Jaromir Myslivecek

INHIBITORY LEARNING AND MEMORY IN NEWBORN RATS

Firstly, the noetic value of the ontogenetic approach to the problems of learning and memory is emphasized; then the heterochrony and uneven time course of the development of neural systems are accentuated, which fully holds for the basic cognitive functions. Contrary to a broadly accepted opinion, that inhibitory learning develops later in the ontogeny, using a special method of passive avoidance (with gentle air flow inciting the new-born animal to move), the ability of new-born rats to learn an inhibitory reaction even several hours after delivery and remember it for 24 hr has been proven; special control experiments have excluded any possibility that it is a non-specific reaction. To get it, the specific features of the neonatal organism are to be considered and its functional capabilities not to be overlooked. This conditioned reaction as well as its 24 hr memory develops with a temporary reversal during several postnatal days, needing decreasing numbers of trials to meet the criteria. In the analysis of their mechanisms, it has been shown that adequate functioning of peripheral receptor zones providing afferent inputs from somatosensory areas of the conditioned stimulus is considerably involved in their establishment. Increased dendritic branching has been found in hippocampus and Meynert nucleus the following day after learning in the neonatal period. Special attention is devoted to the involvement of transmitters and/or modulators; the action of acetylcholine, noradrenaline, dopamine and nitric oxide has been discovered during the first postnatal hours; their application after meeting criteria displays a time and age dependent effect with a general characteristic of memory improvement. Neonatal learning under nitric oxide influence changes nitric oxide-synthase content in the brain. Increasing dopamine and nitric oxide availability in the brain improves both learning and memory, and their joint application positively alleviates these phenomena further. Dopamine and its D1 receptor agonists counterbalance decreased nitric oxide after nitric oxide synthase blockade; increased nitric oxide in brain and dopamine receptor antagonists similarly counterbalance each other.

The Effects of Short‐Term Immobilization Stress on Muscarinic Receptors, β‐Adrenoceptors, and Adenylyl Cyclase in Different Heart Regions

Abstract: Heart muscarinic receptors (MR) and β‐adrenoceptors (BAR) belong to a large family of G‐protein‐coupled receptors. Although the role of catecholamines in the stress has been under keen investigation for many years, the effects of immobilization on this pair of receptors, considering their almost completely opposite actions in the heart, are not yet known. We have studied the effects of short‐term immobilization (for 120 min) with different times of decapitation after the end of the immobilization period (0, 3, and 24 h) on MR, BAR (β1‐AR and β2‐AR using radioligand binding studies), and adenylyl cyclase (AC; using high‐pressure liquid chromatography detection of cAMP) in different heart regions (left and right atria with or without cardiac ganglion cells [auriculae], septum, and left and right ventricles). The effects of one immobilization period were first apparent after 24 h. Stress brought about a downregulation of MR and BAR with decrease in AC activity. These effects were regionally specific and were predominantly expressed in the right atria, which is rich in ganglia cells, and in the right ventricles. Our results indicate that stressful stimuli can influence not only BAR, but MR, and that AC activity can also be affected. This finding is in good agreement with our previous hypothesis that parallel changes are possible in the number of this pair of receptors on cell membranes.

Near‐complete adaptation of the PRiMA knockout to the lack of central acetylcholinesterase

J. Neurochem. (2012) 122, 1065–1080.

The detection of the non-M2 muscarinic receptor subtype in the rat heart atria and ventricles

Mammal heart tissue has long been assumed to be the exclusive domain of the M2 subtype of muscarinic receptor, but data supporting the presence of other subtypes also exist. We have tested the hypothesis that muscarinic receptors other than the M2 subtype are present in the heart as minor populations. We used several approaches: a set of competition binding experiments with pirenzepine, AFDX-116, 4-DAMP, PD 102807, p-F-HHSiD, AQ-RA 741, DAU 5884, methoctramine and tripinamide, blockage of M1 muscarinic receptors using MT7 toxin, subtype-specific immunoprecipitation experiments and determination of phospholipase C activity. We also attempted to block M1–M4 receptors using co-treatment with MT7 and AQ-RA 741. Our results show that only the M2 subtype is present in the atria. In the ventricles, however, we were able to determine that 20% (on average) of the muscarinic receptors were subtypes other than M2, with the majority of these belonging to the M1 subtype. We were also able to detect a marginal fraction (6 ± 2%) of receptors that, based on other findings, belong mainly to the M5 muscarinic receptors. Co-treatment with MT7 and AQ-RA 741 was not a suitable tool for blocking of M1–M4 receptors and can not therefore be used as a method for M5 muscarinic receptor detection in substitution to crude venom. These results provide further evidence of the expression of the M1 muscarinic receptor subtype in the rat heart and also show that the heart contains at least one other, albeit minor, muscarinic receptor population, which most likely belongs to the M5 muscarinic receptors but not to that of the M3 receptors.

Blphasic changes in the density of muscarinic and beta-adrenergic receptors in cardiac atria of rats treated with diisopropylfluorophosphate

Chronic treatment with organophosphate inhibitors of cholinesterases is known to bring about down-regulation of muscarinic acetylcholine receptors in the heart while its effect on the functionally antagonistic beta-adrenergic receptors is not known. We describe experiments in which rats were exposed to daily injections of diisopropylflurophosphate (DFP) and the density of muscarinic and beta-adrenergic binding sites in their cardiac atria was measured according to the binding of (3H)quinuclidinyl benzilate ((3H)QNB) and (-)-4-(3-tert-butylamino-2-hydroxy)-propoxy-(5,7-3H) benzimidazol-2-one ((3H)CGP 12177) as subtype non-specific muscarinic and beta-adrenergic ligands, respectively. Biphasic course of changes was discovered with both ligands. With the dosage scheme applied, the density of beta-adrenoceptors was augmented 24 h after the first dose of DFP and decreased to about one half of control values after 5 days of treatment with DFP. The density of muscarinic receptors was augmented after two days of treatment with DFP and decreased to about one half of control values after 5 days of treatment. Comparatively small changes in the heart rate were observed during the treatment, they reflected changes in the muscarinic and beta-adrenergic receptor density. The finding of DFP-induced changes in the binding of (3H)CGP 12177 suggests that the mechanisms responsible for the control of the density of muscarinic and beta-adrenergic receptors in the heart cells are interconnected but potential roles of other factors involved in in vivo experiments deserve further analysis. The transient increase in the density of muscarinic receptors after two days of DFP treatment appears related to published data on transient stimulation by cholinergic agonists of the transcription of mRNAs for muscarinic receptors.

Heterologous regulation of muscarinic and beta-adrenergic receptors in rat cardiomyocytes in culture

Previous work indicated that hyperstimulation of muscarinic receptors brings about profound changes not only in the density of the muscarinic receptors, but also of the beta-adrenoceptors in rat heart atria in vivo. We have now investigated whether a similar receptor cross-regulation occurs in cardiomyocytes in vitro. Cardiomyocytes from 3-4 day old rats were exposed to chemical agents on days 5-6 in culture. Densities of muscarinic and beta-adrenergic receptors were measured according to the binding of N-[3H]methylscopolamine and [ H]CGP 12177, respectively, to cell surface membranes and cell homogenates. Exposure of cells to the muscarinic agonist carbachol (1 mmol/l) brought about a profound decrease in the number of muscarinic receptors. The number of beta-adrenoceptors displayed biphasic changes, being augmented after 24 h (by 20-45% on the cell surface and by 29% in the homogenate) and diminished after 48 h and 72 h (after 48 h, decrease by 44-75% on the cell surface and by 36% in the homogenate). These effects of carbachol were not prevented by dimethylaminopropyl-bis-indolylmaleimide, the inhibitor of protein kinase C. Exposure of cells to the beta-adrenoceptor agonist isoprenaline (0.1 mmol/l) strongly diminished the number of beta-adrenoceptors on the cell surface and in the homogenate. The density of muscarinic receptors on the cell surface was diminished by 24-43% after 24 h exposure to isoprenaline and unchanged after 48 h, whereas the concentration of muscarinic receptors in the homogenate was unchanged after 24 h and increased by 20% after 48 h. The isoprenaline-induced decrease in the density of cell surface muscarinic receptors could not be simulated by forskolin and was not abolished by the protein kinase A inhibitors Rp-cAMPS and HA-1004. Dibutyryl cyclic AMP diminished the density of cell surface muscarinic receptors more than that of the beta-adrenergic receptors. Our data reveal a novel phenomenon of a biphasic change (an increase followed by a loss) in the density of beta-adrenoceptors during exposure of cardiocytes to carbachol. Activation of beta-adrenoceptors brings about less conspicuous changes in the density of muscarinic receptors. The observed phenomena of receptor cross-regulation cannot be explained by simple activations of protein kinases A and C.

Adrenergic and calcium modulation of the heart in stress: From molecular biology to function

There is strong evidence about the importance of catecholamines and calcium signaling in heart function. Also, interaction of these two systems is well documented. Catecholamines signal through adrenergic receptors, and further activate calcium transport either from the extracellular space, or from the intracellular calcium stores. This review summarizes current knowledge on catecholamine production in the heart, with special focus on the final enzyme in the catecholamine synthesizing pathway, phenylethanolamine N-methyltransferase (PNMT), in different cell types in the heart. Further, signaling through different types of adrenergic receptors in physiological conditions and after exposure to different stressors is discussed. Also, part of this review considers activation of an intracellular calcium transport system via inositol 1,4,5-trisphosphate receptor and to possible functional consequences in control and stress conditions.

Quantitative changes of dendrites in rat dentate gyrus and basal nucleus of Meynert after passive avoidance training in the neonatal period

Quantitative morphological analysis of the number of granule cell dendritic spines, as well as total dendritic length and dendritic branching of neurons in the dentate gyrus and the nucleus of Meynert was done in 11-day-old rats after passive avoidance training in the neonatal period. Learning improved stepwise and its neuromorphological sequels were characterized by a statistically significant enhanced number of dendritic spines, due to an increase of thin spines, enhanced dendritic branching in both structures, and increased total dendritic length in the dentate gyrus compared with the controls.

Regulation of Adrenoceptor and Muscarinic Receptor Gene Expression after Single and Repeated Stress

Although stress is tightly connected with elevated levels of catecholamines, stress effects on target structures of catecholamine action—adrenoceptors (ARs)—has not been deeply studied yet. Similarly, very little is known about changes of muscarinic receptors (MRs) during stress. We determined changes in these receptors in the individual parts of the heart (right atria and ventricles) of animals (rats and mice) exposed to a single and repeated immobilization stress. Changes of tissue catecholamines, β2‐AR gene expression, protein levels, and binding sites were determined in rat right ventricles, and changes in β1‐, β2‐, and β3‐AR gene expression were followed in murine right atria. Tissue catecholamines were elevated, while β2‐AR mRNA levels and β2‐AR proteins and binding were decreased, in rat right ventricles. In murine right atria, β1‐ and β2‐AR gene expression was elevated, while β3‐AR mRNA levels and M2‐MR were reduced. Taken together, our data show that interaction of AR and MR is important for the organism coping with stress and that different heart regions reveal distinct reactions to stress.

Sexual dimorphism in stress-induced changes in adrenergic and muscarinic receptor densities in the lung of wild type and corticotropin-releasing hormone-knockout mice

We tested the hypothesis that single and repeated immobilization stress affect densities of α1-adrenoceptor (α1-AR) and β-AR subtypes, muscarinic receptors (MR), adenylyl cyclase activity (AC) and phospholipase C activity (PLC) in lungs of male and female wild type (WT) and corticotropin-releasing hormone gene (CRH-knockout (KO)) disrupted mice. We found sex differences in the basal levels of α1-AR subtypes (females had 2–3 times higher density of receptors than males) and MR (males had twice the density found in females). In marked contrast, β-AR subtype densities did not differ between sexes. CRH gene disruption decreased all three studied receptors in intact mice (to 20–50% of WT) in both sexes (except β1-AR in females). Stress induced sexually dimorphic responses, while all α1-AR subtypes decreased in females (to 30% of control approximately), only α1A-AR level diminished (about 50%) in males. β1-AR decreased in males (to about 40%) but remained stable in females. β2-AR diminished in females (to about 20–60%) and also in males (to about 30–60%). MR decreased in both sexes (approximately to 50%). AC activity diminished in males (to < 50%) while PLC activity was not changed. In CRH-KO mice, the stress response was severely diminished. Paradoxically, the receptor response to stress was less affected by CRH-KO in males than in females. AC activity did not change in CRH-KO mice. In conclusion, in mice the stress reaction is sexually dimorphic and an intact hypothalamo-pituitary–adrenocortical system is required for the normal reaction of pulmonary adrenergic and MR to stress.