[Age-related changes in AKT and mTOR expression in mediobasal hypothalamus neurons with aging.]

The hypothalamus is a regulatory center that controls homeostasis, reproduction, circadian rhythms, and the endocrine system. It is also involved in the regulation of aging. Expression of protein kinase B (AKT), the mammalian target of rapamycin (mTOR), and phosphorylated forms of AKT (pAKT) and mTOR (pmTOR) was studied by Western blotting in 3-, 12-, and 24-month-old male rats in the arcuate (ARN), dorsomedial (DMN) and ventromedial (VMN) nuclei of the hypothalamus. The results showed that the components of AKT/mTOR signaling change differently in the mediobasal hypothalamic nuclei of rats with age. The expression of AKT and pAKT decreased in the ARN, DMN, and VMN with aging; the expression of mTOR and pmTOR increased in the ARN, but decreased in the DMN and VMN in aged rats. The results obtained can serve as a basis for future studies of the mechanisms of age-related diseases and their pharmacological treatment.

Sirtuin 1 Expression in the Rat Ventromedial and Dorsomedial Hypothalamic Nuclei during Ageing.

We studied the expression of sirtuin 1 (Sirt1) in the dorsomedial and ventromedial nuclei of the hypothalamus in young (2 months) and old (2 years) rats by immunohistochemical methods and Western blotting. In aged males and females, a decrease in Sirt1 expression in dorsomedial nucleus was observed. In ventromedial nucleus, the expression of Sirt1 did not change with age. The results confirm the hypothesis that aging is associated with a decrease in the content of sirtuins in the hypothalamic nuclei.

Changes of the Expression of Neuronal NO-Synthase in Rat Sympathetic Ganglia during Ontogeny.

Expression of neuronal NO synthase in the sympathetic cranial cervical ganglion and stellate ganglion in rats during postnatal ontogeny was studied by immunohistochemistry and Western blotting. In the sympathetic ganglia, neuronal NO synthase-immunoreactive neurons were absent in all rats. In the stellate and cranial cervical ganglia, the expression of neuronal NO synthase and the density of immunoreactive fibers increased in early postnatal ontogeny from the moment of birth to the age of 30 days and then decreased. Thus, we observed heterochroneous expression of neuronal NOS in the preganglionic somata in the spinal cord and in the preganglionic fibers in the sympathetic ganglia during ontogeny.

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.

[Developmental changes of neurotransmitter properties in sympathetic neurons].

Neurochemical composition of metasympathetic nervous system is characterized by a large variation. The main part of the intramural ganglionic neurons is cholinergic. Along with cholinergic neurons, there are ganglionic neurons containing serotonin, histamine, GABA, and several peptides: cholecystokinin, dynorphin, enkephalin, galanin, gastrin-releasing peptide (bombesin in mammals), neuropeptide Y, neurotensin, somatostatin, tachykinins, neurokinin A, vasoactive intestinal polypeptide and calcitonin gene related peptide. Gases as NO, CO, H2S, also act as neurotransmitters. Separate groups of neurons differ in the content of neuronal calcium-binding proteins, such as calbindin, calretinin and parvalbumin and neurofilaments: low molecular weight, a medium molecular weight and high molecular weight. Neurons of the enteric ganglia are the most different by their neurochemistry. There is a species difference in the ganglia of large animals and humans there are more combinations of chemical transmitters. Synthesis of neurotransmitters takes place even in the embryonic period and by the time of birth the most of neurons contain acetylcholine. In postnatal ontogenesis, the proportion of neurons expressing the NO-synthase decreases in the enteric and cardiac intramural ganglionic neurons. The functional significance of these changes is unclear.

Changes in the Neurochemical Composition of Motor Neurons of the Spinal Cord in Mice under Conditions of Space Flight.

Expression of choline acetyltransferase, 200-kDa neurofilament protein, 28-kDa calbindin, neuronal NO synthase, caspase 3, and Ki-67 in the motor neurons of spinal cord segments T3-T5 in male C57Bl/6 mice after 30-day space flight in the Bion-M1 biosatellite was studied by immunohistochemical methods. Under conditions space flight, the size of motoneurons increased, the number of neurons containing choline acetyltransferase and neurofilaments, decreased, and the number of calbindin-positive neurons increased; motoneurons, expressing neuronal NO synthase and caspase 3 appeared, while Ki-67 was not detected. Fragmentation of neurons with the formation structures similar to apoptotic (residual) bodies was observed in individual caspase 3-positive motoneurons.

[CHANGES OF SPINAL MOTOR NEURONS IN MICE AFTER А SPACE FLIGHT].

The expression of choline acetyltransferase (ChAT), neurofilament (NF) protein 200 kDa, calbindin (CAB) 28 kDa, neuronal NO-synthase (nNOS), caspase 3, Ki-67 was studied in motor neurons from TIII­TV segments of the spinal cord in C57/ BL6 male mice by immunohistochemical methods 12 h after a 30 days-long space flight on the Bion-M1 biosatellite. Mice living under standard vivarium conditions served as a control. The motoneurons of experimental animals demonstrated the reactive changes that were manifested by the increase of their size, decrease in the number of subpopulations expressing ChAT and NF, increase of subpopulations containing CAB, appearance of motor neurons expressing nNOS, caspase-3, and the absence of Ki-67. The lack of proliferating gliocytes, the preservation of nucleolar component indicate the absence of necrobiotic changes characteristic of necrosis. The cytoplasmic vacuolization was observed in thionine-stained CAB-immunoreactive neurons. The disintegration of motor neurons into fragments resembling the apoptotic bodies was observed in single cells that contained caspase-3.

[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.

[Subpopulation of calbindin-immunoreactive interneurons in the dorsal horn of the mice spinal cord].

In the dorsal horn of the spinal cord in the plates I-IV on the thoracic and lumbar levels different subpopulations of interneurons immunoreactive for calbindin 28 kDa (CAB IR), which are specific to each plate. In the area of the medial edge of the dorsal horn, we have found a special subpopulation of CAB IR interneurons whose morphometric characteristics differ from CAB IR interneurons subpopulations of said plates. The number of CAB IR interneurons was maximal in the plate II at all levels of the spinal cord. Leveled differences are more CAB IR interneurons and larger area of the cross sections at the lumbar level.

[Calbindin-containing neurons of the ventral horn of murine spinal cord gray matter].

The study was performed in 4 C57black/6 mice to examine the neurons located in T(II), L(IV), L(V) and L(VI) segments of the spinal cord (SC) ventral horn, containing 28 kD calbindin (CAB) and 200 kD neurofilament (NF) proteins. To demonstrate immunoreactive neurons, the cells were labeled with antibodies against CAB and double labeled with antibodies against CAB and NF. The total cell population was demonstrated using NeuroTrace Red Fluorescent Nissl Stain. Results have shown that CAB-immunopositive neurons were identified in ventromedial area of the ventral horn at all SC levels and were represented by Renshaw cells. CAB-positive interneurons located in the medial area of the ventral horn were present only in SC lumbar segments. CAB-positive motorneurons that were identified in the medial area of the ventral horn, were present in one SC segment (L(IV)) and were also found to contain a NF protein.

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.

[Neurochemical properties of sensory neurons in the development].

Sensory neurons represent various groups of neurons differ on their morphological, immunohistochemical and receptor characteristics. The most of large neurons with myelinated Aδ fibers contain neurofilament 200 (NF200), some small afferent neurons can bind the isolectin B4 (IB4). Also, sensory neurons may include different types of tyrosine kinases (trkA, B and C) and neurotransmitters. Neuropeptides are generally located in small and medium-sized neurons. The proportion of neurons which contain trkA decreases and the percentage of NF200-, IB4-, substance P- and CGRP-positive neurons increases during the early development. Development of different types of sensory neurons fulfill under control of neurotrophins.

[Age-dependent changes of neurochemical properties of sensory neurons].

Sensory neurons represent various groups of neurons differ on their morphological, immunohistochemical and receptor characteristics. The most of large neurons with myelinated Adelta fibers contain neurofilament 200 kD (NF200), some small afferent neurons can bind the isolectin B4 (IB4). Also, sensory neurons may include different types of tyrosine kinases (trkA, B and C) and neurotransmitters. Neuropeptides are generally located in small and medium-sized neurons. The proportion of neurons which contain trkA decreases and the percentage of NF200-, IB4-,substance P- and CGRP-positive neurons increases during the early development. Development of different types of sensory neurons fulfill under control of neurotrophins.

[Peculiarities of age changes of NF200(+)-neurons in the sensory ganglia of different segmental levels after chemical deafferentation].

Morphological features of the neurons containing neurofilaments with molecular mass of 200 kD (NF200+), were studied in the sensory ganglia of thoracic and lumbar spinal nerves in rats (n = 80) during the first year of their life. Capsaicin treatment (150 mg/kg) of the newborn animals resulted in the change of age dynamics of NF200+ neurons. This was reflected by a reduction of NF200+ neuron numbers and their cross-sectional areas in both ganglia. Segmental differences included greater reduction of NF200+ neuron number in the sensory ganglion of lumbar spinal nerve in both early and late developmental periods.

[Substance P-immunopositive neurons in rat sensory ganglion of the spinal nerve in postnatal development].

Afferent neurons containing substance P (SP) were studied immunohistochemically in the sensory ganglion of the spinal nerve in 30 rats aged 10-90 days. The results obtained indicated that SP-immunoreactive neurons are present in thesel ganglia from the moment of birth. During the development, the percentage of SP-containing neurons decreased till day 10. SP-immunoreactive neurons were represented by the cells of very small or small size.

[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.