyond maintenance of the cell population.

Modulation of rat parasympathetic cardiac ganglion phenotype and NGF synthesis by adrenergic nerves

Cardiac function is regulated by interactions among intrinsic and extrinsic autonomic neurons, and the mechanisms responsible for organizing these circuits are poorly understood. Parasympathetic neurons elsewhere synthesize the neurotrophin NGF, which may promote postganglionic axonal associations where parasympathetic axons inhibit sympathetic transmitter release. Previous studies have shown that parasympathetic NGF content and neurochemical phenotype are regulated by sympathetic innervation. In this study we assessed contributions of sympathetic input on cardiac ganglion neuronal phenotype and NGF expression. Because cardiac ganglia are reported to contain putative noradrenergic neurons, we eliminated sympathetic input both surgically (extrinsic) and chemically (extrinsic plus intrinsic). In controls, most cardiac ganglion neurons expressed vesicular acetylcholine transporter, frequently colocalized with vesicular monoamine transporter, but lacked catecholamine histofluorescence. Most cardiac ganglion neurons expressed NGF transcripts, and 40% contained mature and 47% proNGF immunoreactivity. Guanethidine treatment for 7 days decreased numbers of neurons expressing vesicular acetylcholine transporter, NGF transcripts and NGF immunoreactivity, but did not affect proNGF or vesicular monoamine transporter immunoreactivity. Stellate ganglionectomy had comparable effects on neurochemical phenotype and mature NGF immunoreactivity, but proNGF expression was additionally reduced. These findings show that individual cardiac ganglion neurons display markers of both cholinergic and noradrenergic transmission. Sympathetic noradrenergic innervation maintains levels of cholinergic but not noradrenergic marker protein. Sympathetic innervation also promotes cardiac ganglion neuronal NGF synthesis. Because chemical blockade of all noradrenergic transmission is no more effective than extrinsic sympathectomy, local intrinsic noradrenergic transmission is not a factor in regulating ganglion neuron phenotype.

Long-Term, Homologous Prolactin, Administered through Ectopic Pituitary Grafts, Induces Hypothalamic Dopamine Neuron Differentiation in Adult Snell Dwarf Mice

Pituitary prolactin (PRL) secretion is inhibited by dopamine (DA) released into the portal circulation from hypothalamic tuberoinfundibular DA (TIDA) neurons. Ames (df/df) and Snell (dw/dw) dwarf mice lack PRL, GH, and TSH, abrogating feedback and resulting in a reduced hypophysiotropic TIDA population. In Ames df/df, ovine PRL administration for 30 d during early postnatal development increases the TIDA neuron number to normal, but 30 d PRL treatment of adult df/df does not. The present study investigated the effects of homologous PRL, administered via renal capsule pituitary graft surgery for 4 or 6 months, on hypothalamic DA neurons in adult Snell dw/dw mice using catecholamine histofluorescence, tyrosine hydroxylase immunocytochemistry, and bromodeoxyuridine immunocytochemistry. PRL treatment did not affect TIDA neuron number in normal mice, but 4- and 6-month PRL-treated dw/dw had significantly increased (P < or = 0.01) TIDA (area A12) neurons compared with untreated dw/dw. Snell dwarfs treated with PRL for 6 months had more (P < or = 0.01) TIDA neurons than 4-month PRL-treated dw/dw, but lower (P < or = 0.01) numbers than normal mice. Periventricular nucleus (area A14) neuron number was lower in dwarfs than in normal mice, regardless of treatment. Zona incerta (area A13) neuron number was unchanged among phenotypes and treatments. Prolactin was unable to induce differentiation of a normal-sized A14 neuron population in dw/dw. Bromodeoxyuridine incorporation was lower (P < or = 0.01) in 6-month PRL-treated normal mice than in 6-month PRL-treated dwarfs in the subventricular zone of the lateral ventricle and in the dentate gyrus, and lower (P < or = 0.05) in 4-month untreated dwarfs than in 4-month untreated normal mice in the median eminence and the periventricular area surrounding the third ventricle. Thus, a PRL-sensitive TIDA neuron population exists in adult Snell dwarf mice when replacement uses homologous hormone and/or a longer duration. This finding indicates that there is potential for neuronal differentiation beyond early developmental periods and suggests plasticity within the mature hypothalamus.

Reply to Kreier and Buijs: no sympathy for the claim of parasympathetic innervation of white adipose tissue

to the editor: Kreier and Buijs ([16][1]) have questioned our work ([14][2]) demonstrating the lack of parasympathetic nervous system (PSNS) innervation of white adipose tissue (WAT) in laboratory rats and mice, as well as in Siberian hamsters. We appreciate the opportunity to clarify and extend our

Prolactin replacement must be continuous and initiated prior to 21 d of age to maintain hypothalamic dopaminergic neurons in hypopituitary mice.

The prolactin (PRL) deficit in mice homozygous for the spontaneous Ames dwarf (df) mutation coincides with a marked reduction in the number of PRL-regulating tuberoinfundibular dopaminergic (TIDA) neurons. The TIDA deficit develops after 14 21 d postnatally and may be prevented by PRL replacement initiated at 12, but not at 60, d of age. The present study was designed to define further the developmental period during which PRL can prevent the deficit in the number of TIDA neurons in df/df mice, as well as to evaluate whether exposure to PRL neonatally affects the response to PRL by TIDA neurons in later development. To address the first aim, litters of df/df and normal (DF/df) mice were treated daily with ovine PRL (50 microg intraperitoneally), starting at 12, 21, or 30 d of age. To address the second aim, DF/df and df/df animals treated with PRL for 30 d starting at 12 d of age were subjected to PRL withdrawal (15 d of saline vehicle treatment), followed by PRL retreatment. All brains were evaluated using both catecholamine histofluorescence and tyrosine hydroxylase (TH) immunocytochemistry. Total numbers of TH-immunostained cells were counted in area A12 (TIDA neurons) and in A13 (medial zona incerta). Qualitatively, catecholamine fluorescence in A12 perikarya and terminals in df/df mice was enhanced by PRL treatment initiated at 12 or 21, but not at 30, d of age. TH immunostaining intensity was enhanced in all df/df PRL-treated groups, compared with saline treatment. However, total numbers of TH-positive TIDA neurons were reduced significantly in df/df mice treated with PRL beginning at 21 or 30 d, as well as with saline at 12 d, compared with similarly treated DF/df groups and with df/df animals treated with PRL beginning at 12 d (p < 0.01 for all comparisons). Among dwarf mice treated with PRL beginning at 12 d, followed by PRL withdrawal, the numbers of TH-positive TIDA neurons were greater than those of saline-treated dwarfs, but less than those in DF/df mice (p < 0.05 for both comparisons). In dwarfs retreated with PRL after withdrawal, the TIDA population was also smaller than that in normal animals (p < 0.05), although it was larger than in vehicle-treated dwarfs of the same age (p < 0.05).

Colocalization of gamma-aminobutyric acid-like immunoreactivity and catecholamines in the feeding network of Aplysia californica.

Functional consequences of neurotransmitter coexistence and cotransmission can be readily studied in certain experimentally favorable invertebrate motor systems. In this study, whole-mount histochemical methods were used to identify neurons in which gamma-aminobutyric acid (GABA)-like immunoreactivity (GABAli) was colocalized with catecholamine histofluorescence (CAh; FaGlu method) and tyrosine hydroxylase (TH)-like immunoreactivity (THli) in the feeding motor circuitry (buccal and cerebral ganglia) of the marine mollusc Aplysia californica. In agreement with previous reports, five neurons in the buccal ganglia were found to exhibit CAh. These included the paired B20 buccal-cerebral interneurons (BCIs), the paired B65 buccal interneurons, and an unpaired cell with projections to both cerebral-buccal connectives (CBCs). Experiments in which the FaGlu method was combined with the immunohistochemical detection of GABA revealed double labeling of all five of these neurons. An antibody generated against TH, the rate-limiting enzyme in the biosynthesis of catecholamines, was used to obtain an independent determination of GABA-CA colocalization. Biocytin backfills of the CBC performed in conjunction with TH immunohistochemistry revealed labeling of the rostral B20 cell pair and the unpaired CBI near the caudal surface of the right hemiganglion. THli was also present in a prominent bilateral pair of caudal neurons that were not stained with CBC backfills. On the basis of their position, size, shape, and lack of CBC projections, the lateral THli neurons were identified as B65. Double-labeling immunohistochemical experiments revealed GABAli in all five buccal THli neurons. Finally, GABAli was observed in individual B20 and B65 neurons that were identified using electrophysiological criteria and injected with a marker (neurobiotin). Similar methods were used to demonstrate that a previously identified catecholaminergic cerebral-buccal interneuron (CBI) designated CBI-1 contained THli but did not contain GABAli. Although numerous THli and GABAli neurons and fibers were present in the cerebral and buccal ganglia, additional instances of their colocalization were not observed. These findings indicate that GABA and a catecholamine (probably dopamine) are colocalized in a limited number of interneurons within the central pattern generator circuits that control feeding-related behaviors in Aplysia.

Deletion of tyrosine hydroxylase gene reveals functional interdependence of adrenocortical and chromaffin cell system in vivo.

Catecholamines are produced in the medulla of the adrenal gland and may participate in the intraglandular regulation of its cortex. We analyzed the adrenal structure and function of albino tyrosine hydroxylase-null (TH-null) mice that are deficient in adrenal catecholamine production. Adrenal catecholamines were markedly reduced, and catecholamine histofluorescence was abrogated in 15-day-old TH-null mice. Chromaffin cell structure was strikingly altered at the ultrastructural level with a depletion of chromaffin vesicles and an increase in rough endoplasmic reticulum compared with wild-type mice. Remaining chromaffin vesicles lined up proximally to the cell membrane in preparation for exocytosis providing a "string-of-pearls" appearance. There was a 5-fold increase in the expression of proenkephalin mRNA (502.8 +/- 142% vs. 100 +/- 17.5%, P = 0.016) and a 2-fold increase in the expression of neuropeptide Y (213.4 +/- 41.2% vs. 100 +/- 59.9%, P = 0.014) in the TH-null animals as determined by quantitative TaqMan (Perkin-Elmer) PCR. Accordingly, immunofluorescence for met-enkephalin and neuropeptide tyrosine in these animals was strongly enhanced. The expression of phenylethanolamine N-methyl transferase and chromogranin B mRNA was similar in TH-null and wild-type mice. In TH-null mice, adrenocortical cells were characterized by an increase in liposomes and by tubular mitochondria with reduced internal membranes, suggesting a hypofunctional state of these steroid-producing cells. In accordance with these findings, plasma corticosterone levels were decreased. Plasma ACTH levels were not significantly different in TH-null mice. In conclusion, both the adrenomedullary and adrenocortical systems demonstrate structural and functional changes in catecholamine-deficient TH-null mice, underscoring the great importance of the functional interdependence of these systems in vivo.

Absence of cholinergic sympathetic innervation from limb muscle vasculature in rats and mice

Although the existence of cholinergic sympathetic vasodilatory innervation in limb muscle vasculature is well established for some species, previous pharmacological studies have failed to reveal the presence of such innervation in rats. Recently, Schafer and colleagues [Schafer, M.K., Eiden, L.E., Weihe, E., 1998. Cholinergic neurons and terminal fields revealed by immunohistochemistry for the vesicular acetylcholine transporter. II. The peripheral nervous system. Neuroscience 84(2), 361–376] reported that vesicular acetylcholine transporter immunoreactivity (VAChT-IR), a marker for cholinergic terminals, is present in the innervation of the microvasculature of rat hindlimb skeletal muscle and concluded that rats possess cholinergic sympathetic innervation of limb muscle vasculature. Because of our interest in identifying targets of cholinergic sympathetic neurons, we have analyzed the transmitter properties of the innervation of muscle vessels in rat and mouse limbs. We found that the innervation of vasculature in muscle is noradrenergic, exhibiting robust catecholamine histofluorescence and immunoreactivity for tyrosine hydroxylase (TH) and the peptide transmitters, neuropeptide Y (NPY) and occasionally vasoactive intestinal peptide (VIP). In contrast, cholinergic phenotypic markers, VAChT-IR and acetylcholinesterase (AChE) activity, are absent. Neuron cell bodies in sympathetic ganglia, retrogradely labeled with injections of tracer into limb muscles, also lacked VAChT but contained TH-IR. The innervation of large extramuscular feed arteries in hindlimbs was also devoid of cholinergic markers, as were the cell bodies of sympathetic neurons innervating extramuscular femoral arteries. These results, like those of previous physiological studies, provide no evidence for the presence of cholinergic sympathetic innervation of muscle vasculature in rats or mice.

Catecholamine Synthesis is Mediated by Tyrosinase in the Absence of Tyrosine Hydroxylase

Catecholamine neurotransmitters are synthesized by hydroxylation of tyrosine to L-dihydroxyphenylalanine (L-Dopa) by tyrosine hydroxylase (TH). The elimination of TH in both pigmented and albino mice described here, like pigmented TH-null mice reported previously (Kobayashi et al., 1995; Zhou et al., 1995), demonstrates the unequivocal requirement for catecholamines during embryonic development. Although the lack of TH is fatal, TH-null embryos can be rescued by administration of catecholamine precursors to pregnant dams. Once born, TH-null pups can survive without further treatment until weaning. Given the relatively rapid half-life of catecholamines, we expected to find none in postnatal TH-null pups. Despite the fact that the TH-null pups lack TH and have not been supplemented with catecholamine precursers, catecholamines are readily detected in our pigmented line of TH-null mice by glyoxylic acid-induced histofluorescence at postnatal day 7 (P7) and P15 and quantitatively at P15 in sympathetically innervated peripheral organs, in sympathetic ganglia, in adrenal glands, and in brains. Between 2 and 22% of wild-type catecholamine concentrations are found in these tissues in mutant pigmented mice. To ascertain the source of the catecholamine, we examined postnatal TH-null albino mice that lack tyrosinase, another enzyme that converts tyrosine to L-Dopa but does so during melanin synthesis. In contrast to the pigmented TH-null mice, catecholamine histofluorescence is undetectable in postnatal albino mutants, and the catecholamine content of TH-null pups lacking tyrosinase is 18% or less than that of TH-null mice with tyrosinase. Thus, these extraordinary circumstances reveal that tyrosinase serves as an alternative pathway to supply catecholamines.

Catecholamines in paraganglia associated with the hepatic branch of the vagus nerve: effects of 6-hydroxydopamine and reserpine

Paraganglia are clusters of cells containing catecholamines (CA), mainly norepinephrine (NE) and dopamine (DA). The presence of epinephrine (E), on the other hand, has only been determined by indirect methods in retroperitoneal paraganglia of newborn and aged rats. Because their location, paraganglia associated with the hepatic branch of the vagus nerve may be a possible source of CA for the liver. The main purposes of the present study were to determine CA levels and whether E can be found in the omentum minus which includes paraganglia associated with the hepatic branch of the vagus nerve, and then to study the effects of 6-hydroxydopamine and reserpine on their CA content. Twenty-four female Wistar rats were randomly ascribed to three groups receiving two intraperitoneal injections of either 6-hydroxydopamine, reserpine or saline. Twenty-four hours after the last administration the rats were anesthetized and a portion of the omentum minus was obtained. Left adrenal medulla and a liver fragment were also collected as controls. The samples were processed to be analyzed by high performance liquid chromatography and catecholamine histofluorescence. The results confirm previous reports about the presence of considerable amounts of norepinephrine and dopamine in paraganglia. Norepinephrine and dopamine in the omentum like the adrenal medulla were significantly depleted by reserpine but not by 6-hydroxydopamine treatment, suggesting that some other sources in addition to sympathetic terminals are responsible for CA in the omentum. On the contrary, both drugs reduced liver NE, consistent with the localization of this amine mainly to hepatic sympathetic terminals. Histofluorescence of the omentum revealed 2–4 paraganglia per tissue fragment. Paraganglia associated with the hepatic branch of the vagus nerve contain also E. The presence of perihepatic sources of extra-adrenal CA, and more specifically E, could be of physiological significance.

Responsiveness of human infant cerebral arteries to sympathetic nerve stimulation and vasoactive agents.

Responses of segments of basilar and middle cerebral arteries of eight human infants to activation of perivascular nerves and to vasoactive drugs were studied using a resistance artery myograph. The infants ages ranged from 23 wk of gestation to 34 postnatal days. Neurogenic vasoconstriction occurred in all segments and at 8 Hz was 12.7 +/- 3.5% (11%) of tissue maximum and was blocked by phentolamine (10(-6) M). There was no evidence of a neurogenic dilator response. Catecholamine histofluorescence was seen in nerves in the adventitia at all ages studied. Norepinephrine ED50 was 7.6 +/- 1.8 x 10(-7) M, and its maximum effect was 43.1 +/- 5.7% of tissue maximum. Both neural and norepinephrine responses were greater than those of the proximal parts of adult human middle cerebral arteries obtained postmortem and surgically removed adult human pial arteries. Electron microscopy demonstrated that neural density at the adventitiomedial junction in the infant vessels was greater than in the pial arteries. Constrictor responses to serotonin and prostaglandin F2 alpha were minimal in the two infants of 23 and 24 wk of gestation but were clearly present in the older infants. Histamine and acetylcholine were potent vasodilators. Indomethacin potentiated agonist-induced contraction. In a limited number of trials angiotensin II, neuropeptide Y, caused contraction and bradykinin, relaxation. It is concluded that there is a quantitative similarity between the studied responses of infant cerebral artery segments and human pial arteries of similar diameter. However, sympathetic nerves may potentially play a more important role in the regulation of cerebrovascular tone in the infant compared with the adult, and during the gestational period examined these vessels possess an indomethacin-sensitive system that buffers agonist tone.

Hyperinnervation of the Airways in Transgenic Mice Overexpressing Nerve Growth Factor

Neuropeptides released from sensory nerve endings are potential mediators of airway inflammation in asthma and lung injury induced by inhalation of respiratory irritants. To develop an in vivo model for assessing the contribution of neurogenic inflammation in these processes, we have generated transgenic mice with altered innervation of the lung. To generate mice with an increased innervation of the airways, we placed the gene that encodes nerve growth factor (NGF) under control of the lung-specific Clara-cell secretory protein (CCSP) promoter. Two lineages of CCSP-NGF transgenic mice overexpressed NGF in the lung and developed a hyperinnervation of the airways. Immunohistochemistry for substance P, a substance P enzyme immunoassay, and catecholamine histofluorescence indicated that both tachykinin-containing sensory fibers and sympathetic fibers were increased around the airways of CCSP-NGF mice. Treatment of CCSP-NGF mice with the sympathetic-specific neurotoxin 6-hydroxydopamine (6-OHDA) eliminated the sympathetic component of the airway innervation, leaving a specific hyperinnervation by tachykinin-containing sensory fibers. CCSP-NGF mice were more sensitive than normal mice to capsaicin-induced increases in respiratory system resistance, demonstrating that the increased sensory innervation led to a change in airway function. We conclude that NGF overexpression from a lung-specific promoter produces anatomic and functional changes in lung innervation, and that CCSP-NGF mice will be useful for studying the role of neurogenic inflammation in airway disease.

Age-related changes of the noradrenergic innervation of rat tracheo-bronchial tree and pulmonary vasculature

Age-related changes of the noradrenergic innervation of the tracheo-bronchial tree and of pulmonary vasculature were investigated in male Wistar rats of 3 months (young), 12 months (adult) and 24 months (old/aged), using catecholamine histofluorescence techniques associated with image analysis and by high pressure liquid chromagraphy with electrochemical detection. In young rats, blue–green fluorescent nerve fibres supply tracheo-bronchial smooth muscle and tracheal and bronchial glands, which are innervated by a delicate network of nerve fibres rich in varicosities. Pulmonary artery and vein are sparsely innervated. They are supplied with nerve fibres distributed in the vasa vasorum or the adventitia and the outer tunica media. The higher noradrenaline concentrations were found in the trachea and extraparenchymal bronchi, followed by pulmonary vein and pulmonary artery. The density and pattern of noradrenergic innervation of the tracheo-bronchial tree, or of the pulmonary vasculature, were similar in young and adult rats. In aged rats, a loss of noradrenergic innervation involving primarily the supply to the smooth muscle of the tracheo-bronchial tree was observed. Histofluorescence techniques demonstrated a higher sensitivity than noradrenaline assay in detecting changes of the sympathetic innervation of the tracheo-bronchial tree and of the pulmonary vasculature. The possible significance of reduced noradrenergic innervation of the tracheo-bronchial tree in aged rats is discussed.

Stimulatory effect of human, but not bovine, growth hormone expression on numbers of tuberoinfundibular dopaminergic neurons in transgenic mice.

Mice transgenic for heterologous and ectopic GH expression serve as models for studying the feedback effects of elevated nonregulated GH on hypothalamic hypophysiotropic neurons as well as on peripheral function. For example, hypothalamic somatostatin expression has been shown to be increased markedly in mice bearing either bovine (b) or human (h) GH transgenes. Human, but not bovine, GH has lactogenic properties in mice, and appears to stimulate PRL-inhibiting tuberoinfundibular dopaminergic (TIDA) neurons. The present study was designed to determine the effect of a lifelong excess of hGH on dopamine (DA) expression in and numbers of TIDA neurons. Male mice of four transgenic lines were examined. The transgenic animals bore constructs of either bGH or hGH fused to either metallothionein (MT) or phosphoenolpyruvate carboxykinase (PEPCK) promoters; brains of transgenic mice were compared morphologically with those of nontransgenic littermates. Formaldehyde-induced catecholamine histofluorescence and tyrosine hydroxylase (TH) immunocytochemistry were examined in alternate brain sections; cell number was quantified for TIDA neurons (area A12) and a nonhypophysiotropic diencephalic DA area, the medial zona incerta (A13). Body weights were higher (P < 0.01) in PEPCK-GH than in MT-GH transgenic mice, as were serum levels of heterologous GH in those lines. In MT-hGH, but not MT-bGH or PEPCK-bGH, transgenic mice, A12 perikaryal fluorescence was enhanced, and ME fluorescence was reduced compared with those in control animals. The reduced ME DA is likely to reflect stimulation of TIDA neurons, because A12 TH-immunoreactive neuron number was increased by 34% in MT-hGH mice compared with that in controls (P < 0.05). In mice bearing the PEPCK-hGH construct, A12 TH neuron number was increased 47% (P < 0.001) compared with that in littermate controls. There were no differences in A13 cell number among animals, and A12 cell numbers in mice expressing bGH did not differ from control values. These results suggest that although extremely high levels of circulating bGH do not stimulate TIDA neurons, lifelong high levels of hGH have a stimulatory and graded effect on developmental differentiation of these cells for TH and DA production, supporting the concept of PRL as a trophic factor for TIDA neurons.

Stimulatory Effect of Human, but not Bovine, Growth Hormone Expression on Numbers of Tuberoinfundibular Dopaminergic Neurons in Transgenic Mice

Mice transgenic for heterologous and ectopic GH expression serve as models for studying the feedback effects of elevated nonregulated GH on hypothalamic hypophysiotropic neurons as well as on peripheral function. For example, hypothalamic somatostatin expression has been shown to be increased markedly in mice bearing either bovine (b) or human (h) GH transgenes. Human, but not bovine, GH has lactogenic properties in mice, and appears to stimulate PRL-inhibiting tuberoinfundibular dopaminergic (TIDA) neurons. The present study was designed to determine the effect of a lifelong excess of hGH on dopamine (DA) expression in and numbers of TIDA neurons. Male mice of four transgenic lines were examined. The transgenic animals bore constructs of either bGH or hGH fused to either metallothionein (MT) or phosphoenolpyruvate carboxykinase (PEPCK) promoters; brains of transgenic mice were compared morphologically with those of nontransgenic littermates. Formaldehyde-induced catecholamine histofluorescence and tyrosine hydroxylase (TH) immunocytochemistry were examined in alternate brain sections; cell number was quantified for TIDA neurons (area A12) and a nonhypophysiotropic diencephalic DA area, the medial zona incerta (A13). Body weights were higher (P < 0.01) in PEPCK-GH than in MT-GH transgenic mice, as were serum levels of heterologous GH in those lines. In MT-hGH, but not MT-bGH or PEPCK-bGH, transgenic mice, A12 perikaryal fluorescence was enhanced, and ME fluorescence was reduced compared with those in control animals. The reduced ME DA is likely to reflect stimulation of TIDA neurons, because A12 TH-immunoreactive neuron number was increased by 34% in MT-hGH mice compared with that in controls (P < 0.05). In mice bearing the PEPCK-hGH construct, A12 TH neuron number was increased 47% (P < 0.001) compared with that in littermate controls. There were no differences in A13 cell number among animals, and A12 cell numbers in mice expressing bGH did not differ from control values. These results suggest that although extremely high levels of circulating bGH do not stimulate TIDA neurons, lifelong high levels of hGH have a stimulatory and graded effect on developmental differentiation of these cells for TH and DA production, supporting the concept of PRL as a trophic factor for TIDA neurons.

Decreased receptivity of pathway connective tissue to sympathetic nerve ingrowth in the developing rat

Sympathetic axons can form atypical pathways to denervated orbital targets in neonatal rats but not in rats aged 30 or more days. The objective of this study was to determine if connective tissue pathways that carry sympathetic nerves lose their ability to sustain axonal sprouting during the early postnatal period. Regions of periorbital sheath known to contain large numbers of sympathetic axons that travel to distal orbital targets were excised from rats (sympathectomized 3 days previously) on postnatal days 6–7, 14–15, 30–31, and 48–49 and placed in anterior chambers of adult host rats. Tissues were removed 3, 6, or 10 days post-transplant and sympathetic ingrowth was analyzed by catecholamine histofluorescence in whole-mount or cryosectioned specimens. Connective tissue transplants from 6–15-day-old donors showed significant fiber ingrowth by 3 days in oculo, and innervation was maximal by 6 days. In contrast, sprouting into 30–49-day-old tissue was significantly slower, with most transplants lacking fibers at 3 days, and with small numbers of short fibers present at 6 days. We conclude that maturational changes occur in periorbital connective tissue pathways in the early postnatal period which make them less receptive to ingrowth by sympathetic nerves. The findings that connective tissue pathways are better substrates for sympathetic sprouting in the neonatal rat supports the view that developmental changes in these tissues are likely to contribute to the impaired reinnervation of orbital targets by contralateral neurons in juvenile and adult rats. © Elsevier Science B.V. All rights reserved.

The sensory branch of the pudendal nerve is the major route for adrenergic innervation of the penis in the rat.

Multiple pathways have been proposed for the course of adrenergic fibers to the penis and, although it is generally recognized that the pudendal nerve (PudN) is the most important, there is little quantitative information available. We used image analysis of catecholamine histofluorescence to quantify the effect of various nerve lesions on the adrenergic innervation of the rat penis. In addition to the denervation studies and as a direct test of whether penile adrenergic fibers traversed the pelvic plexus, penile neurons in the sympathetic chain were first labeled with a retrograde dye placed in the penis. The cavernous nerve of these animals was later exposed to another dye with different spectral characteristics. Interruption of the sensory branch of the PudN reduced adrenergic innervation of cavernosal smooth muscle by 86% (+/- 2.5%). Vascular fibers of the deep penile and helicine arteries were also severely reduced but not entirely eliminated. Interruption of the motor branch of the PudN had a lesser and more variable effect on penile adrenergic innervation: a 21.2% (+/- 6.8%) decrease in cavernosal muscle innervation but no obvious affect on vasomotor fibers. Combining the nerve lesions with phenol degeneration of perivascular fibers of the pudendal vessels further reduced but did not entirely eliminate adrenergic fibers in the cavernosal muscle and penile vessels. The dramatic reduction of adrenergic innervation of the penis after section of the PudN, especially the sensory branch, and the absence of double-labeled neurons in the sympathetic chain suggest that the PudN nerve is the major, if not the exclusive, pathway by which adrenergic fibers reach penile erectile tissue of the rat.