I. V. Romanova

The Dopaminergic System of the Telencephalo-Diencephalic Areas of the Vertebrate Brain in the Organization of the Sleep–Waking Cycle

The aim of the present work was to study the involvement of the dopaminergic system of the telencephalic and diencephalic areas of the vertebrate brain in the organization of the sleep–waking cycle in cold-blooded and warm-blooded vertebrates. Immunohistochemical studies of tyrosine hydroxylase content, this being the key enzyme in dopamine synthesis, in the striatum, supraoptic and arcuate nuclei, and zona incerta of the hypothalamus of sturgeon and mammals (rats) of three age groups (14 and 30 days and adults), in conditions of tactile and sleep-deprivation stressors. In fish, transient stress was followed by the detection of tyrosine hydroxylase-immunoreactive cells in all parts of the brain. In prolonged stress, tyrosine hydroxylase-immunoreactive cells and fibers were not found in the forebrain, though they were well represented in the hypothalamic nuclei. In 14-day-old rat pups, 2-h sleep deprivation increased the tyrosine hydroxylase content of fibers in the caudate nucleus and cells in the zona incerta of the hypothalamus, while 30-day-old animals subjected to 6-h sleep deprivation showed increases in tyrosine hydroxylaseimmunoreactive material contents in cells in the paraventricular nucleus and decreases in the quantity in fibers. In adult rats, the arcuate nucleus and zona incerta showed decreases in the content of tyrosine hydroxylase-immunoreactive material on the background of sleep deprivation, with increases during postdeprivation sleep. These data are discussed in the light of the phylo- and ontogenetic development of the neurosecretory and neurotransmitter functions of the dopaminergic system in the evolutionarily ancient diencephalic and evolutionarily young telencephalic areas of the vertebrate brain as major systems triggering and maintaining the functional states of the body during the sleep–waking cycle.

The dopaminergic nigrostriatal system in sleep deprivation in cats

The dynamics of changes in electrophysiological measures of the sleep-waking cycle were analyzed in Wistar rats after 6 h of sleep deprivation by gentle waking and subsequent 9-h post-deprivation sleep. A delayed sleep “overshoot” reaction was observed 2.5–3 h after sleep deprivation, as a moderate increase in the proportions of slow-wave and fast-wave sleep in the sleep-waking cycle. Immunohistochemical studies were performed in relation to changes in the sleep-waking cycle, with the aim of identifying changes in the quantities of immunoreactive dopamine D1 and D2 receptor material and tyrosine hydroxylase, the key enzyme in dopamine synthesis in the nigrostriatal system. In conditions of sleep deprivation, the caudate nucleus showed increases in the quantities of dopamine D1 and D2 receptor material, while there was a simultaneous decrease in the amount of immunoreactive material in the substantia nigra. Post-deprivation sleep was accompanied by decreases in the quantities of immunoreactive D1 receptor material and increases in D2 receptor material in the caudate nucleus, with an increase in the quantity of immunoreactive tyrosine hydroxylase in the substantia nigra. These data provide evidence of the active role of the dopaminergic nigrostriatal system which, along with other CNS transmitter systems, supports telencephalic-diencephalic interactions, in the sleep-waking cycle.

[Immunohistochemical investigation of Bcl-2 and p53 levels in rat hypothalamus after sleep deprivation].

Immunohistochemical study of changes in the Bcl-2 and p53 peptide optical density levels was carried out in neurons of Wistar rat paraventricular (PVN), supraoptic (SON), and median (MeN) hypothalamic nuclei with taking into account changes in electrophysiological parameters after sleep deprivation (SD; awakening by the gentle handling method) and the subsequent postdeprivative sleep (PDS). The Bcl-2 amount was increased in all nuclei after both SD and PDS. The level of p53 was increased in PVN and SON after SD and PDS, but in MeN —only after PDS. No morphological features of apoptosis were revealed in these nuclei. The obtained data indicate an active role of p53 and Bcl-2 peptides in regulation of neuronal activity in hypothalamus during change of the wakefulness-sleep cycle.

Chaperone Hsp70 Content in Dopaminergic Neurons of the Substantia Nigra Increases in Proteasome Dysfunction

Decreases in the activity of the ubiquitin-proteasome system (UPS), which uses up to 90% of cell protein, are regarded as a key mechanism in the development of age-related conformational diseases (Parkinson’s disease (PD), Alzheimer’s disease, and others). Studies in a model of the preclinical stage of PD in Wistar rats showed that the specific UPS inhibitor lactacystin induced degeneration of 24% of dopaminergic neurons in the compact zone of the substantia nigra (czSN), which was almost the same as the proportion of neurons (23%) which in control conditions did not contain the chaperone 70-kDal Heat Shock Protein (Hsp70), which has neuroprotective properties. Of the 77% of neurons which contained Hsp70 in control conditions, 15% lost it in response to lactacystin (which may reduce their resistance to neurotoxin). However, 62% of surviving dopaminergic neurons in the czSN showed a 47% increase in Hsp70 content (which may protect them from degeneration). The increase in Hsp70 content (in czSN tissue) was verified by immunoblotting. Confocal microscopy studies identified partial colocalization of Hsp70 with the key dopamine (DA) synthesis enzyme tyrosine hydroxylase. Thus, moderate weakening of UPS function in czSN, typical of this model of the preclinical stage of PD, was characterized by an increase in the activity of the chaperone system. It is suggested that this process is directed to restoring UPS activity and preserving the dopaminergic neuron population.

Functional Interaction between the Dopamine and Melanocortin Systems of the Brain

The melanocortin system of the brain plays a key role in regulating energy metabolism and feeding behavior and is involved in forming memories; impairments to this sytem lead to metabolic disorders and cognitive deficit. The dopamine system of the brain controls motor activity and the functions of the nervous, endocrine, and cardiovascular systems, and is also involved in regulating peripheral metabolism and feeding behavior, as well as generating the effects of rewards and reinforcements. The fact that many major physiological functions are regulated by the melanocortin and dopamine systems points to tight functional interactions between them in various brain structures. These interactions are based on the colocalization of components of the melanocortin and dopamine signal systems in neurons and on interactions between them, supporting crosstalk between the melanocortin and dopamine pathways in the CNS. This review provides a wide-ranging analysis of our current understanding of the interaction between the melanocortin and dopamine systems of the brain at both the structural and functional levels.

An Immunohistochemical Study of Cart Peptide in the Striatonigral Projections in Dopamine Deficiency

Studies in Wistar rats identified increases in the optical density of CART peptide both in nucleus accumbens neurons and their processes in the substantia nigra on the background of a 28% reduction in the number of dopaminergic neurons in the substantia nigra (in a model of lactacystin-induced proteasome dysfunction). In in vitro experiments, incubation of nigroaccumbal sections in medium containing α-methylparatyrosine – a blocker of dopamine synthesis – for 4 h decreased the optical density of tyrosine hydroxylase, the rate-limiting step in dopamine synthesis in substantia nigra neurons. On the background of blockade of dopamine synthesis, there were increases in the optical density of CART peptide in nucleus accumbens neurons and in their processes in the substantia nigra. Data on the activation of CARTergic neurons in striatonigral projections provide evidence that CART peptide is involved in the brain’s compensatory mechanisms in dopamine insufficiency and of its role as a modulator of the functional activity of brain dopaminergic neurons.

Phylo- and Ontogenetic Establishment of Dopamine Regulation of the Sleep–Waking Cycle in Vertebrates

Results obtained from comparative immunohistochemical studies of dopamine-containing neurons and fibers in the telencephalic and diencephalic parts of the brain in cold-blooded animals (frogs) and warmblooded (14- and 30-day-old rat pups and adult) vertebrates are presented. The dynamics of quantitative changes in tyrosine hydroxylase and D1- and D2-immunoreactive structures during the sleep–waking cycle were studied using a sleep deprivation model. Morphofunctional correlations were seen in the nature of the responses of the dopaminergic neurotransmitter system to sleep deprivation during phylo- and ontogenesis. In addition, the effects of dopamine agonists and antagonists on the sleep–waking cycle were studied in frogs and young rats. Dopamine and its agonist apomorphine were found to promote increases in the state of immobility of the cataplexy type (this being a homolog of sleep) in frogs, while in rats it promoted increases in waking and catalepsy. The D1 receptor antagonist SCH 23390 induced increases in the quantity of waking and the state of immobility of the catatonia type in frogs, while the D2 receptor antagonist promoted increases only in the state of immobility of the catalepsy type in the sleep–waking cycle. In one-month-old rats, administration of the dopamine antagonist haloperidol initially induced increases in the proportion of the cataleptic stage, which was followed by onset of deep slow-wave sleep. We address the question of the formation of the regulatory role of the dopaminergic system in the sleep–waking cycle during phylo- and ontogenesis, when dopamine shows a transition from predominantly diencephalic neurosecretory influences to predominantly telencephalic neurotransmitter influences.

Interaction of the Dopaminergic and Vasopressinergic Systems in Sleep Deprivation in Rats

Rats were subjected to 6-h sleep deprivation and were given three injections of the dopamine D1 receptor blocker SCH 39166. Immunohistochemical investigations revealed increases in D1 and D2 dopamine receptor and glutamate immunoreactivity on the background of sleep deprivation and a 2-h post-deprivation period; the level of AMPA glutamate receptors increased on the background of sleep deprivation and decreased during the post-deprivation period. These data are assessed in terms of the dynamics of changes in immunoreactive vasopressin levels in the supraoptic and paraventricular nuclei of the hypothalamus in this experiment, as well as in an analogous experiment without blocker administration.

Analysis of development of morphophunctional interconnections between CART- and AGRPergic neurons with dopaminergic structures in ontogenesis of mammals

Morphogenesis and, probably, formation of structural-functional interaction between the CART- and dopaminergic neurons in rats were found to occur as early as during embryonic development. Meanwhile, formation of the AGRPergic system and its structural-functional connections was observed in the course of postnatal ontogenesis. Analysis of literature and the results of our own investigation show that differentiation of dopaminergic neurons in hypothalamus and midbrain occurs concurrently with development of the CARTergic neurons and in the absence of AGRP. Considering our data on the stimulatory effect of the CART-peptide and the inhibitory effect of AGRP on dopaminergic brain neurons, we believe that the leading component in formation of functional interactions of these systems is the later maturing AGRPergic system.

Effect of retinol on interaction of the protein period1, oxytocin, and GABA at the prenatal period of formation of the circadian clock-mechanism in rats

In mammals, clock-gene proteins are expressed in the neurons of hypot halamic suprachiasmatic nucleus and that of other CNS structures, in the muscles, visceral organs and vessels, thus forming circadian rhythms of many functions. Little is known about the factors of formation of the circadian mechanism at the prenatal period. In the rats, E20 stage is characterized by a high level of oxytocin and selective expression of the first protein of clock-genes PER1. The goal of present study was to check the suggestion on the positive feedback between PER1 and oxytocin at the prenatal period, as well as to elucidate a possible role of PER1 in the regulation of oxytocin and GABA interactions at the period of formation of the cerebral circadian mechanism of clockgenes. With aid of western-blotting, we analyzed the nuclear and cytoplasmic fractions of anterior hypothalamus homogenate from the pregnant females and rat embryos (E20). Retinol metabolites through their nuclear receptor RORa are known to be bound to promoters of oxytocin and per 1 genes. Next day after administration of retinol to the females, a rise in PER1 content was noted in their cytoplasm, whereas in their embryos PER1 content was elevated in the nucleus. In the embryo cytoplasm there was a significant rise in production of oxytocin receptors and a decrease in the level of enzymes of GABA synthesis (glutamate decarboxylases 67 and 65). The results indicate the oxytocin- and retinol-dependent increase in the PER1 expression and the subsequent change in the ratio of oxytocin and GABA efficiency at the prenatal stage of formation of the circadian clockmechanism in the rat embryo anterior hypothalamus.