Phenotype of intraadrenal ganglion neurons during postnatal development in rat.

The postnatal development of intraadrenal ganglion neurons was studied in rat by using indirect immunohistochemistry and in situ hybridization. The large neuropeptide tyrosine (NPY)-expressing ganglion neurons (type I ganglion neurons) matured postnatally, with marked increases in acetylcholinesterase (AChE)-, neurofilament 10 (NF10)-, and tyrosine hydroxylase (TH)-like immunoreactivities (LIs) paralleled by increasing levels of mRNAs encoding NPY, low-affinity neurotrophin receptor (LANR), and tropomyosin kinase receptor (trk). The smaller vasoactive intestinal polypeptide (VIP)-immunoreactive (IR) ganglion neurons (type II ganglion neurons) expressed increasing levels of VIP mRNA postnatally and also contained immunoreactive nitric oxide synthase (NOS) and its mRNA. These type II ganglion neurons appeared to be relatively mature already at postnatal day (P2) and did not express detectable levels of LANR or trk mRNAs. The cell size of both the type I and type II ganglion neurons increased about 2.5-fold postnatally. The type I ganglion neurons formed more densely packed clusters with increasing age, whereas the type II ganglion neurons were spread out in small groups or individually, mainly in the peripheral parts of the medulla, and appeared to fulfill their migration into the medulla and/or to the inner regions of the cortex early postnatally, possibly after establishing contact with their cortical targets. We suggest that the type I ganglion neurons represent sympathetic ganglion neurons of the same origin as the chromaffin cells and that they mature mainly postnatally. The development of the type II (VIP/NOS) ganglion neurons takes place earlier; however, their phenotype remains more uncertain.

Developmental expression of GAP43 mRNA in chromaffin cells and intra-adrenal neurons.

Growth associated protein 43 (GAP43) mRNA was found in scattered groups of chromaffin cells prenatally, and in chromaffin noradrenaline cells from postnatal day 6 (P6). The distinct adult distribution of GAP43 mRNA in chromaffin cells appeared between P10 and P16. High levels of GAP43 mRNA were found in intra-adrenal ganglion neurons also expressing neuropeptide tyrosine (NPY) mRNA (type I ganglion neurons), and at prenatal and early postnatal stages in large medullarly clusters of NPY mRNA containing cells, probably representing type I ganglion neurons precursors. The expression of GAP43 mRNA in noradrenaline-producing chromaffin cells and type I ganglion neurons suggests an important role for GAP43 in these cells both during development and in adult life.

PACAP in the adrenal gland--relationship with choline acetyltransferase, enkephalin and chromaffin cells and effects of immunological sympathectomy.

Using indirect immunohistochemistry and immunological sympathectomy pituitary adenylate cyclase activating polypeptide (PACAP)-like immunoreactivity (LI) was studied in the adult rat adrenal gland. All PACAP-positive fibres contained choline acetyltransferase (ChAT)-LI and were found in high numbers among noradrenaline chromaffin cells, whereas enkephalin (ENK)/ChAT-immunoreactive (IR) fibres predominantly innervated adrenaline chromaffin cells. After immunological sympathectomy no PACAP-, ChAT- or ENK-IR fibres remained, strongly suggesting a preganglionic origin. A small number of PACAP-IR fibres was also observed in the subcapsular regions both in controls and in sympathectomized animals, presumably representing sensory fibres. These results define a subpopulation of PACAP-containing cholinergic preganglionic fibres in the adult rat adrenal medulla lacking ENK and innervating noradrenaline chromaffin cells. PACAP was also expressed in a few adrenaline chromaffin cells after immunological removal of the preganglionic innervation, suggesting an additional, hormonal role.

The cholinergic innervation of the adrenal gland and its relation to enkephalin and nitric oxide synthase.

Using a monoclonal antibody against rat brain choline acetyltransferase (ChAT) the cholinergic innervation of the adult rat adrenal gland was visualized. Almost all ChAT-positive fibres contained nitric oxide synthase (NOS), whereas enkephalin (ENK) was exclusively found in ChAT fibres among adrenaline chromaffin cells. The ChAT/NOS/ENK fibres disappeared after immunological sympathectomy, indicating a preganglionic origin. ChAT was not found in the superficial peptide- and NOS containing fibre plexus in the adrenal cortex or in small or large intra-adrenal ganglion neurones under control conditions. Even after colchicine treatment only one single ChAT-positive small ganglion neurone was found. It is possible, therefore that some small intra-adrenal ganglion neurones, which express NOS- and VIP-like immunoreactivities, are noncholinergic, nonadrenergic neurones.

Neuronal markers, peptides and enzymes in nerves and chromaffin cells in the rat adrenal medulla during postnatal development.

Neuronal markers, peptides and enzymes were analyzed in the rat adrenal medulla during the postnatal period, i.e., when the 'functional' splanchnic innervation is assumed to 'mature'. Nerve fibers were present on day 2 as indicated by neurofilament 10 (NF10)- and growth associated protein 43 (GAP43)-like immunoreactivities (LIs). Acetylcholinesterase (AChE)- and enkephalin (ENK)-immunoreactive (IR) fibers, presumably of preganglionic nature, increased in number and intensity during the postnatal period. In contrast, calcitonin gene-related peptide (CGRP)- and galanin (GAL)-IR fibers were almost fully developed on day 2. Thus, the presumably sensory innervation of the adrenal gland seems to precede the development of the autonomic nerves. The AChE- and ENK-IR fibers may exert a suppressive effect on ENK-, CGRP- and neurotensin (NT)-LIs in chromaffin cells, since the levels of these peptides were high in the early postnatal period and then decreased. On the other hand, GAL-LI in chromaffin cells was low also in young rats, while GAP43-IR cells were observed at all stages. Neuropeptide tyrosine (NPY) was expressed in many chromaffin cells at all stages and its turnover rate seemed to decrease towards the adult stage. The expression of the catecholamine synthezising enzymes changed only marginally during development. These results indicate that the preganglionic fibers, but not the sensory axons, in the splanchnic nerve are involved in the developmental control of expression of some, but not all, peptides in the chromaffin cells and that these changes thus may reflect the maturation of a 'functional' transmission.

Perinatal distribution of galanin and galanin receptor-1 mRNA in the rat hindbrain.

In situ hybridisation was used to determine the distribution and levels of mRNA for galanin precursor preprogalanin (ppGAL) and galanin receptor-1 (GAL-R1) in the rat hindbrain before and after birth. Quantification of mRNA levels was performed from E21. Also, immunohistochemistry was used to study GAL-like immunoreactivity (GAL-LI) prenatally. On E16, no expression of ppGAL mRNA could be detected in any areas examined, whereas on E19 low transcript levels were observed. GAL-LI, however, was seen at relatively high levels in nerve fibres already on E16, mainly in the areas receiving primary afferents. Also, GAL-R1 mRNA was expressed at high levels in discrete areas of the hindbrain on E16. On E21 ppGAL mRNA was found in the locus coeruleus (LC), the nucleus of the solitary tract, the dorsal motor nucleus of the vagus (nX), the lateral reticular nucleus (LRn) and superficially along the ventral medullary surface. Expression increased postnatally in all these areas except in nX and LRn. GAL-R1 mRNA, on the other hand, was found to be expressed at high levels on E21 in the LC, where levels then decreased on P1. Expression of GAL-R1 mRNA was also found in other areas of the brainstem, but here no changes were detected around birth. These findings demonstrate that ppGAL and GAL-R1 mRNAs, as well as GAL-LI, are present in the brainstem in the rat fetus and that the changes in expression after birth could be of importance for the newborn in the transition from pre- to postnatal life.

Birth-related expression of c-fos, c-jun and substance P mRNAs in the rat brainstem and pia mater: possible relationship to changes in central chemosensitivity.

In situ hybridization was used to characterize respiration-related areas of the brainstem activated around the time of birth as well as their postnatal sensitivity to CO2. Levels of mRNA corresponding to the immediate early genes (IEG), c-fos and c-jun, and of substance P precursor, ppt-A, were determined in rat fetuses (E21) and neonatal pups (1 h, 1 day and 6 days after normal birth) and after exposure to hypercapnia (12% CO2 for 1 h). Transient increases in c-fos mRNA were observed in the central chemoreceptor area of the ventral medullary surface (VMS), in the lateral reticular nucleus (LRN), in the nucleus of the solitary tract (NTS), and in the nucleus raphé pallidus (RPA) 1 h after birth. Increased expression of c-fos mRNA in the VMS could also be evoked by hypercapnia and this response was particularly pronounced 1 day after birth. On the other hand, c-jun mRNA could be detected already at E21 in the hypoglossal nucleus (XII) and LRN and these levels were not significantly altered at 1 h after birth. There was, however, an increase in the expression of c-jun mRNA in the pia mater surrounding the brainstem after birth. At 1 day after birth, c-jun mRNA levels had decreased in the LRN and pia mater, and later on (6 days after birth) in XII. Furthermore, the ppt-A mRNA level in NTS increased immediately after birth and remained high 1 and 6 days later. These results suggest that (a) the central chemoreceptor area of the VMS, as well as the NTS, LRN, RPA and pia mater are activated following birth; (b) the VMS, but not the other structures examined, can be activated immediately after birth by hypercapnia; and (c) increased expression of ppt-A mRNA may be related to the transition of respiratory control at birth.

Effects of immunological sympathectomy on postnatal peptide expression in the rat adrenal medulla.

Administration of monoclonal antibodies against acetylcholinesterase (AChE-mabs) to adult rats leads to a selective degeneration of the acetylcholine esterase-(AChE), choline acetyltransferase-(ChAT) and enkephalin-(ENK) positive preganglionic fibres of the splanchnic nerve innervating the adrenal gland. Here we used this approach of immunological sympathectomy, performed at postnatal day 2 (P2), in an attempt to study the development role of the preganglionic fibres in the adrenal medulla in more detail. Analysis was performed at P16 and revealed that the effect of this treatment varied considerably between animals, as judged by the number of remaining AChE-, ChAT- and ENK-positive fibres. The number and intensity especially of ENK fibres in the adrenal medulla correlated negatively with the number and staining intensity of ENK-immunoreactive chromaffin cells, suggesting a 'dose-response' relationship. Thus, the high early postnatal levels of ENK-like immunoreactivity generally persisted in chromaffin cells of adrenals with a successful immunosympathectomy, i.e. in those adrenals that lacked AChE-, ChAT- and ENK-positive nerves. In contrast, calcitonin gene-related peptide-like immunoreactivity in nerves and chromaffin cells was not affected. Large and strongly AChE-positive intra-adrenal ganglion neurones, recently termed type I ganglion neurones, were present also after AChE-mab treatment and had an apparently normal morphology. These results indicate a role for preganglionic fibres in the developmental regulation of ENK in the chromaffin cells. However, these fibres appear less important for the postnatal development of the type I ganglion neurones.

Birth-related up-regulation of mRNA encoding tyrosine hydroxylase, dopamine beta-hydroxylase, neuropeptide tyrosine, and prepro-enkephalin in rat adrenal medulla is dependent on postnatal oxygenation.

The transynaptic splanchnic regulation of the adrenal medulla is not functional in the newborn rat. Thus, synthesis and release of adrenal catecholamines and peptides are assumed to be regulated by nonneuronal mechanisms at this stage. In a previous study we reported 4-5-fold increases in the levels of mRNA encoding tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and neuropeptide tyrosine (NPY) in the rat adrenal medulla immediately after birth. In the present in situ hybridization study the importance of postnatal oxygenation for the birth-related increase in expression of these mRNA, and of preproenkephalin (ENK) mRNA was investigated in rat pups. We report here that maternal hypoxia (11-13% O2) leads to 2-3-fold increases (p < 0.01) in fetal adrenal TH, DBH, and NPY mRNA levels on the day before birth. These mRNA then further increased 1-5-2-fold (p < 0.01) 12 h after birth in room air. Only ENK mRNA levels increased (5-fold; p < 0.01) when pups were born in 11-13% O2; however, still less (p < 0.01) than after birth in room air when it increased nine times (p < 0.01). Thus, the birth-related increases in TH, DBH, NPY, and ENK mRNA levels in the rat adrenal medulla are dependent on postnatal oxygenation.

Functional and developmental studies of the peripheral arterial chemoreceptors in rat: effects of nicotine and possible relation to sudden infant death syndrome.

The drive on respiration mediated by the peripheral arterial chemoreceptors was assessed by the hyperoxic test in 3-day-old rat pups. They accounted for 22.5 +/- 8.8% during control conditions, but only for 6.9 +/- 10.0% after nicotine exposure, an effect counteracted by blockade of peripheral dopamine type 2 receptors (DA2Rs). Furthermore, nicotine reduced dopamine (DA) content and increased the expression of tyrosine hydroxylase (TH) in the carotid bodies, further suggesting that DA mediates the acute effect of nicotine on arterial chemoreceptor function. During postnatal development TH and DA2R mRNA levels in the carotid bodies decreased. Thus, nicotine from smoking may also interfere with the postnatal resetting of the oxygen sensitivity of the peripheral arterial chemoreceptors by increasing carotid body TH mRNA, as well as DA release in this period. Collectively these effects of nicotine on the peripheral arterial chemoreceptors may increase the vulnerability to hypoxic episodes and attenuate the protective chemoreflex response. These mechanisms may underlie the well-known relation between maternal smoking and sudden infant death syndrome.

Expression and release of neuroregulators during development: monoamines and neuropeptides.

The turnover of catecholamines (CA) was determined in the adrenal medulla and brain of rat fetuses and pups. In general we found a considerable increase soon after birth. The expression of mRNA for CA-synthesizing enzymes was also considerably enhanced in the adrenals shortly after birth. Furthermore, we demonstrated increased expression of neuropeptides after birth, increased synthesis of mRNA encoding for neuropeptide Y in the adrenals 24 h after birth; and considerable activation of the substance P gene in a respiratory nuclei of rabbit pups which had been breathing for 2 h as compared with fetuses at term.

Changes in levels of mRNA coding for catecholamine synthesizing enzymes and neuropeptide Y in the adrenal medulla of the newborn rat.

We have measured levels of mRNA coding for the catecholamine synthesizing enzymes tyrosine hydroxylase (TH), dopamine beta-hydroxylase (D beta H), phenylethanolamine N-methyltransferase (PNMT) and for neuropeptide Y (NPY) in rat adrenal medulla by using in situ hybridization histochemistry. Ages of one day before birth (E21), 12 h, 24 h, 2 days and 4 days after birth and in adults were studied. TH, D beta H and NPY mRNA levels increased markedly postnatally. Twelve hours after birth the levels of mRNA for TH, D beta H and NPY were, respectively, 512 +/- 18%, 370 +/- 24% and 253 +/- 21% of E21 levels. At 24 h of age NPY mRNA level was 437 +/- 73% of fetal value. In contrast, the levels of mRNA coding for PNMT increased more slowly and reached 196 +/- 9% of E21 level on postnatal day four and was further increased in adult rats.

Ventilatory response to hyperoxia in newborn rats born in hypoxia--possible relationship to carotid body dopamine.

1. The influence of postnatal hypoxia on regulation of breathing and turnover rate of carotid body dopamine was examined in newborn rats. The percentage change in frequency, tidal volume and ventilation elicited by transient hyperoxia was assessed by flow plethysmography in unanaesthetized pups. The alteration in ventilation was taken as an index of peripheral chemoreceptor activity. 2. The rats were born and reared in hypoxia. The inspired oxygen fraction (FI,O2) was 0.12-0.14 until 2 days after delivery when the rats were placed into room air and the ventilatory chemoreflex was tested. At 4 days of age, i.e. 2 days after termination of hypoxia, the rats were tested again. The ventilatory data were compared with those from a previous study in normoxic rats. 3. We found a smaller decrease in ventilation (8.8 +/- 3.9%, mean +/- S.D.) in the hypoxic rats at 2 days of age compared with normoxic rats (22.7 +/- 6.4%; P < 0.001). In contrast, at 4 days of age there was no difference in ventilatory response between the posthypoxic rats (19.2 +/- 4.6%) and normoxic pups (18.6 +/- 4.9%). 4. The turnover rates of dopamine in carotid bodies were determined at 0-6, 6-12, 12-24 h and 2 days after birth in hypoxic rats and in 2-day-old posthypoxic rat pups at different time intervals after termination of hypoxia. Postnatal hypoxia sustained a high turnover rate which decreased after termination of the hypoxia. 5. We propose that the weak chemoreflex in hypoxic rat pups is brought about by a high release of carotid body dopamine.

Birth-related changes of expression and turnover of some neuroactive agents and respiratory control.

Respiratory movements are partially inhibited before birth and central and peripheral chemoreceptors are probably less sensitive. The transition to continuous breathing might be due to the switch-off of inhibitory neuromodulators and the switch-on of excitatory neuroactive agents. This hypothesis has been explored mainly in rats. One example is dopamine which was found to have a high turnover in carotid bodies in newborn pups prior to decreasing rapidly. It is suggested to inhibit the carotid bodies before birth. In the brain, the noradrenergic system has been found to be activated at birth, possibly promoting neural transmission and arousal. Increased expressions of mRNA for c-fos, tyrosine hydroxylase and some neuropeptides have also been found.

Ontogeny of aromatic L-amino acid decarboxylase-containing tubule cells in rat kidney.

Dopamine plays an important role in regulation of renal sodium transport. Proximal tubule cells produce dopamine after decarboxylation of L-DOPA via the enzyme aromatic L-amino acid decarboxylase (AADC). The presence and cellular localization of AADC-like immunoreactivity (-LI) and AADC mRNA were examined during pre- and postnatal development in rat kidney by indirect immunofluorescence and in situ hybridization histochemistry. Few scattered condensations of AADC-immunoreactive (-IR) tubule cells forming a lumen were detected on gestational day 18. From gestational day 21, many AADC-IR tubule cells were observed in the inner cortex, whereas the outer cortex lacked AADC-LI. Within 24 hours of birth, AADC-IR cells in the inner cortex could be identified as proximal tubule cells. During day 3 and 5 there was an increase in number of AADC-IR proximal tubule cells in the inner cortex, leaving less amount of AADC-negative cells in the outer cortex. Starting from day 8, AADC-IR cells could be seen in the outer cortex. An apparent decrease in AADC-immunofluorescence intensity was observed at 40 days, and at 80 days virtually no AADC-LI could be demonstrated. Intrarenal levels of dopamine showed a tendency to increase between 3 and 20 days, and showed significant decreases between 20 to 40 days and between 40 to 80 days. AADC mRNA was not detected in the kidney at 18 hours after birth, but could be observed in the inner cortex at 6 days. At 12, 19 and 40 days AADC mRNA was seen in the entire cortex.(ABSTRACT TRUNCATED AT 250 WORDS)