Morphology of the principal divisions of heart conducting system of rats]

Light optic and electron microscopic investigation of the sinuauricular node node (spu), atrioventricular node (pzhu) and bundle of His (pzhp) has been carried out in 23 hearts of intact non-inbred male rats. Original techniques of oriented embedding of elements of the conductive system for their electron microscopic identification have been suggested. A morphological classification of specialized cardiac myocytes has been worked out basing on differences in their form and size, number of myofibrils and degree of their regulation. On its base three type of specialized myofibrils have been revealed in the conductive system. Topography of these cells has been described within spu, pzhu and pzhp. The suggested ultrastructural classification of specialized cardiac myocytes is compared with the data obtained for the cardiac conductive system in other types of mammals.     

Ultrastructural analysis of collagen fibers in the conductive and working myocardium of the sinoatrial region of the human heart

Qualitative and quantitative electron microscopic analysis of collagenous fibers has been performed in the sinusal node (SN) and perinodal working myocardium of the right atrium (RA) in the hearts of 22 men, died at the age of 23-63 years. Five of them died from alcoholic cardiomyopathy, 8--from coronary cardiac disease and 9--from noncardiac causes and craniocerebral trauma (group of comparison). In 21 cases seven types of ultrastructural changes of the collagenous fibers have been revealed in the SN (changes in the form, size and degree of their osmiophilia). As a whole the volumetric density of the collagenous fibers in the selection makes 24.4 +/- 1.6% from the volume of the SN conductive myocardium and 5.7 +/- 0.8% from the volume of the RA perinodal working myocardium. Content of the collagenous fibers in the SN does not depend on the death cause, age of the dead or on degree of the cardiac hypertrophy. Content of the collagenous fibers in the RA is 2.2 times higher at a sudden coronary death, than in the group of comparison. With age in the RA the volumetric density of the collagenous fibers increases by 1.9 times, and with rise in the cardiac mass, it increases by 2.2 times. The data obtained are discussed in order to understand the role of the collagenous fibers in functioning of the sinoauricular area both normal and at the cardiac pathology.     

Ultrastructure of the vocal muscle and its morphological differences from the myocardium]

A comparative ultrastructural investigation of the M. vocalis in mammals has been carried out. Morphological differences between the vocal muscle and cardiac tissue are reported; a distinct classification of the M. vocalis according to a typisation of skeletal muscle fibers is presented. In all species investigated (man, dog, cat, guinea-pig and rat) the general ultrastructure of the sarcomeres as well as their mitochondrial content and the innervation pattern allow to classify the M. vocalis as to belong to the "fast twitch (white) skeletal muscle fibers. A single innervation was found with large motor endplates containing numerous synaptic infoldings. Structural specializations known to be characteristic for cardiac tissue, e.g. intercalated discs, T-tubules at the level of the Z-band and nuclei in a midst postion of the muscle cell could not be observed. The m. vocalis, therefore, cannot be considered to have histologically any relationship with cardiac tissue. The vocal muscle is described as a special type of skeletal muscle very similar to the extraocular muscles. The electron microscopic findings are discussed with respect to current theories of phonation. The myoleastic theory of phonation can be favoured according to our ultrastructural results.     

The development of innervation in the rat atrioventricular node

Summary The problem of development of the innervation of the rat atrioventricular node has been investigated by electron microscopy. Nerve bundles appear in relation to the node as early as the second postnatal day and vesiculated axons are seen throughout the entire node by the fourth day. Intimate contacts between nodal cells, axons and terminal varicosities are frequently observed.Use of the 5-hydroxydopamine tracer technique has enabled the identification of both cholinergic and adrenergic axons. It is concluded that the node has a dual innervation although cholinergic endings far outnumber those classified as adrenergic on the sixth postnatal day.These results are quite different to earlier findings made at the light microscope level and the discrepancies are discussed with respect to the histochemical techniques used. The suggestion that nodal differentiation is induced by nerves is considered in relation to the differences in cholinesterase activity exhibited by nodal cells during normal development and following neonatal sympathectomy.     

Visualization and functional characterization of the developing murine cardiac conduction system

The cardiac conduction system is a complex network of cells that together orchestrate the rhythmic and coordinated depolarization of the heart. The molecular mechanisms regulating the specification and patterning of cells that form this conductive network are largely unknown. Studies in avian models have suggested that components of the cardiac conduction system arise from progressive recruitment of cardiomyogenic progenitors, potentially influenced by inductive effects from the neighboring coronary vasculature. However, relatively little is known about the process of conduction system development in mammalian species, especially in the mouse, where even the histological identification of the conductive network remains problematic. We have identified a line of transgenic mice where lacZ reporter gene expression delineates the developing and mature murine cardiac conduction system, extending proximally from the sinoatrial node to the distal Purkinje fibers. Optical mapping of cardiac electrical activity using a voltage-sensitive dye confirms that cells identified by the lacZ reporter gene are indeed components of the specialized conduction system. Analysis of lacZ expression during sequential stages of cardiogenesis provides a detailed view of the maturation of the conductive network and demonstrates that patterning occurs surprisingly early in embryogenesis. Moreover, optical mapping studies of embryonic hearts demonstrate that a murine His-Purkinje system is functioning well before septation has completed. Thus, these studies describe a novel marker of the murine cardiac conduction system that identifies this specialized network of cells throughout cardiac development. Analysis of lacZ expression and optical mapping data highlight important differences between murine and avian conduction system development. Finally, this line of transgenic mice provides a novel tool for exploring the molecular circuitry controlling mammalian conduction system development and should be invaluable in studies of developmental mutants with potential structural or functional conduction system defects.     

Capsaicin-sensitive nerves influence the release of atrial natriuretic factor by atrial stretch in the rat.

Although many factors may modulate the release of atrial natriuretic factor (ANF), the primary mechanism has been demonstrated to be atrial stretch. Recent studies have led to the suggestion that the peptidergic innervation of the heart, through the release of peptides, may be involved in the control of ANF secretion. We have examined the influence of chronic capsaicin treatment on three models of atrial stretch that release ANF. This treatment inhibited ANF released through in vivo blood volume expansion and through balloon inflation in the right atrium of in vitro isolated perfused hearts. Immunohistochemical and electron microscopical analysis confirmed the absence of innervation of the heart by calcitonin gene related peptide and substance P immunoreactive nerve fibres and apparent lack of effect on atrial granules in capsaicin treated rats. We conclude that capsaicin-sensitive cardiac innervation is a component modulating the release of ANF, stimulated by atrial stretch in the rat.     

Reflexotherapeutic effects on biologically active points of the concha auriculae

By means of histochemical method, for revealing cholinergic nervous structures, and electron microscopy, innervation of biologically active points (BAP) and that of neutral areas of the rabbit ear skin has been studied, normal and after acu-, electro- and electroacupuncture. The BAP have more intensive vascularization and innervation, their specific feature is presence of well pronounced nervous fasciculi. The latter are formed by 6-10 fibers 1-6 mcm in diameter. The diameter of the fasciculi is within the limits 25-30 mcm up to 40-45 mcm. Under the electron microscopic investigation myelin and amyelin fibers are revealed in the nervous fasciculi. In the area of the epidermal basal layer and in the epidermis itself, single nerve terminals are found; they are considered as the point pain receptors. After acu-, electro- and electroacupuncture, intensity of the nervous fibers staining increases, thus demonstrating an increment of acetylcholine esterase activity. After insertion of acupuncture needles and after electrical irritation, the changes in the nervous and cellular elements in the BAP areas are studied electron microscopically. After the effects mentioned, mast cells situating in the BAP area become, as a rule, degranulated. After repeated electroacupuncture irritation of the BAP, an inflammatory focus appears with peculiarities specific for the given process. The reflexotherapeutic effect is supposed to be transferred via the nervous system. The mechanical irritation of the nerve fasciculi and the accompanying traumatization of the surrounding cellular elements initiate the mechanism of reflexotherapy.     

Simulation of the diffusion of acetylcholine in the neuroeffector junctions of the sinus node

Traditionally, the diffusion of acetylcholine (ACh) from a neuron to cardiac muscle in a neuroeffector junction has been modeled as radial diffusion from a nerve ending into a spherical homogeneous medium. Various microscopic structures in the heart may or may not influence the spatial distribution of ACh within neuroeffector junctions. To determine the effect of microscopic anatomy on the diffusion of ACh in neuroeffector junctions, we simulated the diffusion of ACh in a two-dimensional inhomogeneous geometry that was based on micrographs of neuroeffector junctions in the sinus node. ACh was released at sites adjacent to a neuron. Simulations showed that the times of peak concentration after release and the peak concentrations per se were distributed symmetrically above and below and to the right and left of the neuron, but not radially about the neuron. We conclude that the diffusion of ACh in the neuroeffector junctions of the sinus node cannot be predicted well by a mathematical model that assumes radial diffusion in a spherical and homogeneous medium.     

Distribution of vasoactive intestinal polypeptide-like immunoreactivity in the mammalian heart

Summary The distribution of vasoactive intestinal polypeptide (VIP) immunoreactivity has been studied in the mammalian heart and compared with that of neurotensin and substance P by use of light-microscopic peroxidase-antiperoxidase immunohistochemistry. VIP-immunoreactive cell bodies are present in intracardiac ganglia in various locations. VIP-immunoreactive nerve fibers predominate in the atria and the conduction system but are rare in the ventricles and occur in cardiac ganglia, endocardium, and epicardium. VIP-ergic nerves supply the coronary vasculature having a preference for the microvasculature and the nodal cells of the sinuatrial node. The large vessels of the heart and periarterial cardiac glomera also receive a VIP-immunoreactive nerve supply. There is partial co-distribution with neurotensin- and substance P-immunoreactive nerve fibers but no co-location in identical nerve fibers is detectable. The VIP-ergic cardiac innervation, which is probably predominantly intrinsic, may stem from postganglionic parasympathetic neurons and is less substantial than the more homogeneous neurotensin-ergic and substance P-ergic nervous supply which is probably extrinsic. The occurrence of an extrinsic VIP-ergic cardiac innervation cannot be excluded however. The differential histotopography of the multitarget cardiac nerves containing the cardiovascular active peptides VIP, neurotensin and substance P may suggest multiple and complex peptide-peptide and peptide-classical transmitter interactions. These may contribute to the regulation of various cardiac functions.     

Histochemical and ultrastructural data on the adrenergic and cholinergic innervation of the epididymis in the mouse

The adrenergic and cholinergic innervation of the epididymal duct in the mouse has been investigated using histochemical methods and electron microscopy. This study demonstrates that the epididymal innervation of the mouse does not really differ from that of other mammals. Cholinergic nerves are mainly vascular, even in the cauda where cholinesterase activity is increased within the tubular musculature. Catecholamine fibres ensure the motricity of the wall of the canalicular system, particularly the terminal segment where the smooth muscle fibres are specifically differentiated.     

Morphological and functional studies on the serotoninergic innervation of the inferior olive

The inferior olive of the cat has, with fluorescence histochemistry, been shown to receive a rich serotoninergic innervation. The distribution of this innervation agrees with the topography of spinal afferent termination as well as the olivo-cerebellar climbing fiber projection. This indicates that different olivary compartments are under different serotoninergic influence. The serotoninergic innervation of the dorsal accessory nucleus (DAO) of the inferior olive of the rat has been identified with electron microscopic radioautography after labelling with 3H-serotonin. The serotoninergic varicosities contain microcanaliculi, tubular-vesicular organelles and large granular vesicles. Few of the serotoninergic varicosities engage in typical synaptic junctions. However, non-junctional varicosities often display other ultrastructural indications of polarity and directed transmitted release. Electrophysiological results indicate that the harmaline-induced tremor, as well as the tremor component of the "serotonin-syndrome", depends on the serotoninergic innervation of the inferior olive. Thus, the sensitivity of different olivary compartments to the induction of rhythmic, synchronous activity by harmaline parallels the distribution of serotoninergic innervation. Neurotoxic destruction of the serotoninergic innervation leads to decreased sensitivity to harmaline. Further, the serotonin receptor agonist 5-methoxy-N,N-dimethyltryptamine, as well as monoamine oxidase inhibition + L-tryptophan loading, leads to rhythmic mass climbing fiber activity in the cerebellum and whole body tremor. A neuromodulatory effect of serotonin on the olivary action potential mechanisms is proposed.     

Nerve elements of the heart of the rat after right-sided vagotomy

A quantitative electron microscopical investigation of the sinus node of the atrioventricular His' bundle and of the perinodal working myocardium in intact rats and 7, 15 and 30 days after right-sided vagotomy has revealed variable character of changes in the neural elements of the zones mentioned. Certain ultrastructural rearrangements in the neural apparatus are described; they reflect a combination of destructive and regenerative processes in the heart under vagotomy. Thirty days after the operation, regeneration of the neural elements in the sinoauricular area is of restorative and in the atrioventricular area--of excess character. The data are presented on dynamics of changes in the diameter of the amyelinated neural fibers in the cardiac areas investigated. Participation of both nervi vagi in innervation of the main and additional pacemakers of the organ is discussed.     

Morphological study of the heart innervation of bats Myotis daubentoni and Eptesicus serotinus (Microchiroptera: Vespertilionidae) during hibernation

The capability of bats to have heart rates fewer than 10 beats/min during hibernation and greater than 700 beats/min during flight surprises biologists and cardiologists. Cardioacceleration of hibernating bats is considered to be a function of their intracardiac nervous system. In the present study we investigated the morphology of the heart innervation of ten M. daubentoni and four E. serotinus bats during their natural hibernation in order to determine which intracardiac structures may be involved in cardioacceleration during their short-term (in av. 15-30 min) arousal from hibernation. The primary conclusions were as follows: (1) The innervation pattern of bats differs from many mammals in that bats have: (a) a subepicardiac nerve plexus which is vastly developed and contains a large number of intrinsic ganglia on both atria and ventricles, and (b) very small diameter axons within the unmyelinated nerve fibres, from 0.15 to 0.7 microm. (2) During hibernation an intercellular space of the sinoatrial node of M. daubentoni bats was in part filled with a cottony substance which can presumably be considered to be a temporary barrier between the conductive cardiomyocytes and nerve fibres. (3) In the hibernating bats, the acetylcholine vesicles were aggregated in the synaptic bulbs away from the presynaptic membrane. Possibly, the aggregation of the acetylcholine vesicles is capable of modifying cholinergic influences on the heart activity of hibernating bats. (4) The dense cores of catecholamine synaptic vesicles within, adrenergic axon terminals were seldomly observed in hibernating bats. Therefore, catecholamines probably do not play a crucial role in the cardioacceleration of hibernating bats.     

Characterization of a protein kinase from human seminal plasma.

Localization of creatine kinase in isolated cardiac cells nuclei has been studied. Carefully purified cardiac nuclei preparations contain creatine kinase electrophoretically similar to the mitochondrial creatine kinase isoenzyme. Histochemical electron microscopic investigations have shown that nuclear creatine kinase is localized inside nuclei closely to chromatin.     

The 5-HT immunoreactive innervation of the Helix procerebrum

In the procerebrum of terrestrial snails, 5-HT is a key modulatory substance of the generation of synchronous oscillatory activity and odor learning capability. In this study, we have analyzed the characteristics of the 5-HT-immunoreactive (5-HT-IR) innervation of the distinct anatomical regions of the procerebrum of Helix pomatia, applying correlative light- and electron microscopic immunocytochemistry. A dense network of 5-HT-IR innervation was demonstrated in the cell body layer, meanwhile a varicose fiber system of different density occurred in the different neuropil regions. At the ultrastructural level, labeled varicosities were found to contact both procerebral cell bodies, and different unlabeled axon profiles in the neuropils. The labeled structures established mostly close non-specialized membrane contacts with the postsynaptic profiles. The overall dense distribution of 5-HT-IR innervation supports a general modulatory role of 5-HT in processing different olfactory events.     

Trypanosoma cruzi: acetylcholine content and cholinergic innervation of the heart in rats.

The acetylcholine content, as well as the density and distribution of cholinergic innervation were studied in the hearts of rats after inoculation with the Y strain of Trypanosoma cruzi. Cholinergic innervation was studied by histochemical techniques using acetylcholinesterase in the sinoatrial node, in fragments of the right atrium and auricular appendages. Acetylcholine was assayed on a strip of guinea pig ileum suspended in Tyrode's solution. Twenty days after inoculation, the cardiac content of acetylcholine dropped 40% and the cholinergic innervation was markedly reduced in 80 to 100% of the rats. However, at 70 and 97 days the cardiac content of acetylcholine was not different from that of the controls. The cholinergic innervation in the heart of the animals sacrificed 98 and 180 days after inoculation was normal in 60 to 66% of the rats. The results indicate that the cardiac parasympathetic innervation is damaged during the acute phase of experimental Chagas' disease, but returns to normal during the chronic phase in most animals.     

Electrical activation of the ventricular myocardium of the crocodile Crocodylus johnstoni: a combined microscopic and electrophysiological study.

We mapped the sequence of ventricular depolarization in the crocodile Crocodylus johnstoni. We also attempted to find specialized conduction tissue within the ventricular myocardium. Electrical recordings with miniature multi-point electrodes revealed two strands of rapidly conducting tissue (channels) within the interventricular septum, suggestive of conductive tissue pathways. From these septal channels, wavefronts of excitation swept around each ventricle. Electrical recordings did not indicate that there was conductive tissue in the wall of either ventricle. Similarly, microscopic studies of the septal channels provided no indication of specialized conductive tissue. We suggest that the channels of early septal depolarization provide the crocodile heart with a high speed depolarization pathway functionally analogous to a rudimentary conductive system.     

Differentiation of tactile corpuscles (Merkel's) in the beak of Coturnix coturnix quail during ontogeny

Succession of the Merkel's corpuscles appearance and the nervous fibers connected with them in the germ of the beak has been studied by means of light (silver nitrate impregnation) and electron microscopic techniques. Appearance of the Merkel's corpuscles in the subepithelial connective tissue is preceded by sprouting of nervous fibers into the area. From the very beginning of their differentiation the tactile corpuscles are in contact with nervous terminals. Sensory innervation is a necessary condition for differentiation of the Merkel's corpuscles and tactile bodies in the bird quail.     

Fine structures of interphase nuclei : III. Replication site analysis of DNA during the S period of Crepis capillaris

The replication sites and morphological steps of chromosomal condensation during S period in the nuclei of Crepis capillaris root tip cells have been studied with light and electron microscopic autoradiography. From light microscopic autoradiographic observations, the S period can be divided with three portions, early S, mid S, and late S period. Labelled nuclei for each portion of the S period have also been found by using electron microscopic autoradiography. With electron microscopic autoradiography it has been found that in early, mid, and late S period, the replication sites are distributed in the electron transparent regions, interspersed with dense chromatin masses of variable size which are distributed throughout the nucleus. The time-dependent behavior of the label indicates that when compared with either mid or early replicated DNA, a majority of this chromatin, which contains predominantly late replicated DNA, is the earliest chromatin to be organized into the condensed chromatin. They are organized into the condensed chromatin within 15 min after the termination of replication.     

Adrenergic innervation of the kidneys in man and various laboratory animals

By means of the neurohistochemical method for slice incubation in 2% solution of glyoxylic acid, innervation of the kidneys of a 57-year-old man after a sudden cardiac death has been investigated, as well as innervation of the kidneys in white rat, rabbit, guinea pig and cat. A rich adrenergic innervation in the organ's blood vessels has been revealed. In particular, adrenergic nervous fibers have been found along the course of afferent glomerular arterioles. Together with innervation of the proximal and distal convoluted tubules, a high density of the terminal adrenergic nervous plexus is revealed along the course of the nephron loops. Adrenergic nervous plexuses of high density are found in the area of the initial part of the urinary excretory pathways and their connection with nervous plexuses of the kidney itself.