Age-dependent changes of the autonomic neurons expressing vanilloid TRPV1 receptors

Expression of vanilloid receptors in sympathetic and afferent ganglionic neurons was studied in rats of different ages (newborn, 10-day old, 20-day old, 30-day old, 60-, 180-day old) using immunohistochemical methods. The results obtained indicate that the majority of the afferent neurons in the nodose ganglion of vagus nerve (GNVN) and in the spinal ganglia (SG) were TRPV1-positive from birth onwards. The percentage of neurons containing TRPVT receptors in SG slightly increased with age up to 30 days postnatally. In the GNVN, the percentage of TRPV1-positive neurons was higher in comparison with the SG in all age groups. The vast majority of the sympathetic neurons were TRPV1-positive from birth onwards, and the percentage of TRPV1-immunoreactive neurons substantially decreased during further development. In 20-day old and older animals, we observed only few TRPV1-immunoreactive neurons in sympathetic ganglia. Finally, the percentage of neurons containing these types of neurons, become similar to adult animals to the end of the first 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.     

Comparing the somal size and nuclear positions of the monkey stellate and coeliac ganglion cells

The stellate and coeliac ganglia of 2 Macaque monkeys were cut serially at 1 micron thickness and analysed. Results from the analysis of 82 stellate and 60 coeliac ganglion cells in 1 monkey show that in cross-sections, the neuronal nuclei may be eccentric, centric or nearly centric and remain so throughout the longitudinal extent of the neuron. In both ganglia, the majority of neurons possess eccentric nuclei, but in the coeliac ganglion, the percentage of neurons with centric and/or nearly centric nuclei is higher (41.7%) than that in the stellate ganglion (26.3%). While 5% of neurons in the coeliac ganglion are binucleated, no binucleated neurons were found in the stellate ganglion. The somal size ranges of the stellate (10-39 microns) and the coeliac (14.5-45 microns) ganglion neurons as obtained from both monkeys are quite close. The percentage frequency distribution of the stellate ganglion neurons in monkey 1 was also quite similar to that of the coeliac ganglion neurons. It is concluded that different neuronal size is not likely to be associated with different target organs.     

Connections of the cat stellate ganglion with target organs during postnatal ontogenesis

The enzyme horseradish peroxidase was administered into trachea, esophagus, heart, sternocleidomastoid muscle, and biceps of newborn, 10-, 20-day old kittens, and adult cats. The labeled neurons were located on the topical basis of the stellate ganglion. In newborn kittens, the largest cells take part in the heart innervation. In animals of other ages, the largest cells take part in innervation of the sternocleidomastoid muscle. The findings suggest that, in postnatal ontogenesis, neuronal organisation of the stellate ganglion is changing parallel to an enhancement of the neurons' size and ganglion section area.     

Phenotypic alterations of neuropeptide Y and calcitonin gene-related peptide-containing neurons innervating the rat temporomandibular joint during carrageenan-induced arthritis

The aim of this study was to identify immunoreactive neuropeptide Y (NPY) and calcitonin gene-related peptide (CGRP) neurons in the autonomic and sensory ganglia, specifically neurons that innervate the rat temporomandibular joint (TMJ). A possible variation between the percentages of these neurons in acute and chronic phases of carrageenan-induced arthritis was examined. Retrograde neuronal tracing was combined with indirect immunofluorescence to identify NPY-immunoreactive (NPY-IR) and CGRP- immunoreactive (CGRP-IR) neurons that send nerve fibers to the normal and arthritic temporomandibular joint. In normal joints, NPY-IR neurons constitute 78±3%, 77±6% and 10±4% of double-labeled nucleated neuronal profile originated from the superior cervical, stellate and otic ganglia, respectively. These percentages in the sympathetic ganglia were significantly decreased in acute (58±2% for superior cervical ganglion and 58±8% for stellate ganglion) and chronic (60±2% for superior cervical ganglion and 59±15% for stellate ganglion) phases of arthritis, while in the otic ganglion these percentages were significantly increased to 19±5% and 13±3%, respectively. In the trigeminal ganglion, CGRP-IR neurons innervating the joint significantly increased from 31±3% in normal animals to 54±2% and 49±3% in the acute and chronic phases of arthritis, respectively. It can be concluded that NPY neurons that send nerve fibers to the rat temporomandibular joint are located mainly in the superior cervical, stellate and otic ganglia. Acute and chronic phases of carrageenan-induced arthritis lead to an increase in the percentage of NPY-IR parasympathetic and CGRP-IR sensory neurons and to a decrease in the percentage of NPY-IR sympathetic neurons related to TMJ innervation.Key words: trigeminal ganglion, otic ganglion, superior cervical ganglion, arthritis, temporomandibular joint.     

Melanogenesis in cultures of peripheral nervous tissue. II. Environmental factors determining the fate of pigment-forming cells

Spinal ganglia from 4- to 7-day [Stage 23–30; Hamburger and Hamilton (1951) J. Morphol.88, 49–92] chicken embryos were cultured in vitro to investigate the effect of various environmental conditions on cell differentiation. Culture morphology (i.e., degree of dispersion of the explanted ganglia, survival of neurons, and outgrowth of axons) was observed to depend upon several factors including: (1) the age of the explanted ganglia, (2) the presence or absence of nerve growth factor (NGF), and (3) the nature of the substratum on which the cultured tissue resides. These observations enabled us to disturb the association of neurons with the other cells in ganglion cultures and thereby modulate the differentiation of adventitious melanocytes. Thus, in medium permissive for melanogenesis, melanocytes appear when the association between neurons and small stellate nonneuronal cells in the ganglion is disrupted. This disruption is most extensive (1) when young (Stage 26–27, 5-day) ganglia are explanted on plastic substrata, in the initial absence of NGF, and (2) when cells from enzyme-dissociated ganglia are cultured on plastic substrata. In comparable media, pigment cell differentiation is not observed when the association between neurons and small stellate cells is preserved. Such associations tend to endure (1) in developmentally older (Stage 30+, 7- to 8-day) ganglia or (2) when ganglia are cultured on agar or fibroblast substrata. We conclude that loss of association between neurons and the nonneuronal cells in young ganglia is necessary for the latter to undergo melanogenesis in vitro.     

Autoradiographic study of 3H-DOPA uptake by superior cervical and stellate ganglia of spontaneously hypertensive rats during the prehypertensive stage

The functional state of sympathetic ganglia in spontaneously hypertensive rats (SHR) was compared with that of ganglia in normotensive Wistar Kyoto rats (WKY) by examining catecholamine synthetic activity by light microscopic autoradiography 3H-L-dihydroxyphenyl alanine (3H-DOPA). The number of silver grains over the perikarya of ganglion cells in the superior cervical (SCG) and stellate ganglia (SG) of newborn, 10-day-old and 30-day-old animals was counted on photographic enlargements. There were significantly more silver grains over ganglion cells in SHR compared with those in age-matched WKY at almost all incorporation times at all ages examined in SCG, at all incorporation times in newborn rats, and at incorporation times of 15 and 60 min in SG of 10-day-old rats. The increased incorporation of the label by both sympathetic ganglia was more marked in newborn than in 30-day-old animals. These results indicate that catecholamine synthetic activity in these ganglion cells is increased in SHR from the newborn stage, suggesting that a congenital hyperfunction of sympathetic ganglia occurs in SHR.     

DNA content of the mitochondria of the developing rat brain

Studies have been made on the content of DNA in the mitochondria from the brain of 1, 5, 10, 15, 21, 60, and 400 days old rats. Isolated DNA preparations contained 7% of RNA and 3% of proteins. It was shown that the amount of DNA in 21- and 60-day rats is 4 times higher that than in newborn ones. DNA content of the mitochondria increases during the first 3 weeks of postnatal life of rats.     

Iron metabolism and erythropoiesis in the prenatal and early postnatal periods of the rat

Spectrophotometric studies have been made on iron balance in the liver, spleen, bone marrow, and blood serum of 15-, 17- and 20-day rat embryos, as well as 1-90 days old rats. It was shown that foetal period is the main one for the formation of iron-accumulating function of the liver. Anaemic period in 14-28-day rat puppies results from insufficient hemoglobin synthesis, rather than from iron deficiency in the organism. The latter is observed in 35-60-days old rats. Maturation of the spleen as the organ involved in reutilization of iron was noted in 42-60-day rats.     

3H-GABA uptake and acetylcholinesterase activity in dissociated cell cultures of the medial hypothalamus.

Medial hypothalamic tissue of 1 to 4 days old rats was dissociated and cultured in vitro for 8--10 days. Neuronal perikarya were demonstrated by supravital methylene blue staining and electron microscopy. Synapses with typical vesicles and subsynaptic thickening were also observed. 3H-GABA was taken up into a small percentage of the cells in the cultures. Neuron-like perikarya and long processes accumulated the label while many neurons contained much less activity. Some astroglial and oligodendroglial cells and processes also accumulated GABA. A few neurons in these cultures contained acetylcholinesterase. It is concluded that neurons concentrating GABA and containing acetylcholinesterase are present in the hypothalamus of rats of 1 to 4 days of age and can be maintained in dissociated cell culture.     

DNA repair in mammalian nerve cells. II. DNA synthesis in neurons, induced by gamma-irradiation of isolated rat neocortex slices

DNA synthesis was studied in the neocortex neurons of 0-, 14- and 60-day old rats after gamma-irradiation in vitro of isolated slices of the neocortex on determining 3H-thymidine incorporation into DNA. Gamma-irradiation (20 Grays) increases levels of DNA synthesis for all the age groups of animals examined. The induced DNA synthesis in neurons of newborn rats is higher than that in 14- and 60-day old animals. Specificity of DNA synthesis in the process of nerve cell differentiation is discussed.     

Horseradish peroxidase localization of sympathetic postganglionic and parasympathetic preganglionic neurons innervating the monkey heart

The localization of the sympathetic postganglionic and parasympathetic preganglionic neurons innervating the monkey heart were investigated through retrograde axonal transport with horseradish peroxidase (HRP). HRP (4 mg or 30 mg) was injected into the subepicardial and myocardial layers in four different cardiac regions. The animals were euthanized 84-96 hours later and fixed by paraformaldehyde perfusion via the left ventricle. The brain stem and the paravertebral sympathetic ganglia from the superior cervical, middle cervical, and stellate ganglia down to the T9 ganglia were removed and processed for HRP identification. Following injection of HRP into the apex of the heart, the sinoatrial nodal region, or the right ventricle, HRP-labeled sympathetic neurons were found exclusively in the right superior cervical ganglion (64.8%) or in the left superior cervical ganglion (35%). Fewer labeled cells were found in the right stellate ganglia. After HRP injection into the left ventricle, labeled sympathetic cells were found chiefly in the left superior cervical ganglion (51%) or in the right superior cervical ganglion (38.6%); a few labeled cells were seen in the stellate ganglion bilaterally and in the left middle cervical ganglion. Also, in response to administration of HRP into the anterior part of the apex, anterior middle part of the right ventricle, posterior upper part of the left ventricle, or sinoatrial nodal region, HRP-labeled parasympathetic neurons were found in the nucleus ambiguus on both the right (74.8%) and left (25.2%) sides. No HRP-labeled cells were found in the dorsal motor nucleus of the vagus on either side.     

Substance P-like immunoreactivity in the trigeminal ganglion. A fluorescence, light and electron microscope study

The trigeminal ganglion of rat and guinea pig was studied for the presence of immunoreactive substance-P using fluorescence, light and electronmicroscopy. In untreated animals substance P containing cells, with a diameter of 15 to 50 micron, were distributed throughout the ganglion and comprised 10-30% of all ganglion cells. Colchicine, injected intraventricularly to inhibit intra-axonal transport, had no effect on the number of substance P cells; but when the drug was injected directly into the posterior root of the ganglion, the proportion of these cells increased to as much as 50%. In the electron microscope, immunoreactive substance-P was confined to ganglion cells classified as B type according to the arrangement of subcellular organelles, and to unmyelinated nerve fibers. Subcellularly the immunoreactivity appeared in cytoplasmic vesicles, as well as dispersed in the nerve fibers and the perikarya of neurons. The great number of substance P immunoreactive ganglion cells suggests that they do not comprise a well defined subpopulation of the B-cells. However, the immunoreactivity was restricted to a distinct ultrastructural type of neurons with unmyelinated nerve fibers, suggesting that they also may share some distinct functions.     

Distribution and changes with age of nitric oxide synthase-immunoreactive nerves of the rat urinary bladder, ureter and in lumbosacral sensory neurons

In the distal parts of the urinary tract, nerves containing nitric oxide (NO) are either postganglionic parasympathetic nerves, with cell bodies in the major pelvic ganglia, or sensory nerves with cell bodies in the lumbosacral dorsal root ganglia. We have used indirect immunohistochemical techniques to examine the distribution and regional variation of nerves immunoreactive for neuronal nitric oxide synthase (NOS) in the urinary bladder, distal ureter and in neurons in lumbosacral dorsal root ganglia (L1-L2 & L6-S1) of young adult (3 months) and aged (24 months) male rats. Semi-quantitative estimations of nerve densities were made of NOS fibres innervating the dome, body and base of the urinary bladder and distal ureter. Quantitative studies were also used to examine the effects of age on the percentage of dorsal root ganglion neurons immunoreactive for NOS. The dome and the body regions, in both age groups, contained no NOS-immunoreactive axons. The bladder base and distal ureter in young adults showed sparse to moderate numbers of fibres immunoreactive to NOS within the urothelium and in the subepithelium and muscle coat. In the aged rat there were slight reductions in the densities of NOS-immunoreactive nerves in all three regions. In the lumbosacral dorsal root ganglia, the percentage of NOS-immunoreactive neuronal profiles showed a significant reduction from 4.6 +/- 0.2% in young adult to 2.7 +/- 0.2% (means +/- S.E.M) in aged rats. These findings suggest that the effects of NO on the bladder and distal ureteric musculature and also its expression in dorsal root ganglion neurons are affected in aged rats and that the micturition reflex may be perturbed as a result.     

Age-related changes in sensory neurons containing calcitonin gene-related peptide under conditions of afferentation deficit in rats

Morphological features of calcitonin gene-related peptide (CGRP)-immunoreactive neurons were studied in the sensory ganglia of the vagus and thoracic nerves in 3-, 10-, 20-, 30-, 60-, 90-, and 180-day-old rats under conditions of chemically-induced deafferentation. We found that, in rats, CGRP-containing neurons appeared in both ganglia immediately after they were born and their number decreased with aging. Most of CGRP-immunoreactive neurons were small in size, i.e., up to 600 ??m². Administration of capsaicin modified age-related changes in the number of CGRP-immunopositive neurons. In the thoracic nerve ganglion, the mean square of these cells and their number substantially decreased, whereas, in the vagus nerve ganglion, positive cells were not observed.     

Postnatal development of the rat intrinsic cardiac nervous system: a confocal laser scanning microscopy study in whole-mount atria

We used confocal laser scanning microscopy and fluorescent immunohistochemistry to study the developmental pattern and distribution of specific neuronal phenotypes within the intrinsic cardiac nervous system in whole-mount atrial preparations from newborn to 5 week old rats. Individual ganglia and neuronal cell bodies were localized by means of two general neuronal markers: protein gene product 9.5 (PGP) and microtubule-associated protein two (MAP). In rats < or =2 weeks old there were two main subpopulations of intrinsic neurons located in the intraatrial septum and around the origin of the superior vena cava. The more abundant was a population of strongly tyrosine hydroxylase (TH) immunoreactive (IR) neurons (10-40 microm in diameter) most of which were also PGP-IR. The second, less numerous (approximately 60-70% than the TH-IR group) type of neurons exhibited ChAT-IR which colocalized with MAP-IR. Towards the end of the second postnatal week and during the third, the ganglia containing these neurons became more numerous and their localization also included tissues around the origins of the inferior vena cava and the pulmonary veins, as well as both atrial walls close to the AV junction. During the second and third postnatal weeks, when the extrinsic innervation of the adrenergic and cholinergic phenotypes largely increases, the intrinsic innervation also changed greatly, and around the 21st postnatal day it appeared to acquire mature characteristics. The TH-IR neurons changed their characteristics and formed two types of ganglia. The larger ganglia containing large cells (20-40 microm in diameter) expressed TH-IR mostly close to their inner body surface (approximately 80-90% of identified neurons). Most of these neurons also expressed neuropeptide Y (NPY)-IR, specifically around their nuclei. The second type of small strongly TH-IR neurons (approximately 10% of all identified neurons) were contained in smaller groups (20-50 cells) which were usually embedded into much larger ganglia (100-400 cells), containing large (20-50 microm) neurons. Unlike all other intrinsic neurons, these small TH-IR cells did not exhibit any PGP-IR or MAP-IR. The number of ChAT-IR neurons increased at this stage, reaching approximately 90% of the neurons identified by the general neuronal markers. These neurons were surrounded by a rich network of cholinergic varicose nerve fibers, some of which were likely of an extrinsic origin. We have also identified relatively small ganglia expressing immunoreactivity to vasoactive intestinal polypeptide (VIP), and to substance P (SP). The presented data indicate that the phenotypes of intrinsic neurons in the rat heart change greatly during the first month of postnatal development. This may be at least partially related to the development and maturation of functional extrinsic nervous control of the heart.     

The chicken trigeminal ganglion. I. An anatomical analysis of the neuron types in the adult

An anatomical analysis of the chicken trigeminal ganglion was made using light microscopy on specimens prepared by usual chemical fixation or freeze-drying methods and by electron microscopy. Two types of neurons were consistently seen, dark and light cells. Dark cells contained a dense cytoplasm with Nissl substance distributed evenly throughout, whereas light cells had a less dense cytoplasm containing clumps of Nissl substance. The Nissl bodies in light cells contained only a few small cisternae of granular endoplasmic reticulum as compared with many stacked cisternae in Nissl bodies of dark cells. The ratio of dark to light cells was approximately 62:38 in all regions of the ganglion. Dark cells were consistently smaller than light cells. In the seven-day old chick, the mean diameters of the dark and light neurons were 21.4 μ and 29.5 μ respectively; in the adult the values were 29.9 μ and 39.7 μ respectively. It is concluded that the dark and light cells belong to two distinct neuronal cell populations.     

Properties and kinetics of a population of B-lymphocytes during rat ontogenesis]

Cells bearing surface immunoglobulins (Ig+-cells) detected by the indirect immunofluorescent method and cells forming rosettes (RFC) with sheep erythrocytes coated with antibody and complement (EAC rosettes) were found in the liver and the spleen on the 15th and the 20th days of prenatal life of rats. The percentage of Ig+-cells and RFC in the liver was high and remained unchanged and at about the same level during the whole postnatal life. The spleen and the bone marrow displayed an increase of the Ig+-cells and RFC increased throughout the 1st month of postnatal life with the maximum at the 30th day after birth; a sharp decrease occurred in old animals. In the thymus the Ig+-cells and the RFC were either absent or present in very small amounts only at some periods of study. Ig+-cells with "capping" were discovered in the spleen and bone marrow on the 5th--10th days of postnatal life; their count increased considerably in 30-day and adult rats. Such cells were absent in the lymphoid tissues of old 40-month rats.     

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.     

Differentiation of noradrenergic traits in the principal neurons and small intensely fluorescent cells of the parasympathetic sphenopalatine ganglion of the rat

Catecholamine synthetic enzymes are found in many cranial parasympathetic principal neurons, and in the small intensely fluorescent (SIF) cells that populate parasympathetic as well as sympathetic ganglia. While there is evidence that the acquisition of noradrenergic properties in sympathetic neuron precursors depends on factors that these cells encounter in the trunk environment, the mechanisms that direct the development of noradrenergic traits in cranial parasympathetic neurons and SIF cells are not understood. The present study examines the time course of appearance of tyrosine hydroxylase (TH) immunoreactivity in the principal neurons and SIF cells of the rat sphenopalatine ganglion. We show that the sphenopalatine ganglion of normal adult rats contains both a small population of TH-immunoreactive principal neurons and many SIF cells. The TH-immunoreactive principal neurons do not synthesize or store detectable catecholamines, even though the majority of sphenopalatine ganglion neurons do contain 1-amino acid decarboxylase catalytic activity. Sphenopalatine ganglion principal neurons do not accumulate detectable levels of exogenous catecholamines. This observation suggests that they lack a high affinity norepinephrine uptake system. In contrast to what has been observed previously for sympathetic neurons, the appearance of TH immunoreactivity in sphenopalatine neurons is not temporally correlated with the cessation of neural crest cell migration. The first TH-immunoreactive neurons do not appear in the sphenopalatine ganglion until Embryonic Day 16.5, 2 days after the ganglion has condensed and process outgrowth has begun. The number of sphenopalatine neurons that express TH immunoreactivity increases dramatically between Embryonic Day 18.5 and Postnatal Day 1, but then decreases. In fact, the percentage of sphenopalatine neurons that express TH immunoreactivity is almost fivefold higher in newborn than in adult rats. SIF cells cannot be definitively identified in the sphenopalatine ganglion until after Embryonic Day 18.5. The time course of appearance of TH immunoreactivity in sphenopalatine ganglion cells raises the possibility that TH expression is stimulated in these cells by factors encountered either at their condensation site or at their target, such as glucocorticoids or nerve growth factor. The relatively late appearance of SIF cells in the sphenopalatine ganglion argues against the hypothesis that SIF cells are the precursors of all autonomic neurons.