Cardiac ganglia of Japanese quail — distribution and morphology

= = = = = = = = = ANNALS Of ANATOMY

Cardiac ganglia of Japanese quail - distribution and morphology T. Kuder and A. Tekieli Department of Comparative Anatomy, Institute of Biology, Pedagogical University, Konopnickiej Str. 15, PL-Kielce 25-406, Poland

Summary. The cardiac ganglia in Japanese quail were studied with the use of histological, histochemical and ultrastructural techniques. Histological investigations revealed the presence of a number of cholinergic ganglia in the fatty tissue of the epicardium. They were grouped in plexo-ganglionic forms localised in three regions: 1) on the ventral surface of the cardiac atria, 2) on the ventral surface of the cardiac ventricle, 3) on the dorsal surface of the cardiac ventricle. These plexoganglia are structures composed of many ganglia differing in size (from 77 ).lm to 577 11m length and from 53 ).lm to 163 ).lm width), connected by fascicles of nerve fibres. The cells of cardiac ganglia have single, round or oval nuclei with one or several dense nucleoli. There were myelinated and unmyelinated fibres in the intercellular spaces. Rough endoplasmic reticulum (RER) and free ribosomes were localised mainly in the perinuclear part of the cytoplasm. In the peripheral part, RER was less abundant, but mitochondria were more numerous in this part of the cytoplasm. In the peripheral parts of the neurones, axo-somatic synapses were usually observed. Profiles of the end sections ofaxons contained two kinds of synaptic vesicles: small, agranular ones and among them large ones with a dense core.

Up to now the investigations have focused mainly on morphology, topography and cytoarchitectonics of cardiac neurones. The results of these investigations show that cardiac ganglia contain parasympathetic postganglionic neurones (Baluk and Gabella 1987; Bojsen-Moller and Traum-Jensen 1971; Furukava et al. 1990; Hancock et al. 1987; Michell 1956). Cardiac ganglia have not yet been described in the Japanese quail. The present study concerning the morphology, topography and ultrastructure of the cardiac ganglia in this species has been undertaken to complete data and carry out the comparative analysis.

Key words: Cardiac ganglia - Japanese quail - Topography - Histology - Ultrastructure - Synaptic vesicles

Histological study. Four of the hearts were fixed in 3% formalin, then embedded in paraffin and next cut on a microtome into 5 11m sections. The hearts were sectioned from the base of the heart. The sections were stained with hematoxylin and eosin using the method of Hall (Carter 1980).

Introduction

Electron microscopy. Four of the hearts were fixed by immersion in 3% glutaraldehyde in phosphate buffer at pH 7.25 and postfixed in 1% OS04. The material was embedded in Durcupan ACM Fluka and cut into ultrathin sections. The sections were stained with uranyl acetate and lead citrate by Reynolds' method (1963) and examined with a Tesla BS 500 microscope.

Cardiac ganglia have been described in many species of vertebrates: Amphibians, Reptiles, Avians and Mammals. Correspondence to: T. Kuder

Ann Anat(1999) 181: 467-473 © Urban & Fischer Verlag http://www.urbanfischer.de/journals/annanat

Materials and methods Twelve adult Japanese quails of both sexes were used for the experiments. After the quails had been anaesthetised with Vetbutal the hearts were taken out. Histochemical study. Four of the hearts were fixed in 10% formalin and then treated by a whole mount using the histochemical method of Koelle-Friedenwald (1949) modified by Gienc (1976, 1977) for use in macromorphological specimens.

0940-9602/99/181/5-467 $12.00/0

Results Histochemical study. Histochemical investigations revealed the presence of a number of cholinergic ganglia in the fatty tissue of the epicardium in all the quails examined. The ganglia were grouped in three agglomerations. Each of them was composed of many ganglia different in size and interconnected by fascicles of nerve fibres. In this way the agglomerations of ganglia were formed into a system of plexo-ganglionic forms. Figures 1 A and 1 B show the schematic distribution of all the groups of cardiac ganglia and the fascicles of postganglionic fibres mentioned. One of these plexo-ganglionic forms was located on a ventral surface of the cardiac atria and contained 10-19 small ganglia each composed of a few dozen ganglionic cells, and 1 or 2 bigger ganglia. The ganglia were located near the entry of the pulmonary veins and left superior vena cava. The ganglia were joined with each other and also with the ganglia from the ventral surface of the cardiac ventricle by fascicles of nerve fibres. The second plexo-ganglionic form was located on the ventral surface of a heart, near the interventricular septum (Figs. 1 A, 2). It spread from the coronary sulcus to the apex of heart. This plexo-ganglion contained a few dozen (30-50) small ganglia, connected by fascicles of nerve fibres. The fascicles containing the small ganglia, arranged like a rosary, were located along the anterior interventricular septum (Fig. 2). These small ganglia were of various dimensions (77 !lm to 577 !lm in length

and from 53 !lm to 163 !lm in width). The average length of a ganglion was 254!lm and the average width was 111 !lm. The third plexo-ganglionic form was observed on the dorsal superior surface of the cardiac ventricle (Figs. 1 B, 3). It was always smaller then the above mentioned (the second) plexo-ganglion. It was located in the vicinity of the coronary sulcus and it spread from the aorta and the pulmonary trunk to the upper part of the ventricular wall. The plexo-ganglion contained a few dozen (15-25) small ganglia joined together by nerve fibres. The size of these ganglia was similar to those mentioned above, which formed the plexo-ganglion on the dorsal surface of the heart (Fig. 3). Histological study. The results of histological investigations showed that the cardiac ganglia were composed of a number of cell agglomerations connected by the fascicle of nerve fibres (Fig. 4). Usually they consisted of 7 to 15 cells on cross-section, but more than 20-cell ganglia were also observed (Fig. 5). The cells were distributed regularly on the ganglion and occupied about 25-50% of the transverse sectional area. Nerve fibres occupied the remaining part of section. Occasionally single neurones (ganglion cells) were observed along the nerve fibre fascicles connecting the ganglia. The cells of cardiac ganglia in the quail were of oval or elliptical shape. The diameter of the cardiac ganglia neurones varied from 17!lm to 35 !lm. The neurocytes had single, round or oval nuclei with one or several dense nucleoli in transverse section.

Fig. l. A: Scheme of distribution of two plexoganglionic forms on a ventral surface of a heart (on atria - 1, on ventricles - 2). A - aorta, ACCD - arteria carotica communis dextra, ACCS - arteria carotica communis sinistra, AD - atrium dextrum, APD - arteria pulmonaris dextra, APS - arteria pulmonaris sinistra, ASD - arteria subclavia dextra, AS - atrium sinistrum, ASS - arteria subclavia sinistra, AuD - auricula dextra, AuS - auricula sinistra, SC - sulcus coronarius, TBD - truncus brachiocephalicus dexter, TBS - truncus brachiocephalicus sinister, VCC - vena cava caudalis, VCCD - vena cava cranialis dextra, VCCS - vena cava cranialis sinistra, VD - ventriculus dexter, VJD - vena jugularis dextra, VPD - vena pulmonaris dextra, VPS - vena pulmonaris sinistra, VS - ventriculus sinister, VSS - vena subclavia sinistra, VSD - vena subclavia dextra. B: Scheme of plexo-ganglionic form (3) on the dorsal surface of a heart. For abbreviations, see Fig. 1 A.

1A

468

18

.. ....- •.. i !

it .'. ~

;

.

'

~

..

"~

. " ,t

...>. .t ...' ... .. , ,

. .,'.... ' . . ,\~': \

"

~

.'

• , .' ~

~

-\

t

'0 (

.

• J

:

'.

.

..

" . . ": .... .

".,

-< ..... " -. . "

,

I , "

\

, t'

~..

I~'

. .'," ~ ""

.'

: ..

t

•

....

"

.'

"

,

.... ,...

" I

Fig. 2. The plexo-ganglion from the ventral inferior surface of the heart of a Japanese quail. Thiocholine method. Magn. approx. 25x. Fig. 3. Multifonn ganglia from the plexo-ganglion on ventral surface of ventricles. Thiocholine method. Magn. approx. 145 x. Fig. 4. The thick fascicles of nerve fibres (NF) with two cell aggregations (arrows) from ventral surface of ventricles. H & E method. Magn approx. 15 x. Abbreviation: VS - ventriculus sinister Fig. 5. Very elongated ganglion, near the coronary artery, from the dorsal surface of a heart. H & E method. Magn. approx. 200x. Abbreviation: eM - cardiac muscle

469

Electron microscopy. The ultrastructural examination showed that all the neurones were multipolar and completely surrounded by satellite cells. In the perinuclear part of the cytoplasm, large amounts of rough endoplasmic reticulum (RER) were observed (Fig. 6). It was occasionally organised in a form corresponding to a Nissl's body. The cisterns of the RER were short and usually arranged in parallel. Moreover, great numbers of free ribosomal parts (most commonly in groups of 4-8) were present throughout the cytoplasm in this region. The mitochondria have • various shapes and sizes, but most common were oval or elongated profiles of 300-1250 nm in diameter and transverse crests. They were localised mainly in the peripheral part of the cytoplasm (Fig. 7). The Golgi complex was poorly developed. In the vicinity of the Golgi complex, vesicles with a dense core and 100 nm in diameter were found. There were a few lysosomes, mostly primary ones measuring about 200 nm in diameter (Fig. 6). There were myelinated and unmyelinated nerve fibres in the intercellular spaces. In the peripheral parts of neurones, chemical synaptic junctions were often observed. These were usually axosomatic synapses, but axo-dendritic were also visible. The synaptic profiles contained mostly two kinds of synaptic vesicles: the small, agranu-

Fig. 6. Perinuclear region of the cytoplasm of cardiac ganglion. Magn. approx. 30000 x. Abbreviation: N - nucleus, NM - nuclear membrane, M -mitochondria, L - lysosome, RER - rough endoplasmic reticulum II

Fig. 7. Peripheral part of a cardiac ganglion with many mitochondria. Magn. approx. 40000 x Fig. 8. Synaptic profiles with two kinds of synaptic vesicles: agranular (AV) and granular (GV). Magn. approx. 50000x.

470

Fig. 9. The end section of an axon (arrows) in the intercellular spaces of cardiac muscle (CM). Magn. approx. 30000x. Abbreviation: CL - capillary lumen

lar ones of 25-45 nm in diameter and the large ones with a dense core of 50-90 nm in diameter (Fig. 8). Generally, small agranular vesicles were numerous. In the intercellular spaces of cardiac muscles the end section ofaxons also containing two kinds of synaptic vesicles were observed (Fig. 9).

Discussion The results of the histochemical and the histological investigations demonstrate that cardiac ganglia in Japanese quail are embedded in epicardial fat. They form a system of plexo-ganglionic forms, including more or less a dozen ganglia which were diverse in size and shape. Becker et al. (1997) described a so-called retrocardiac ganglion in the mouse heart, which contained about 90 neurones in transverse section, but no defined location of this ganglion was given. Such a plexo-ganglionic form is characteristic for the quail. As far as mammals are concerned we can notice a similar situation, but their plexuses are less organised and smaller than in birds. The number of cells in mammalian ganglia is bigger than those in birds. For example, in the mouse heart two plexo-ganglionic forms were observed in the fatty tissue of the epicardium of the right atrium. These plexo-ganglions usually consist of 4-5 big, spindle-shaped ganglia and 5-6 smaller ones (Kuder and Soja 1997; Kuder and Tekieli, in print). The largest ganglion in the mouse investigated was localised in the depression between the right cardiac auricle and the aorta. A similar localisation was observed in a number of other species (Ardell and Randall 1986; Davies et al. 1952; Gagliardi et al. 1988; Randall et al. 1987; Randall et al. 1990; Smith 1971). The nerve cells were sometimes found in myocardial tissue (Kuder and Soja 1997; Kuder and Tekieli, in print; Michell 1956; Robb 1965). Some authors have suggested that localisation of cardi-

ac ganglia in specific regions of heart could be the cause of a different functional property of these parts of the heart (Ardell and Randall 1986; Armour and Hopkins 1990; Bluemel et al. 1990; Furukava et al. 1990; Randall et al. 1987; Yuan et al. 1993; Yuan et al. 1994). This was confirmed by histochemical experiments which revealed various neurotransmitters in cardiac ganglia cells (BojsenMoller and Traum-Jensen 1971; Moravec et al. 1990). Cardiac ganglia were considered as parasympathetic, which does not rule out a presence of adrenergic cells. The parasympathetic nature of these ganglia was confirmed, among other things, by histochemical investigation (Jacobowitz 1967; Abraham 1969; Taylor and Smith 1971; Edwards et al. 1995; Becker et al. 1997). Edwards et al. (1995) observed bundles of nerve fibres leaving the vagal nerve (X) and going to the atrial ganglia in a guinea-pig heart. Similar junctions have also been observed in quails (Fitzgerald 1969; Kuder and Tekieli, in print; Langenfeld 1992). Hardwick et al. (1995) suggest that the parasympathetic neurones of the guinea-pig cardiac ganglion receive inputs from peptidergic, afferent fibres and that this input provides a pathway for potential local reflex control of cardiac function. Langenfeld (1992) described five cardiac plexuses III birds: 1) 2) 3) 4) 5)

plexus plexus plexus plexus plexus

cardiacus cranialis cardiacus cranialis cardiacus ventralis cardiacus ventralis atrium ventralis.

dexter, sinister, dexter, sinister,

We have demonstrated only three cardiac plexuses in our investigations: 1) on the ventral surface of the cardiac atria (it corresponds to plexus 5 of Langenfeld), 2) on the ventral surface of the ventricles (it corresponds to plexuses 3 and 4 of Langenfeld), 3) on the dorsal surface of the ventricle (it corresponds to plexuses 1 and 2 of Langenfeld).

471

The ultrastructural results presented show general similarity to other cardiac and parasympathetic ganglia. (De Stefano et al. 1993; Ellison and Hibbs 1976; Kuder 1990; Kuder et al. 1995; Yuan et al. 1994). In our investigations we found only chemical synapses, while De Stefano et al. (1993) showed in ciliary neurones in chicken and in quails the presence of a mixed synaptic junction - chemical and electrical. This kind of synaptic junction was previously demonstrated in the ciliary ganglion of the chicken (Cantino and Mugnaini 1975) and in pigeon (Marwitt et al. 1971). The preganglionic endings observed in our investigations form synapses mainly of an axo-somatic type. Similar observations were done by De Stefano et al. (1993) in quail ciliary neurones and by Kuder (1990) in the pterygopalatine neurones of the rat. An interesting problem is the presence of two kinds of vesicles in the synaptic profiles in quail cardiac neurones: the small agranular 25-45 nm in diameter and the large, with a dense core 50-90 nm in diameter. In general, small, agranular vesicles were numerous. The granular vesicles were also observed in canine cardiac ganglia (Yuan et al. 1994) and in the guinea-pig, cat, rat, and monkey (Ellison and Hibbs 1976). Such granular vesicles were also observed in other parasympathetic ganglia, e. g. in the otic ganglion in the mouse (Kuder et al. 1995), and rabbit (Dixon 1966), and in the pterygopalatine ganglion in the rat (Kuder 1990) and quail and chicken ciliary ganglia (De Stefano et al. 1993). The presence of these kinds of vesicles might suggest that these synaptic endings represent peptidergic neurones.

References Abraham A (1969) Microscopic innervation of the heart and blood vessels in vertebrates including man. Pergamon Press, Oxford Ardell JA, Randall WC (1986) Selective vagal innervation of sinoatrial and atrioventricular nodes in canine heart. Am J Physiol251: 764-773 Armour JA, Hopkins DA (1990) Activity of in situ canine left atrial ganglion neurones. Am J Physiol259: 1207-1215 Baluk P, Gabella G (1987) Fine structure of the autonomic ganglia of the mouse pulmonary vein. J Neurocytol16: 169-184 Becker C, Haberberger R, Fisher A (1997) Neurochemical characterisation of the intrinsic innervation of murine heart and lung. Ann Anat (Suppl) 179: 76 Bluemel KM, Wurster RD, Randall WC, Duff MJ, O'Toole MF (1990) Parasympathetic postganglionic pathways to the sinoatrial node. Am J Physiol 259: 1504-1510 Bojsen-Moller F, Traum-Jensen J (1971) Whole-mount demonstration of cholinesterase-containing nerves in the right atrial wall, nodal tissue and atrioventricular bundle of the pig heart. J Anat 108: 375-386 Cantino D, Mugnaini E (1975) The structural basis for electronic coupling in the avian ciliary ganglion. A study with thin sectioning and freeze-fracturing. J Neurocytol 4: 505-536

Carter DW (1980) Hall's Modified Harris Hematoxylin. Histology 10: 143 Davies F, Francis ETB, King TS (1952) Neurological studies on the cardiac ventricles of mammals. J Anat 86: 302-309 De Stefano ME, Ciofi Luzzatto A, Mugnaini E (1993) Neuronal ultrastructure and somatostatin immunolocalisation in the ciliary ganglion of chicken and quail. J Neurocytol 22: 868892 Dixon JS (1966) The fine structure of parasympathetic nerve cells in the otic ganglia of rabbit. Anat Record 156: 239-252 Edwards FR, Hirst GHS, Klemm MF, Steele PA (1995) Different types of ganglion cell in the cardiac plexus of guinea pigs. J Physiol 486, 453--471 Ellison JP, Hibbs RG (1976) An ultrastructural study of mammalian cardiac ganglia. J Molec Cell Cardiol8: 89-101 Fitzgerald ThC (1969) The Coturnix Quail - Anatomy and Histology. The Iowa State University Press, AMES IOWA Furukava Y, Wallick DW, Martin PJ, Levy MN (1990) Chronotropic and dromotropic responses to stimulation of intracardiac sympathetic nerves to sinoatrial and atrioventricular nodal regions in anaesthetised dogs. Circ Res 66: 1391-1399 Gagliardi M, Randall WC, Bieger D, Wurster RD, Hopkins DA, Armour JA (1988) Activity in neurones in the in situ canine heart. Am J Physiol 225: 789-800 Gienc J (1976) Porownawcza morfologia i topografia niektorych zwojow oraz zazwojowych odcinkow przywspolczulnych drog wydzielniczych dUiych slinianek u doswiadczalnych gryzoni i mittsoiernych w swietle bad an histochemicznych. Zesz Nauk ART Olsztyn Vet 158: 1-50 Gienc J (1977) The application of histochemical method in the anatomical studies on the parasympathetic ganglia and nerve bundles of postganglionic axons in the sublingual region of some mammals. Zool Pol 26: 187-192 Hancock JC, Hoover DB, Hougland MW (1987) Distribution of muscarinic receptors and acetylocholinesterase in the rat heart. J Auton Nerv Syst 19: 59-66 Hardwick JC, Mawe GM, Parsons RL (1995) Evidence for afferent fiber innervation of parasympathetic neurons of the guinea-pig cardiac ganglion. J Auton Nerv Syst 53: 166-174 Jacobovitz D (1967) Histochemical studies of the relationship of chromaffin cells and adrenergic nerve fibres to the cardiac ganglia of several species. J Pharmacol Exp Therap 158: 227240 Koelle GB, Friedenwald JS (1949) A histochemical method for localising cholinesterase activity. Proc Soc Exp BioI Med 70: 617-622 Kuder T (1990) The pterygopalatine ganglion in rat II. Ultrastructural research. Folia Morphol Warszawa 49: 109-118 Kuder T, Slovakova D, Mraz P (1995) Ultrastructure of neurones of the otic ganglion in mouse. Zool Pol 40: 79-90 Kuder T, Soja A (1997) Gross anatomy of the mouse (Mus musculus) cardiac ganglia. Ann Anat (Suppl) 179: 130 Kuder T, Tekieli A (in print) Gross and light microscopic anatomy of the mouse cardiac parasympathetic nervous system. Zool Pol Langenfeld SM (1992) Anatomia kury. PWN, Warszawa-Krakow Marwitt R, Pilar G, Weakly IN (1971) Characterization of two ganglion cell populations in avian ciliary ganglion. Brain Res 25: 317-334 Michell GAG, Brown R, Cookson FB (1953) Ventricular nerve cells in mammals. Nature 172: 812 Michel GAG (1956) Cardiovascular Innervation. E. & S. Livingston, Edinburgh Moravec M, Moravec J, Forsgren S (1990) Catecholaminergic

472

and peptidergic nerve components of intramural ganglia in the rat heart. Cell Tissue Res 262: 315-327 Randall WC, Ardell JL, Wurster RD, Milosavljevic M (1987) Vagal postganglionic innervation of the canine sinoatrial node. J Auton Nerv Syst 20: l3-23 Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell BioI 17: 208-212 Randall WC, Ardell JL, Calderwood D, Milosavljevic M, Goyal SC (1990) Parasympathetic ganglia innervating the canine atrioventricular nodal region. J Auton Nerv Syst 16: 311323 Robb JS (1965) Comparative Basic Cardiology. Grune & Stratton, New York

Smith RB (1971) Observations in nerve cells in human, mammalian and avian cardiac ventricles. Anat Anz 129: 436-444 Tylor 1M, Smith RB (1971) Cholinesterase activity in the human, fetal heart between the 35- and 160-millimetre crown-rump length stages. J Histochem Cytochem 19: 498-503 Yuan BX, Ardell JL, Hopkins DA, Armour JA (1993) Differential cardiac responses induced by nicotine sensitive canine atrial and ventricular neurones. Cardiovasc Res 27: 76G-769 Yuan BX, Ardell JL, Hopkins DA, Losier AM, Armour JA (1994) Gross and microscopic anatomy of the canine intrinsic nervous system. Anat Rec 239: 75-87 Accepted March 8, 1999

473