Tlx3 controls cholinergic transmitter and Peptide phenotypes in a subset of prenatal sympathetic neurons.

The embryonic sympathetic nervous system consists of predominantly noradrenergic neurons and a very small population of cholinergic neurons. Postnatal development further allows target-dependent switch of a subset of noradrenergic neurons into cholinergic phenotype. How embryonic cholinergic neurons are specified at the prenatal stages remains largely unknown. In this study, we found that the expression of transcription factor Tlx3 was progressively restricted to a small population of embryonic sympathetic neurons in mice. Immunostaining for vesicular acetylcholine transporter (VAChT) showed that Tlx3 was highly expressed in cholinergic neurons at the late embryonic stage E18.5. Deletion of Tlx3 resulted in the loss of Vacht expression at E18.5 but not E12.5. By contrast, Tlx3 was required for expression of the cholinergic peptide vasoactive intestinal polypeptide (VIP), and somatostatin (SOM) at both E12.5 and E18.5. Furthermore, we found that, at E18.5 these putative cholinergic neurons expressed glial cell line-derived neurotrophic factor family coreceptor Ret but not tyrosine hydroxylase (Ret(+)/TH(-)). Deletion of Tlx3 also resulted in disappearance of high-level Ret expression. Last, unlike Tlx3, Ret was required for the expression of VIP and SOM at E18.5 but not E12.5. Together, these results indicate that transcription factor Tlx3 is required for the acquisition of cholinergic phenotype at the late embryonic stage as well as the expression and maintenance of cholinergic peptides VIP and SOM throughout prenatal development of mouse sympathetic neurons.

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.

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.

p38α and p38ß mitogen-activated protein kinases determine cholinergic transdifferentiation of sympathetic neurons.

Although the p38 mitogen-activated protein kinases are active in many neuronal populations in the peripheral and central nervous systems, little is known about the physiological functions of p38 in postmitotic neurons. We report that p38 activity determines in vitro and in vivo the switch from noradrenergic to cholinergic neurotransmission that occurs in sympathetic neurons on exposure to the neuropoietic cytokines CNTF and LIF. This transdifferentiation serves as a model for the plastic mechanisms that enable mature neurons to change some of their central functions without passing through the cell cycle. We demonstrate that in postmitotic neurons, p38 and STAT pathways are concurrently activated by neuropoietic cytokine treatment for at least 12 h overlapping with changes in neurotransmitter marker gene expression. Inhibition of p38 blocks the upregulation of the nuclear matrix protein Satb2 and of cholinergic markers by CNTF without affecting STAT3 phosphorylation. Conversely, overexpression of p38α or ß in the absence of cytokines stimulates cholinergic marker expression. The neurotransmitter switch in vitro is impaired in neurons isolated from p38ß(-/-) mice. Consistent with these in vitro results, a substantial loss of cells expressing cholinergic properties is observed in vivo in the stellate ganglion of mature mice deficient in the p38ß isoform.

[Morphological features of the rat stellate ganglion during early postnatal development].

The aim of this work was to study the anatomical characteristics of the stellate ganglion (SG) and the morphometric characteristics of its neurons in rats of different age groups (newborn, 10-, 20-, 30-, 60- and 180-day-old) using anatomical and histological methods. The results obtained indicated that in rats since birth there were three variants of branch origin from the medial margin of SG. No differences were observed in these variants between right and left SG. The sizes of both SG and its neurons increased during the first two months of postnatal development. The density of neurons in SG sections decreased from the moment of birth until the six months of age. The number of SG neurons did not change significantly in the postnatal ontogenesis. Thus, SG in rats is anatomically formed by the moment of birth, while the sizes and morphometric characteristics of SG neurons become finally stabilized by the second month of age.

Development of rat stellate ganglion neurons containing membrane-bound muscarinic receptors and purinoreceptors.

The aim of the study reported here was to investigate the locations and morphometric characteristics of neurons in the stellate ganglion (SG) containing muscarinic cholinoreceptors and purinoreceptors in rat pups of different ages (neonatal, 10, 20, 30, 60, and 180 days) using immunohistochemical methods. The results showed that in all animals, most neurons were immunoreactive to M1 cholinoreceptors and P2X2 and P2X6 purinoreceptors from birth. Sections showed isolated neurons containing P2X3 purinoreceptors, the numbers of which increased from day 10 of life to day 20 and then decreased again. During ontogenesis, the proportions of neurons expressing M1 cholinoreceptors and P2X2 and P2X6 purinoreceptors did not change significantly. Thus, by the moment of birth, muscarinic synaptic transmission already occurred in the SG in rats, while the final set of purinoreceptors in the neurons of this sympathetic ganglion formed by age 30 days.

Rhythmic electrical activity in branches of the stellate ganglion in the cat during postnatal ontogenesis.

Electrical activity in branches of the stellate ganglion was studied in neonatal cats and cats aged 10, 20, and 30 days and two and six months, with subsequent spectral analysis. Maturation of the pattern of the baseline rhythmic activity in branches of the ganglion differed during ontogenesis in the kittens. The neonatal period until the end of the second month of life was accompanied by an increase in the amplitude of electrical oscillations. Synchronous discharges in postganglionic fibers consisted of slow, low-amplitude oscillations at the respiratory frequency and heart rate in neonatal and 10-day-old-kittens. Synchronous spike activity with a frequency of about 10 Hz appeared from day 20 of life. The formation of the pattern of sympathetic nerve fiber discharges was complete by the second month of life.

Immunocytochemical characteristics of neurons in the stellate ganglion of the sympathetic trunk in mice during postnatal ontogenesis.

The neurotransmitter composition of neurons in the stellate ganglion of mice of different ages (neonatal and aged 10, 20, 30, and 60 days) was studied using an immunocytochemical method. Most of the neurons in the mice of these age groups contained tyrosine hydroxylase (TH). Most choline acetyltransferase (CAT)-positive neurons in neonatal and 10-day mice were also TH-positive. From birth and at all subsequent ages, there were increases in the proportion of cells contained TH and neuropeptide Y. The proportion of neurons containing vasoactive intestinal peptide and CAT increased to 10 days of life and then decreased. Somatostatin-and galanin-positive neurons were absent from all animals. Maturation of the set of neurotransmitters in stellate ganglia was complete by age two months.

[Rhythmical electrical activity in the cat stellate ganglion during postnatal ontogenesis].

Electrical activity of the stellate ganglion was studied in newborn, 10-, 20-, 30-day-old, two- and six-month-old kittens using the spectral analysis. The development of sympathetic activity patterns was different during ontogenesis. The amplitude of discharges increased from the period of birth until the second month of kittens' life. In newborn and 10-day-old kittens, synchronous discharges of postganglionic fibers were represented by slow and low frequency impulses with frequencies of breathing and heart rate. ppears in 20-day-old kittens. The formation of the sympathetic discharge patterns ends at the second month of animals life.

Preganglionic inputs to the stellate ganglion of the cat during postnatal ontogenesis.

The localization of preganglionic neurons forming, connections with the stellate ganglion was studied using a method based on retrograde axonal transport of horseradish peroxidase in cats during postnatal ontogenesis from birth to six months of life. The number of preganglionic neurons and spinal cord segments connected to the ganglion increased from birth to 10 days of age, and then decreased. The proportion of preganglionic neurons in different spinal cord nuclei changed during life and became comparable with that in adults by age six months. This was accompanied by an increase in the proportion of cells in the nucleus intermediolateralis thoracolumbaris pars principalis, with a decrease in the number of neurons in the nucleus intermediolateralis thoracolumbaris parts funicularis and in the ventral horns of the spinal cord.

[Immunocytochemical characteristic of neurons of the mouse truncus sympaticus stellate ganglion in postnatal ontogenesis].

Neurotransmitter content in stellate ganglion neurons was studied immunocytochemically in mice of different ages (newborns, 10-, 20-, 30- and 60-days-old). Majority of the stellate ganglion neurons in mice of all the groups studied were tyrosine hydroxylase (TH)-positive. Most of neurons with the immunore-activity to choline acetyltransferase (ChAT) were shown to be also TH-positive in the newborn and 10-day-old animals. The percentage of neurons containing TH and neuropeptide Y increased from birth throughout all the age periods studied. The proportion of vasoactive intestinal peptide (VIP)- and ChAT-positive neurons was maximal in 10-day-old animals and then decreased up to 60 days of age. The somatostatin- and galanin-reactive cells were absent in all the mice. Thus, the maturation of neurotransmitter composition is complete in the mouse stellate ganglion by the end of the second month of life.

Immunocytochemical properties of stellate ganglion neurons during early postnatal development.

Neurotransmitter features in sympathetic neurons are subject to change during development. To better understand the neuroplasticity of sympathetic neurons during early postnatal ontogenesis, this study was set up to immunocytochemically investigate the development of the catecholaminergic, cholinergic, and peptidergic phenotypes in the stellate ganglion of mice and rats. The present study was performed on Wistar rats and Swiss mice of different ages (newborn, 10-day-old, 20-day-old, 30-day-old, and 60-day-old). To this end, double labeling for tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), vasoactive intestinal (poly)peptide (VIP), neuropeptide Y (NPY), galanin (GAL), and somatostatin (SOM) was applied. The results obtained indicate that the majority of the neurons in the stellate ganglion of both species were TH-positive from birth onward and that a large part of these neurons also contained NPY. The percentage of neurons containing TH and NPY invariably increased with age up to 60 days postnatally. A smaller portion of the stellate ganglion neurons contained other types of neuropeptides and showed a distinct chronological pattern. The proportion of VIP- and ChAT-positive neurons was maximal in 10-day-old animals and then decreased up to 60 days of age, whereas the number of SOM-positive cells in rats significantly decreased from birth onward. In newborn rats, VIP-, ChAT- and SOM-positive neurons were largely TH-positive, while their proportions decreased in 10-day-old and older rats. Accordingly, the largest part of VIP-positive neurons also expressed SOM immunoreactivity at birth, after which the number of neurons containing both peptides diminished. The VIP- and SOM-positive cells did not contain NPY in any of the age groups studied. In rats up to 10 days of life, GAL-immunoreactive (-IR) neurons were scarce, after which their number increased to reach a maximal value in 30-day-old animals and then declined again. The SOM-reactive cells had the smallest size in all rats, while the largest neurons were those containing ChAT. In the mouse stellate ganglion, VIP- and ChAT-IR neurons were larger in comparison to NPY- and TH-IR cells. Our study further revealed some species differences: compared to mice the proportion of neurons containing TH and NPY was higher in rats at all ages under study. Furthermore, no GAL-immunostained neurons were found in mice and the number of SOM-positive cells in mice was limited compared to that observed in rats. In conclusion, the development of neurotransmitter composition is complete in rats and mice by their second month of life. At this age, the percentages of immunopositive cells have become similar to those reported in adult animals.

[Preganglionic inputs to cat stellate ganglion in postnatal ontogenesis]. / Preganglionarnye vkhody v zvezdchatyi uzel koshki v postnatal'nom ontogeneze.

Localization of the spinal cord preganglionic neurons projecting to the stellate ganglion, was studied in kittens from the moment of birth till six month of life, using the method based on retrograde axonal transport of horseradish peroxidase. The number of preganglionic neurons and spinal cord segments connected with the stellate ganglion, increased from the moment of birth till 10th postnatal day, decreasing afterwards. The proportion of preganglionic neurons in different nuclei of the spinal cord changed during the development and became comparable to that in adult animals by 6th month. The proportion of neurons in the nucleus intermediolateralis thoracolumbalis pars principalis was found to increase, while the number of neurons in the nucleus intermediolateralis thoracolumbalis pars funicularis and in the spinal cord ventral horns was decreased.

c-ret regulates cholinergic properties in mouse sympathetic neurons: evidence from mutant mice.

The search for signalling systems regulating development of noradrenergic and cholinergic sympathetic neurons is a classical problem of developmental neuroscience. While an essential role of bone morphogenetic proteins for induction of noradrenergic properties is firmly established, factors involved in the development of cholinergic traits in vivo are still enigmatic. Previous studies have shown that the c-ret receptor and cholinergic properties are coexpressed in chick sympathetic neurons. Using in situ hybridization we show now that a loss-of-function mutation of the c-ret receptor in mice dramatically reduces numbers of cells positive for choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) in stellate ganglia of homozygous newborn animals. The number of neurons positive for tyrosine hydroxylase (TH) mRNA, the rate-limiting enzyme of noradrenaline synthesis, is reduced to a smaller degree and expression levels are not detectably altered. Already at embryonic day 16 (E16), ChAT and VAChT-positive cells are affected by the c-ret mutation. At E14, however, ChAT and VAChT mRNAs are detectable at low levels and no difference is observed between wildtype and mutant mice. Our data suggest that c-ret signalling is necessary for the maturation of cholinergic sympathetic neurons but dispensable for de novo induction of ChAT and VAChT expression.

Histochemical features of neurons in the cat stellate ganglion during postnatal ontogenesis.

Changes in the distribution of NADPH-diaphorase (NADPH-d) and acetylcholinesterase (AChE) were studied in neurons of the stellate ganglion in newborn, 10-, 20-day-old, 1-, 2-, 4- and 6-month-old kittens. AChE-positive neurons were revealed in the stellate ganglion (SG) from birth onwards. The number of these neurons increased until 20 days of postnatal life and then declined in 1- and 2-month-old kittens. A small number of weakly stained, NADPH-d-positive cells were found in newborn kittens, while intensely stained neurons first appeared in 10-day-old animals and increased in number up to the second month of life. The size of AChE-positive neurons was larger in comparison with NADPH-d-positive cells in the stellate ganglion of all animals under study. We suggest that putative vasodilator neurons or cells innervating sweat glands exhibit different development patterns from the moment of birth.

Development of the evoked potentials in the thalamus and cerebral cortex after stimulation of the stellate ganglion afferents in kittens.

The primary evoked potentials (EP) were studied in newborn, 10-, 20-day-old, 1-month-old kittens in the thalamus and cerebral cortex after stimulation of the stellate ganglion (SG) afferents. The nerves were irritated with single square-wave pulses with an amplitude of 1.2-1.3 threshold for EP appearance. In all kittens, EP were registered in the same zones as in adult animals. In the thalamus, this zone was represented by the centromedial part of the ventralis posterior lateralis (n. VPL), contralaterally to the stimulation place. In the cerebral cortex, the EP were observed in the cerebral cortex, specifically in the somatosensory zones S1 and S2 also contralaterally to the stimulation place. In postnatal ontogenesis, the latency of evoked potentials decreases, amplitude increases and the form of these potentials transforms from negative in newborn kittens to positive-negative wave in 1-month-old animals. Maturation of the SG afferent pathways exists in parallel with the somatosensory ones and does not finish at 1 month of life.

The development of synaptophysin immunoreactivity in the human sympathetic ganglia.

Using an indirect immunohistochemical method, synaptophysin immunoreactivity (SYN-IR) has been studied in cryostat sections of stellate and thoracic ganglia in human fetuses, neonates, infants and adults. In the course of development, a progressive increase in SYN-IR in axonal terminals and around nerve cells was demonstrated. In contrast, large clusters of small intensely fluorescent (SIF) cells and paraganglionic cells increased in number in fetuses and premature neonates at 24-25 weeks. Such SIF cell clusters varied in form and often occurred at pole or subcapsular areas of sympathetic ganglia close to blood vessels or paraganglia. With increasing gestational age and during infancy, a decrease in sizes of SIF cell groups and paraganglionic cells as well as changes in their distribution were found. The results show that the amount and distribution of SYN-IR is temporally related to the maturation and functional activity of human sympathetic ganglia neurons. It was suggested that numerous SIF cells and paraganglia in human prenatal sympathetic ganglia were both indicative of incomplete cell migration and an important source of regulation of ganglionic microcirculation under the conditions of relative hypoxia and immature nervous regulation.

Sympathetic neurons of the cat stellate ganglion in postnatal ontogenesis: morphometric analysis.

Basic morphometric parameters (the maximal diameter, cross-sectional area), the distribution density of neurons were determined in the zones of the emergence of the basic nerves and in the center of the stellate ganglion (SG) in newborn, 10-, 20-day-old as well as 1- and 2-month-old kittens. Most of the investigated neurons in all animals were oval in their profile. In parallel to the increase of the average diameters, the number of small neurons decreased and the percentage of large neurons increased in postnatal ontogenesis. In all kittens, neurons with larger average size were located in the cranial pole of the ganglion. In 10-day-old kittens and older animals, the average size of neurons was more in the left SG in comparison to the right one. Only in 1-month-old kittens the density of location of neurons in the right ganglion exceeded such parameters of neurons located at the left side. The number of neurons in the SG was not constant in postnatal ontogenesis and reduced from newborn to 20-day-old animals.

Morphological features of neurons innervating different viscera in the cat stellate ganglion in postnatal ontogenesis.

Retrograde axonal transport of horseradish peroxidase (HRP) was used in this study to determine morphological parameters in the stellate ganglion (SG) in newborn, 10-, 20-day- and 1-month-old kittens. Neurons with the largest average size participated in innervation of the heart in newborn kittens and in innervation of the sternocleidomastoid muscle in other animals. The number of neurons sending their axons to target-organs also changed in postnatal ontogenesis. Regardless of the site of HRP injection at animals of all ages labeled neurons in the SG were located in certain zones on a topographical basis. Thus, it is concluded that in postnatal ontogenesis the neuronal organization of the SG changes in parallel to the increase of neuronal sizes and ganglion cross section area and practically finishes at 1 month of age.