Generation of conditional ALK F1174L mutant mouse models for the study of neuroblastoma pathogenesis.

Neuroblastoma, an embryonal tumor arising from the sympathetic ganglia and adrenal medulla, is among the most intractable pediatric cancers. Although a variety of genetic changes have been identified in neuroblastoma, how they contribute to its pathogenesis remains largely unclear. Recent studies have identified alterations of the anaplastic lymphoma kinase (ALK) gene in neuroblastoma; ALK F1174L (a phenylalanine-to-leucine substitution at codon 1174) represents one of the most frequent of these somatic mutations, and is associated with amplification of the MYCN gene, the most reliable marker for the poor survival. We engineered the mouse Alk locus so that ALK F1174L is expressed by its endogenous promoter and can be induced in a spatiotemporally controlled fashion using Cre-loxP system. Although expression of ALK F1174L resulted in enhanced proliferation of sympathetic ganglion progenitors and increased the size of the sympathetic ganglia, it was insufficient to cause neuroblastoma. However, lethal neuroblastoma frequently developed in mice co-expressing ALK F1174L and MYCN, even in a genetic background where MYCN alone does not cause overt tumors. These data reveal that physiological expression of ALK F1174L significantly potentiates the oncogenic ability of MYCN in vivo. Our conditional mutant mice provide a valuable platform for investigating the pathogenesis of neuroblastoma.

HIF-1α is required for development of the sympathetic nervous system.

The molecular mechanisms regulating sympathetic innervation of the heart during embryogenesis and its importance for cardiac development and function remain to be fully elucidated. We generated mice in which conditional knockout (CKO) of the Hif1a gene encoding the transcription factor hypoxia-inducible factor 1α (HIF-1α) is mediated by an Islet1-Cre transgene expressed in the cardiac outflow tract, right ventricle and atrium, pharyngeal mesoderm, peripheral neurons, and hindlimbs. These Hif1aCKO mice demonstrate significantly decreased perinatal survival and impaired left ventricular function. The absence of HIF-1α impaired the survival and proliferation of preganglionic and postganglionic neurons of the sympathetic system, respectively. These defects resulted in hypoplasia of the sympathetic ganglion chain and decreased sympathetic innervation of the Hif1aCKO heart, which was associated with decreased cardiac contractility. The number of chromaffin cells in the adrenal medulla was also decreased, indicating a broad dependence on HIF-1α for development of the sympathetic nervous system.

Regional Differences in the Contributions of TNF Reverse and Forward Signaling to the Establishment of Sympathetic Innervation.

Members of the TNF and TNF receptor superfamilies acting by both forward and reverse signaling are increasingly recognized as major physiological regulators of axon growth and tissue innervation in development. Studies of the experimentally tractable superior cervical ganglion (SCG) neurons and their targets have shown that only TNF reverse signaling, not forward signaling, is a physiological regulator of sympathetic innervation. Here, we compared SCG neurons and their targets with prevertebral ganglion (PVG) neurons and their targets. Whereas all SCG targets were markedly hypoinnervated in both TNF-deficient and TNFR1-deficient mice, PVG targets were not hypoinnervated in these mice and one PVG target, the spleen, was significantly hyperinnervated. These in vivo regional differences in innervation density were related to in vitro differences in the responses of SCG and PVG neurons to TNF reverse and forward signaling. Though TNF reverse signaling enhanced SCG axon growth, it did not affect PVG axon growth. Whereas activation of TNF forward signaling in PVG axons inhibited growth, TNF forward signaling could not be activated in SCG axons. These latter differences in the response of SCG and PVG axons to TNF forward signaling were related to TNFR1 expression, whereas PVG axons expressed TNFR1, SCG axons did not. These results show that both TNF reverse and forward signaling are physiological regulators of sympathetic innervation in different tissues.

Development of non-catecholaminergic sympathetic neurons in para- and prevertebral ganglia of cats.

Expression of vasoactive intestinal peptide (VIP), neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT) and calcitonin gene-related peptide (CGRP) in the sympathetic ganglia was investigated by immunohistochemistry in the superior cervical ganglion (SCG), stellate ganglion (SG) and celiac ganglion (CG) from cats of different ages (newborn, 10-day-old, 20-day-old, 30-day-old and 2-month-old). Non-catecholaminergic TH-negative VIP-immunoreactive (IR) and nNOS-IR sympathetic ganglionic neurons are present from the moment of birth. In all studied age groups, substantial populations of VIP-IR (up to 9.8%) and nNOS-IR cells (up to 8.3%) was found in the SG, with a much smaller population found in the SCG (<1%) and only few cells observed in the CG. The percentage of nNOS-IR and VIP-IR neurons in the CG and SCG did not significantly change during development. The proportion of nNOS-IR and VIP-IR neuron profiles in the SG increased in first 20 days of life from 2.3±0.15% to 8.3±0.56% and from 0.3±0.05% to 9.2±0.83%, respectively. In the SG, percentages of nNOS-IR sympathetic neurons colocalizing VIP increased in the first 20 days of life. ChAT-IR and CGRP-IR neurons were not observed in the sympathetic ganglia of newborn animals and did not appear until 10 days after birth. In the SG of newborn and 10-day-old kittens, the majority of NOS-IR neurons were calbindin (CB)-IR, whereas in the SCG and CG of cats of all age groups and in the SG of 30-day-old and older kittens, the vast majority of NOS-IR neurons lacked CB. We conclude that the development of various non-catecholaminergic neurons in different sympathetic ganglia has its own time dynamics and is concluded at the end of the second month of life.

Development of nNOS-positive neurons in the rat sensory and sympathetic ganglia.

Neurochemical features in sympathetic and afferent neurons are subject to change during development. Nitric oxide (NO) plays a developmental role in the nervous system. To better understand the neuroplasticity of sympathetic and afferent neurons during postnatal ontogenesis, the distribution of neuronal NO synthase (nNOS) immunoreactivity was studied in the sympathetic para- and prevertebral, nodose ganglion (NG) and Th2 and L4 dorsal root ganglia (DRG) from female Wistar rats of different ages (newborn, 10-day-old, 20-day-old, 30-day-old, 2-month-old, 6-month-old, 1-year-old, and 3-year-old). nNOS-positive neurons were revealed in all sensory ganglia but not in sympathetic ones from birth onward. The percentage of nNOS-immunoreactive (IR) neurons increased during first 10 days of life from 41.3 to 57.6 in Th2 DRG, from 40.9 to 59.1 in L4 DRG and from 31.6 to 38.5 in NG. The percentage of nNOS-IR neurons did not change in the NG later during development and senescence. However, in Th2 and L4 DRG the proportion of nNOS-IR neurons was high in animals between 10 and 30days of life and decreased up to the second month of life. In 2-month-old rats, the percentage of nNOS-IR neurons was 52.9 in Th2 DRG and 51.3 in L4 DRG. We did not find statistically significant differences in the percentage of nNOS-IR neurons between Th2 and L4 DRG and between young and aged rats. In NG and DRG of 10-day-old and older rats, a high proportion of nNOS-IR neurons binds isolectin B4. In newborn animals, only 41.3%, 45.3% and 28.4% of nNOS neuron profiles bind to IB4 in Th2, L4 DRG and NG, respectively. In 10-day-old and older rats, the number of sensory nNOS-IR neurons binding IB4 reached more than 90% in DRG and more than 80% in NG. Only a small number of nNOS-positive cells showed immunoreactivity to calcitonin gene-related peptide, neurofilament 200, calretinin. The information provided here will also serve as a basis for future studies investigating mechanisms of the development of sensory neurons.

Synaptic protein and pan-neuronal gene expression and their regulation by Dicer-dependent mechanisms differ between neurons and neuroendocrine cells.

BACKGROUND: Neurons in sympathetic ganglia and neuroendocrine cells in the adrenal medulla share not only their embryonic origin from sympathoadrenal precursors in the neural crest but also a range of functional features. These include the capacity for noradrenaline biosynthesis, vesicular storage and regulated release. Yet the regulation of neuronal properties in early neuroendocrine differentiation is a matter of debate and the developmental expression of the vesicle fusion machinery, which includes components found in both neurons and neuroendocrine cells, is not resolved. RESULTS: Analysis of synaptic protein and pan-neuronal marker mRNA expression during mouse development uncovers profound differences between sympathetic neurons and adrenal chromaffin cells, which result in qualitatively similar but quantitatively divergent transcript profiles. In sympathetic neurons embryonic upregulation of synaptic protein mRNA follows early and persistent induction of pan-neuronal marker transcripts. In adrenal chromaffin cells pan-neuronal marker expression occurs only transiently and synaptic protein messages remain at distinctly low levels throughout embryogenesis. Embryonic induction of synaptotagmin I (Syt1) in sympathetic ganglia and postnatal upregulation of synaptotagmin VII (Syt7) in adrenal medulla results in a cell type-specific difference in isoform prevalence. Dicer 1 inactivation in catecholaminergic cells reduces high neuronal synaptic protein mRNA levels but not their neuroendocrine low level expression. Pan-neuronal marker mRNAs are induced in chromaffin cells to yield a more neuron-like transcript pattern, while ultrastructure is not altered. CONCLUSIONS: Our study demonstrates that remarkably different gene regulatory programs govern the expression of synaptic proteins in the neuronal and neuroendocrine branch of the sympathoadrenal system. They result in overlapping but quantitatively divergent transcript profiles. Dicer 1-dependent regulation is required to establish high neuronal mRNA levels for synaptic proteins and to maintain repression of neurofilament messages in neuroendocrine cells.

Development of neuropeptide Y-containing neurons in sympathetic ganglia of rats.

Expression of neuropeptide Y (NPY) in the sympathetic ganglia was investigated by immunohistochemistry and tract tracing. The distribution of NPY immunoreactivity (IR) was studied in the superior cervical ganglion (SCG), stellate ganglion (SG) and celiac ganglion (CG) from rats of different ages (newborn, 10-day-old, 20-day-old, 30-day-old, 2-month-old, 6-month-old, 24-month-old). We observed that the percentage of NPY-IR neuronal profiles increased during early postnatal development. In the SCG and SG, the percentage of NPY-IR profiles enlarged in the first month of life from 43±3.6% (SCG) and 46±3.8% (SG) until 64±4.1% (SCG) and 58±3.5% (SG). The percentage of NPY-IR profiles in the CG increased during the period between 20days (65±3.8%) and 30days (82±5.1%) of animals' life and did not change in further development. In newborn and 10-day-old rats, a large portion of NPY-IR neurons was also calbindin D28K (CB)-IR in all sympathetic ganglia. The proportion of CB-IR substantially decreased during next 10days in the SCG, SG and CG. NPY-IR was approximately present in a half of the postganglionic neurons innervating muscle vessels of the neck and forearm, and the percentage of labeled NPY-IR profiles did not change during the development. Only single Ki67-IR neurons were also NPY-IR in the SCG, SG and CG in newborns and not in older animals. No NPY+/caspase 3+IR neurons were observed. Finally, the process of morphological changes in the size and percentages of NPY-IR profiles is complete in rats by the first month of life.

NRP1 and NRP2 cooperate to regulate gangliogenesis, axon guidance and target innervation in the sympathetic nervous system.

The sympathetic nervous system (SNS) arises from neural crest (NC) cells during embryonic development and innervates the internal organs of vertebrates to modulate their stress response. NRP1 and NRP2 are receptors for guidance cues of the class 3 semaphorin (SEMA) family and are expressed in partially overlapping patterns in sympathetic NC cells and their progeny. By comparing the phenotypes of mice lacking NRP1 or its ligand SEMA3A with mice lacking NRP1 in the sympathetic versus vascular endothelial cell lineages, we demonstrate that SEMA3A signalling through NRP1 has multiple cell-autonomous roles in SNS development. These roles include neuronal cell body positioning, neuronal aggregation and axon guidance, first during sympathetic chain assembly and then to regulate the innervation of the heart and aorta. Loss of NRP2 or its ligand SEMA3F impaired sympathetic gangliogenesis more mildly than loss of SEMA3A/NRP1 signalling, but caused ectopic neurite extension along the embryonic aorta. The analysis of compound mutants lacking SEMA3A and SEMA3F or NRP1 and NRP2 in the SNS demonstrated that both signalling pathways cooperate to organise the SNS. We further show that abnormal sympathetic development in mice lacking NRP1 in the sympathetic lineage has functional consequences, as it causes sinus bradycardia, similar to mice lacking SEMA3A.

[Age-related development of calbindin-immunopositive neurons of rat sympathetic ganglia].

Neurons of cranial cervical, stellate and celiac sympathetic ganglia containing calbindin (CALB) were studied in rats (n = 60) aged 3-90 days using immunohistochemical method. The results obtained indicate that the largest population of CALB-immunopositive neurons was located in the stellate ganglion. The proportion of CALB-containing neurons in sympathetic para- and prevertebral ganglia decreased during the development. Final formation of CALB-immunopositive group of neurons was observed by the end of the first month of life.

Calbindin-D28k immunoreactivity in sympathetic ganglionic neurons during development.

Expression of CB in the sympathetic ganglia was investigated by immunohistochemistry. The distribution of CB immunoreactivity was studied in the superior cervical ganglion (SCG), stellate ganglion (SG) and celiac ganglion (CG) from rats and cats of different ages (newborn, 10-day-old, 20-day-old, 30-day-old, two-month-old, six-month-old). We observed that the percentage of CB-immunoreactive (IR) neurons decreased during early postnatal development in rats and cats. In all studied ganglia of both species, the percentage of CB-IR neurons was high in newborn and 10-day-old animals and significantly decreased up to 30 days of life. In rats of all ages, the largest percentage of CB-IR neurons was observed in the SG compared to the SCG and CG. In the cat sympathetic ganglia, the number of CB-IR neurons decreased more rapidly during the first two months of life, and only scattered CB-IR neurons were found in the sympathetic ganglia of two-month-old and six-month-old cats. In cats, the highest percentage of CB-IR neurons was observed in the SG, while the lowest percentage was found in the CG. The difference in size between CB+ and CB- neurons equally changed during development. Finally, the changes in the size and percentages of CB-IR neurons were complete in rats at the first month of life, and in cats at the end of the second month.

[Ultrastructure of the caudal mesenteric ganglion neurons during early development in kittens].

Electron microscopy was used to study the peculiarities of the development of nervous elements in the sympathetic caudal mesenteric ganglion (CMG) in the cat from the moment of birth until the end of the second month of life. The discordance in the rate of maturation of both neurons and their endings was observed. In newborn kittens, mature neurons, glial cells and synapses were observed together with many immature ones. In 14-day-old animals, the proportion of immature neurons decreased, while destruction of neurons was observed more frequently in this age. In CMG of the animals of all the age groups, axodendritic synapses were found most frequently and axosomatic synapses were observed more rarely. Finally, the ultrastructure of CMG in kittens become comparable to that of adult animals at the age of 60 days.

Kinesin-12, a mitotic microtubule-associated motor protein, impacts axonal growth, navigation, and branching.

Kinesin-12 (also called Kif15) is a mitotic motor protein that continues to be expressed in developing neurons. Depletion of kinesin-12 causes axons to grow faster, more than doubles the frequency of microtubule transport in both directions in the axon, prevents growth cones from turning properly, and enhances the invasion of microtubules into filopodia. These results are remarkably similar to those obtained in previous studies in which neurons were depleted of kinesin-5 (also called Eg5 or Kif11), another mitotic motor protein that continues to be expressed in developing neurons. However, there are also notable differences in the phenotypes obtained with depleting each of these motors. Depleting kinesin-12 decreases axonal branching and growth cone size, whereas inhibiting kinesin-5 increases these parameters. In addition, depleting kinesin-12 diminishes the appearance of growth-cone-like waves along the length of the axon, an effect not observed with depletion of kinesin-5. Finally, depletion of kinesin-12 abolishes the "waggling" behavior of microtubules that occurs as they assemble along actin bundles within filopodia, whereas inhibition of kinesin-5 does not. Interestingly, and perhaps relevant to these differences in phenotype, in biochemical studies, kinesin-12 coimmunoprecipitates with actin but kinesin-5 does not. Collectively, these findings support a scenario whereby kinesin-12 shares functions with kinesin-5 related to microtubule-microtubule interactions, but kinesin-12 has other functions not shared by kinesin-5 that are related to the ability of kinesin-12 to interact with actin.

Eph/ephrin interactions modulate vascular sympathetic innervation.

Ephs and ephrins are membrane-bound proteins that interact to modulate axon growth and neuronal function. We tested the hypothesis that eph/ephrin interactions affected the growth and function of vascular sympathetic innervation. Using RT-PCR analyses, we detected both classes of ephs (A and B) and both classes of ephrins (A and B) in sympathetic ganglia from neonatal and adult rats. Both classes of ephs (A and B) and both classes of ephrins (A and B) bound to the cell bodies and neurites of dissociated postganglionic sympathetic neurons. Messenger RNAs encoding for both classes of ephs (A and B) and both classes of ephrins (A and B) were also detected in sympathetically innervated arteries from neonatal and adult rats. These data suggest that ephrins/ephs on nerve fibers of postganglionic sympathetic neurons could interact with ephs/ephrins on cells in innervated arteries. We found that ephA4 reduced reinnervation of denervated femoral arteries. Reinnervation in the presence of ephA4-Fc (38.9±6.6%) was significantly less than that in the presence of IgG-Fc (62±10%; n=5; p<0.05; one-tailed unpaired t-test). These data indicate that eph/ephrin interactions modulated the growth of vascular sympathetic innervation. We also found that ephA4 increased basal release of norepinephrine from nerve terminals of isolated tail arteries. These data indicate that eph/ephrin interactions affect the growth and function of vascular sympathetic innervation.

Neurotransmitter composition of neurons in the cranial cervical and celiac sympathetic ganglia in postnatal ontogenesis.

The neurotransmitter composition of neurons in the cranial cervical ganglion (CCG) and celiac ganglia (CG) in rats of different ages (neonatal, 10, 12, 30, and 60 days) was studied by immunohistochemical methods. The results showed that most neurons in these sympathetic ganglia contain tyrosine hydroxylase (TH). Most TH-positive neurons were also neuropeptide Y (NPY)-positive. In all ganglia, the proportions of neurons containing NPY increased from the moment of birth to the end of the first month of life. In the CG, NPY was present in a significantly greater proportion of neurons than in the CCG. Substance P, vasoactive intestinal peptide, and choline acetyltransferase were present in occasional neurons in the CCG and CG from birth. There was no change in the proportion of this type of neuron with age. Definitive establishment of the neurotransmitter composition in the sympathetic ganglia studied here occurred by the end of the first month of life.

NGF protects paravertebral but not prevertebral sympathetic neurons against exposure to high glucose in vitro.

In diabetes, sympathetic neuroaxonal dystrophy occurs in prevertebral celiac/superior mesenteric ganglia (CG/SMG) but not in paravertebral superior cervical ganglia (SCG). Changes in neurotrophic support by NGF occur during postnatal development and are implicated in diabetic neuropathy. Therefore, our aim was to compare the effects of age and NGF on the responses of CG/SMG and SCG neurons to high glucose levels in vitro. Neurons were dissociated from neonatal (5 days) and adult (12 weeks) rat ganglia and maintained in serum-free media containing glucose (10-100 mM) in the presence or absence of NGF (50 ng/ml) for 48 h. Cultures were immunostained for the pan neuronal marker, PGP9.5, and TUNEL. Neurons were assessed for viability, the presence of neurite outgrowth and for TUNEL-positive nuclei as a marker of apoptosis. Glucose caused significant concentration-dependent decreases in both viability and the proportion of neurons developing neurites together with significant increases in TUNEL-positive staining. Neonatal SCG neurons with neurites were significantly more susceptible to high glucose than adult SCG neurons whereas postnatal age had no influence on the response of CG/SMG neurons to high glucose. NGF protected adult SCG but not adult CG/SMG neurite-bearing neurons against the induction of TUNEL staining by high glucose. In the presence of NGF, CG/SMG neurons were markedly more susceptible to high glucose than SCG neurons. The greater susceptibility of CG/SMG neurons to diabetic neuropathy may be due to a selective inability of NGF to protect this particular population of sympathetic neurons against hyperglycaemia.

Lateralization of the connections of the ovary to the celiac ganglia in juvenile rats.

During the development of the female rat, a maturing process of the factors that regulate the functioning of the ovaries takes place, resulting in different responses according to the age of the animal. Studies show that peripheral innervation is one relevant factor involved.In the present study we analyzed the anatomical relationship between the neurons in the celiac-superior mesenteric ganglia (CSMG), and the right or left ovary in 24 or 28 days old female pre-pubertal rats. The participation of the superior ovarian nerve (SON) in the communication between the CSMG and the ovaries was analyzed in animals with unilateral section of the SON, previous to injecting true blue (TB) into the ovarian bursa. The animals were killed seven days after treatment. TB stained neurons were quantified at the superior mesenteric-celiac ganglia.The number of labeled neurons in the CSMG of rats treated at 28 days of age was significantly higher than those treated on day 24. At age 24 days, injecting TB into the right ovary resulted in neuron stains on both sides of the celiac ganglia; whereas, injecting the left side the stains were exclusively ipsilateral. Such asymmetry was not observed when the rats were treated at age of 28 days.In younger rats, sectioning the left SON resulted in significantly lower number of stained neurons in the left ganglia while sectioning the right SON did not modify the number of stained neurons. When sectioning of the SON was performed to 28 days old rats, no staining was observed.Present results show that the number and connectivity of post-ganglionic neurons of the CSMG connected to the ovary of juvenile female rats change as the animal mature; that the SON plays a role in this communication process as puberty approaches; and that this maturing process is different for the right or the left ovary.

NADPH-diaphorase-positive neurons in sympathetic ganglia during postnatal ontogenesis.

The locations and morphometric characteristics of NADPH-diaphorase (NADPH-d)-positive neurons were identified in the cranial cervical (CCG), stellate (SG), and celiac (CG) ganglia in neonatal rats, mice, and cats and animals aged 10, 20, 30, 60, and 180 days. No NADPH-d-positive neurons were found in rats and mice in any of the age groups studied. In kittens, the majority of NADPH-d-positive neurons were located in the SG, with fewer in the CCG and only occasional neurons in the CG, regardless of age. The proportion of NADPH-d-positive neurons in the SG increased during the first 20 days of life and decreased after 30 days, to the end of the second month of life. The proportion of NADPH-d-reactive neurons in the CCG and CG did not change during ontogenesis. The mean sizes of NADPH-d-positive neurons in different ganglia in animals of the same age were not significantly different. These data lead to the conclusion that the development of NADPH-d-positive neurons with age occurs heterochronously and is complete by the end of the second month of life.

[NADPH-diaphorase-positive neurons in the sympathetic ganglia during postnatal ontogenesis].

Localization and the morphometric characteristics of NADPH-diaphorase--(NADPH-d)-positive neurons were studied in the superior cervical ganglion (SCG), stellate ganglion (SG) and the celiac ganglia (CG) in newborn, 10-, 20-, 30-, 60 and 180-day-old rats, mice and cats. No NADPH-d-positive neurons were found in rats and mice of all the age groups studied. In cats, the largest proportion of NADPH-d-positive neurons was found in the SG, the smaller one--in the SCG, while only the individual neurons we detected in the CG, irrespective of the animal age. In the SG, the proportion of NADPH-d-positive neurons increased during the first 20 days of life and then decreased after 30 days till the end of the second month. The content of NADPH-d-positive neurons in the CG and SCG d-remained unchanged during the development. There were no significant differences in the cross-sectional area between the neurons located in the different ganglia of animals from the same age group. It is concluded that the age development of NADPH-d-positive neurons in different sympathetic ganglia occured heterochronously was finished by the end of the second month of life.

Kinesin-5 is essential for growth-cone turning.

Inhibition of kinesin-5, a mitotic motor protein also expressed in neurons, causes axons to grow faster as a result of alterations in the forces on microtubules (MTs) in the axonal shaft. Here, we investigate whether kinesin-5 plays a role in growth-cone guidance. Growth-cone turning requires that MTs in the central (C-) domain enter the peripheral (P-) domain in the direction of the turn. We found that inhibition of kinesin-5 in cultured neurons prevents MTs from polarizing within growth cones and causes them to grow past cues that would normally cause them to turn. We found that kinesin-5 is enriched in the transition (T-) zone of the growth cone and that kinesin-5 is preferentially phosphorylated on the side opposite the invasion of MTs. Moreover, when a growth cone encounters a turning cue, phospho-kinesin-5 polarizes even before the growth cone turns. Additional studies indicate that kinesin-5 works in part by antagonizing cytoplasmic dynein and that these motor-driven forces function together with the dynamic properties of the MTs to determine whether MTs can enter the P-domain. We propose that kinesin-5 permits MTs to selectively invade one side of the growth cone by opposing their entry into the other side.

Development of the NADPH-diaphorase-positive neurons in the sympathetic ganglia.

Changes in the distribution of NADPH-diaphorase (NADPH-d) were studied in neurons of the superior cervical ganglion (SCG), stellate ganglion (SG) and celiac ganglia (CG) in newborn, 10-, 20-day-old, 1-month-old, 2-month-old and 6-month-old rats, mice and kittens. NADPH-d-positive neurons were revealed in all sympathetic ganglia in kittens but not in rodents from birth onwards. In kittens, the largest population of NADPH-d-positive cells was found in the SG, the smallest in the SCG (<1%) and we observed only a few cells in the CG. The proportion of NADPH-d-positive cells in the SG increased from 3.1 +/- 0.15% in newborn kittens to 9.3 +/- 0.63% in 20-day-old animals and decreased further from 8.1 +/- 0.75% in 30-day-old kittens to 3.4 +/- 0.54% in 2-month-old animals. The content of NADPH-d-positive cells in the CG and SCG did not change during development. There were no differences in cross-sectional area between neurons located in different ganglia of animals from the same age group under study. We conclude that the development of NADPH-d-positive neurons in different sympathetic ganglia has its own time dynamics and is completed by the end of the second month of life.