Expression of GAD65/67 and VGLUT2 in Mediobasal Nuclei of Rat Hypothalamus during Aging.

The expression of glutamate decarboxylase GAD65/67, an enzyme of GABA synthesis, and vesicular glutamate transporter 2 (VGLUT2) in the arcuate, dorsomedial, and ventromedial nuclei of the hypothalamus of young (3 months), adult (12 months), and old male rats (24 months) was studied by Western blotting. In old rats, an increase in the expression of GAD65/67 in the arcuate and dorsomedial, VGLUT2 in the arcuate, dorsomedial, and ventromedial nuclei was observed. Thus, an increase in opposite processes of inhibition and excitation is observed in the hypothalamic nuclei during aging.

Changes in the Expression of Calbindin and Calretinin in Interneurons of the Spinal Dorsal Horns Under Conditions of Antiorthostatic Suspension in Mice.

In female C57Bl/6 mice subjected to antiorthostatic suspension of the hind limbs for 30 days, calbindin- and calretinin-containing interneurons of the dorsal horns of the upper thoracic segments of the spinal cord were studied using immunohistochemical methods. In mice of the experimental group, cross-sectional area of calbindin- and calretinin-containing interneurons decreased in laminae I, II, and III and increased in laminae IV and V and in the region of the medial edge of the dorsal horn. After antiorthostatic suspension, expression of calretinin decreased in interneurons of laminae I and II and calbindin expression increased in the interneurons of laminae III, IV, and V. The total number of interneurons in laminae of the spinal cord detected by Nissl staining in the control and experimental groups remained unchanged. After antiorthostatic suspension and in control group, number of interneurons immunoreactive for calbindin and calretinin was maximum in lamina II and minimum in laminae IV and V and in the region of the medial edge of the spinal dorsal horn.

Sympathetic Innervation of Stomach in Postnatal Development.

Sympathetic innervation of the stomach was studied in rats by the method of retrograde axon transport of Fast Blue in postnatal ontogenesis. The number of labeled neurons increased in the first 10 days of life and then did not change until the senescence. All labeled neurons innervating the stomach contain the catecholamine synthesis enzyme, tyrosine hydroxylase. The proportion of labeled neuropeptide Y-immunopositive neurons did not change in the development, the percentage of labeled calbindin-immunoreactive neurons decreased in the first month of life.

[Age-development changes of the sympathetic innervation of the rat stomach.]

Sympathetic innervation of the stomach is carried out by the prevertebral ganglia of the solar plexus. The localization and neurochemical composition of neurons innervating the stomach in postnatal ontogenesis in rats was studied using the method of retrograde axon transport of Fast Blue. In all animals, the celiac ganglia had more labeled neurons compared to the superior mesenteric ganglion. The number of labeled neurons increased in the first 10 days of life and then did not change until the senescence. All labeled neurons innervating the stomach contain the catecholamine synthesis enzyme, tyrosine hydroxylase. The proportion of labeled neuropeptide Y-immunopositive neurons did not change in the development, the percentage of labeled calbindin-immunoreactive neurons decreased in the first month of life.

[Age-related expression of calcium-binding proteins in autonomic ganglionic neurons].

Calbindin 28 kDa (CB), calretinin (CR) and parvalbumin (PB) are belonged to calcium-binding proteins which are widely distributed in the nervous system and selectively expressed in certain population of neurons. These proteins are expressed not only in the central nervous system, but also in the autonomic ganglia. CB and PB are found in the sympathetic ganglia of rodents, CB and CR are found in metasympathetic intramural ganglia. Their functions are poor understood but one can suggest their important role in regulation of the Ca2+ level in the cell. Сalcium-binding proteins are also play an important role in the development of autonomic neurons. There is an increasing of the percentage of CB and CR in the metasympathetic intramural ganglia of small intestine in the early postnatal development, whereas in sympathetic ganglia the percentage of CB is decreased. Possibly, the functional meaning of such changes can be explained by the role of calcium currents in the development of neurons and the synaptic transmission.

[Developmental changes of neurotransmitter properties in sympathetic neurons].

Sympathetic ganglia consist of neurochemically and functionally distinct populations of neurons, characterized by a specific projection pattern and a set of neutransmitters including classical mediators (catecholamines and acetylcholine), neuropeptides and small molecules such as NO, H2S, CO. The majority of the principal ganglionic sympathetic neurons is noradrenergic and expresses tyrosine hydroxylase (TH), i.e., a key enzyme in catecholamine synthesis. In mammals, two third of catecholaminergic neurons also co-localizes neuropeptide Y. A small number of ganglionic sympathetic neurons contains enzyme of acetylcholine synthesis and some neuropeptides, such as somatostatin, vasoactive intestinal (poly)peptide (VIP), calcitonin gene-related peptide (CGRP). Acetylcholine-containing sympathetic neurons in most cases colocalize VIP and/or CGRP. Phenotype of autonomic neurons is regulated by both target-independent and target-dependent mechanisms. The most of transmitters are expressed during embryogenesis. TH appears during embryonic development and the percentage of TH-positive neurons remains virtually identical during ontogenesis. After birth, cholinergic neurons exhibit a noradrenergic phenotype. Expression of different neuropeptides changes in pre- and postnatal development. Neurotransmitter expression in sympathetic neurons is influenced by growth factor signaling via innervated target tissues. Multiple growth factors including bone morphogenetic proteins, neurotrophins, glial cell line-derived neurotrophic factor family ligands and neuropoietic cytokines play instructive role at different stages of neurotransmitter development.

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.

Age-associated changes in sympathetic neurons containing neurofilament 200 kDa during chemical deafferentation.

Neurofilament with a molecular weight of 200 kDa is detected in the rat sympathetic ganglia since birth. The percentage of neurons containing this neurofilament decreases during the first 20 days of life. Just solitary neurofilament-positive neurons are detected in rats at the age of 180 and 360 days. Chemical deafferentation by capsaicin, used as a model of age-associated neuron degeneration, leads to a significant reduction of the level of neurofilament-200-imminopositive neurons in comparison with the control starting from day 10 of life. Presumably, part of the sympathetic ganglionic neurons are capsaicin-positive and their function is afferent.

[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.

[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.

[Age factors in the background electrical activity of the superior cervical ganglion neurons in rats].

Background discharges of single neurons were studied from the superior cervical ganglion in newborn, 10-, 20-day-old, 1-, 2- and 6-month-old rats. In all age groups, the largest proportion of neurons exhibited aperiodic activity. The percentage of neurons with respiratory rhythmic was less. In newborn and 10-day-old rats, the frequency of discharges was low. Discharge frequency increased in 20-day-old rats. In 20-day-old and more adult rats, we found neurons bursting with cardiac frequency. The means of frequency did not statistically differ in 1-, 2- and 6-month-old rats. Thus, the pattern of neuronal activity is formed during the development in 20-day-old rats. Final maturation of this pattern is observed in 1-month-old rats.

Age-related changes in rhythmic electrical activity in the cervical sympathetic trunk in rats and cats.

Baseline electrical activity in the cervical sympathetic trunk was studied in neonatal rats and cats and at ages 10, 20, and 30 days and two and six months, using spectral analysis. Rats from the neonatal period to the end of the first month of life and cats to 20 days of life showed increases at the amplitudes of electrical oscillations. From birth, all animals showed oscillations in the respiratory and cardiac rhythms. From day 20, frequencies with a cardiac component in rats dominated the power spectrum. The proportion of other frequencies, not associated with the cardiac or respiratory rhythms, was smaller. In cats, unlike the situation in rats, there were no age-related changes in the spectral composition of baseline electrical activity. High-frequency oscillations were recorded in cats from birth.

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.

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.

[Age development of the rhythmical electrical activity in the cervical, sympathetic trunk in rats and cats].

Electrical activity of the cervical sympathetic trunk was studied in newborn, 10-, 20-, 30-day-old, two- and six-month-old rats and cats using the spectral analysis. The amplitude of discharges increased from the period of birth until the first moth of life in rats and until 20 days in cats. Frequencies synchronous with cardiac and respiration those were revealed in all animals. Heart-related frequencies were predominant in rats older than 20 days. Other frequencies were lower. In kittens, we did not observe any age-dependent changes of the spectrum of sympathetic nerve discharge. Discharges with high frequencies were observed in cats from the moment of birth.

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.

Efferent innervation of the cervical segment of the trachea in early postnatal ontogenesis.

A method based on retrograde axonal transport of horseradish peroxidase was used to study the efferent innervation of the cervical segment of the trachea in neonatal kittens and kittens aged 10, 20, and 30 days and two months. Labeled neurons in all animals were located in the cranial cervical, middle cervical, and cervicothoracic sympathetic ganglia on the right and left sides, at the level of the medulla oblongata, and in the dorsal and ambiguus nuclei of the vagus nerves. Up to age 30 days, neurons were also seen in the ventral horns of the spinal cord in segments C1 to C5. The number of sympathetic neurons innervating the trachea increased from the moment of birth, reaching a maximum by 10-20 days and then decreasing to age two months. The number of parasympathetic neurons gradually decreased during ontogenesis.

[Development of rat stellate ganglion neurons, containing membrane muscarinic and purinoreceptors].

The aim of this study was to determine the localization and morphometric characteristics of the stellate ganglion (SG) neurons containing muscarinic and purinoreceptors in rats of different ages (newborn, 10-, 20-, 30-, 60-, and 180-day-old) using the immunocytochemical methods. The results obtained indicated that in all the animals studied, the major part of neurons contained immunopositive P2X2, P2X6 purinoreceptors and M1 cholinoreceptors since birth onwards. A few of the neurons containing P2X3 purinoreceptors were detected in all the stages of postnatal development; these neurons were scarce in rats up to 10 days of life, after which their number increased to reach a maximal value in 20-day-old animals and then declined again. No significant changes were found in the proportion of neurons expressing M1 cholinoreceptors and P2X2, P2X6 purinoreceptors during the ontogenesis. Thus, the muscarinic synaptic transmission was already present in SG of rats by the time of their birth, while the final set of purinoreceptors on the neurons of sympathetic ganglion was formed by the age of 30 days.

[Efferent innervation of the cervical region of the trachea during the early postnatal ontogenesis].

Method based on retrograde axonal transport of horseradish peroxidase was used to study the efferent innervation of the cervical trachea in neonatal, 10-, 20-, 30-day-old and two-month-old kittens. In all the animals, labeled neurons were localized in the cranial cervical, middle cervical and cervicothoracic (stellate) sympathetic ganglia both on the right and on the left sides. Neurons were also localized at the level of medulla oblongata, in the dorsal and ambiguus nuclei of the vagus nerve. Labeled neurons were also found in the ventral horns of C(I-)C(V) spinal segments in the animals before 30 days of life. The number of sympathetic neurons, innervating the trachea, cells increased from the moment of birth, reaching a maximum by the age of 10 and 20 days and then declined up to the second month of life. The total number of parasympathetic neurons decreased gradually in the early development.