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Histochemical features of neurons in the cat stellate ganglion during postnatal ontogenesis
Autonomic Neuroscience: Basic and Clinical 106 (2003) 84 – 90 www.elsevier.com/locate/autneu
Histochemical features of neurons in the cat stellate ganglion during postnatal ontogenesis Petr M. Masliukov a,*, Valentin V. Shilkin b, Alexandr D. Nozdrachev c, Jean-Pierre Timmermans d a
Department of Normal Physiology and Biophysics, Yaroslavl State Medical Academy, Revoliucionnaya 5, Yaroslavl 150000, Russia b Department of Human Anatomy, Yaroslavl State Medical Academy, Yaroslavl, Russia c Department of Physiology, St. Petersburg State University, St. Petersburg, Russia d Laboratory of Cell Biology and Histology, Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium Received 15 February 2003; received in revised form 6 April 2003; accepted 7 April 2003
Abstract Changes in the distribution of NADPH-diaphorase (NADPH-d) and acetylcholinesterase (AChE) were studied in neurons of the stellate ganglion in newborn, 10-, 20-day-old, 1-, 2-, 4- and 6-month-old kittens. AChE-positive neurons were revealed in the stellate ganglion (SG) from birth onwards. The number of these neurons increased until 20 days of postnatal life and then declined in 1- and 2-month-old kittens. A small number of weakly stained, NADPH-d-positive cells were found in newborn kittens, while intensely stained neurons first appeared in 10-day-old animals and increased in number up to the second month of life. The size of AChE-positive neurons was larger in comparison with NADPH-d-positive cells in the stellate ganglion of all animals under study. We suggest that putative vasodilator neurons or cells innervating sweat glands exhibit different development patterns from the moment of birth. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Sympathetic nervous system; Sympathetic ganglia; Stellate ganglion; NADPH diaphorase activity; Acetylcholinesterase activity; Postnatal ontogenesis
1. Introduction Postganglionic sympathetic neurons can be distinguished by their characteristic palette of cotransmitters and by morphological features, such as soma size and dendritic morphology (Gibbins, 1992, 1995; Elfvin et al., 1993; Klimaschewski et al., 1996; Gibbins et al., 2000). Vasoconstrictor and vasodilator neurons are known to have different sizes (Gibbins and Matthew, 1996). Histochemical and immunohistochemical techniques revealed that the large majority of principal sympathetic ganglionic neurons contain noradrenaline as their main neurotransmitter (von Euler, 1946; Hartman, 1972). Certain subpopulations of sympathetic neurons are cholinergic. These neurons display particularly high acetylcholinesterase (AChE) activity (Sjo¨qvist, 1963) and contain VIP and calcitonin gene-related peptide (CGRP) (Lundberg et al., 1979; Heym et al., 1993), and, in the cat, additionally substance P (Lindth et al., 1990). Further substances found * Corresponding author. Tel.: +7-852-305763; fax: +7-852-305013. E-mail address: [email protected] (P.M. Masliukov).
in these populations are choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) (Buckley et al., 1967; Weihe et al., 1996; Guidry and Landis, 1998). Some neurons express nitric oxide synthase (NOS) (Bredt et al., 1990; Morris et al., 1995; Klimaschewski et al., 1996). NO may colocalize with acetylcholine and one or more neuropeptides. In the cat, NOS was detected in 99% of presumably sudomotor neurons exhibiting CGRP and VIP immunoreactivity and in 70% of presumably muscle vasodilatator neurons containing VIP but not CGRP (Anderson et al., 1995). Nevertheless, the neurotransmitter features in sympathetic neurons are subject to change during development. It is generally accepted that the catecholaminergic transmitter phenotype in sympathetic neurons develops during embryogenesis (Cochard et al., 1979; Ernsberger and Rohrer, 1996). Some studies describe changes in neuropeptide content in sympathetic ganglionic neurons during maturation (Baetge et al., 1990; Roudenok, 2000; Anderson et al., 2001; Roudenok and Kuhnel, 2001). Sympathetic AChE-positive neurons innervating sweat glands in rats are initially noradrenergic but, after birth, change their neuro-
1566-0702/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1566-0702(03)00051-1
P.M. Masliukov et al. / Autonomic Neuroscience: Basic and Clinical 106 (2003) 84–90 Table 1 Mean values of the basic morphometric parameters of AChE-positive neurons in the SG of kittens of different ages (n = 100 for each age group) Age
Surface area (Am2)
Maximal diameter (Am)
Optical density
Newborn 10-day-old 20-day-old 1-month-old 2-month-old 4-month-old 6-month-old
295.1 F 16.95* 471.1 F 43.18w 453.3 F 18.63w 584.2 F 32.14x 663.2 F 31.44r 651.8 F 25.75r 683.5 F 24.86r
24.7 F 0.58* 30.4 F 0.85w 30.3 F 0.66w 33.1 F 1.05x 35.2 F 0.53r 35.1 F 0.64r 35.3 F 0.81r
14.7 F 0.86 16.4 F 0.75 16.2 F 0.91 14.6 F 0.63 15.9 F 0.56 14.5 F 0.82 16.1 F 0.63
*, w, x, r statistically significant differences ( P < 0.05). Mean values with the same symbol in the same column are not statistically different from each other.
transmitter phenotype from noradrenergic to cholinergic (Ernsberger and Rohrer, 1999). Other data, however, indicate the presence of AChE-positive neurons in the rat stellate ganglion (SG) in newborn puppies (Rumjanceva, 2000). In addition, the way in which neurons with an NADPH-diaphorase (NADPH-d)-positive phenotype develop is still unknown. The majority of studies dealing with the postnatal ontogenesis of cholinergic transmission so far were performed
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using a histochemical method for AChE detection (Sjo¨qvist, 1963; Lundberg et al., 1979; Lindth et al., 1989; Ernsberger and Rohrer, 1999), although it should be kept in mind that nowadays more specific immunocytochemical markers are available. Intense AChE and NADPH-d activity correlates highly with the activity of enzymes involving in acetylcholine metabolism (Sjo¨qvist, 1963) and NOS (Hope et al., 1991; Santer and Symons, 1993) in neurons. The purpose of this study was to investigate the development of AChE- and NADPH-d-positive neurons in the SG of kittens aged 0 –180 days. The SG is known as important center of sympathetic innervation of organs of the thoracic cavity, neck and upper extremity (Gabella, 1976; Nozdrachev, 1983; Phillips et al., 1986). There are some data indicating that sympathetic ganglion consists of different populations of neurons. Neurons in the ganglion can differ by their function and neurotransmitter compound (Gibbins, 1992; Morris et al., 1998). Our previous study indicated that neurons in the cat SG innervating different targets exhibit a clear topography (Masliukov et al., 2000). In the present study, we aimed at correlating the size and neurochemical features of these neurons with their specific topography.
Fig. 1. Microphotographs of AChE-positive neurons in the SG in (1) newborn, (2) 10-day-old, (3) 20-day-old, (4) 2-month-old kittens. Bar, 30 Am.
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2. Materials and methods
buffer for 1 h at 37 jC, after which they were rinsed for 10 min in distilled water.
2.1. Anesthesia and tissue handling 2.3. NADPH diaphorase histochemistry Six newborn (weight 80 –120 g), six 10-day-old (weight 150 – 170 g), six 20-day-old (200 –250 g), six 1-month-old (300 –350 g), six 2-month-old (400 –500 g), six 4-monthold (700 – 1000 g) and six 6-month-old (1.5 –3.0 kg) kittens were used in this study. All animals were killed with a lethal dose of sodium pentobarbital (Nembutal, St. Petersburg, Russia, 300 mg/kg, i.p.), after which the SG were removed from each side in all animals. Ganglia were fixed for 2 h with 4% paraformaldehyde in 0.1 M phosphate buffer (PB) (pH 7.4) and washed twice in 0.1 M PB. For NADPH-d staining, ganglia were transferred to a 0.1 M PB solution containing 15% sucrose (pH 7.4) and stored overnight at 4 jC. Serial sections (30 Am) were prepared with a cryomicrotome. 2.2. AChe histochemistry AChE histochemistry was performed according to the method of Crevier and Belanger (1955). Sections were placed in a reaction medium containing 0.3 mM lead nitrate, 16 mM thioacetic acid in 0.1 M sodium citrate (pH 6.2)
NADPH-d reactivity was detected histochemically (Hope and Vincent, 1989). Frozen sections were washed in 0.1 M phosphate buffered saline for 20 min, 0.05 M Tris buffer (pH 7.6) for 10 min and then incubated for 60 min in the dark at 37 jC in 0.05 M Tris buffer, containing 1 mM hNADPH and 0.5 mM nitroblue tetrazolium and 0.2% Triton X-100 (all purchased from Sigma, St. Louis, USA). The reaction was stopped by adding excess 0.05 mM Tris buffer. 2.4. Data analysis Morphometric analysis (cross-sectional surface area, maximal diameter, optical (from 0 (dark) to 1 (light)) and distribution density) was performed with BIOSCAN (Konako, Minsk) software running on a base IBM PC. The number of neurons in the SG was determined using the formula: N = P/D, where N was the number of neurons/ mm3, P the distribution density in sections of the SG and D the diameter of the cross-sectional area (hypothetically conceived as a circle) (Avtandilov, 1990). The number of
Fig. 2. Microphotographs of NADPH-d-positive neurons in the SG in (1) newborn, (2) 10-day-old, (3) 20-day-old, (4) 2-month-old kittens. Bar, 30 Am.
P.M. Masliukov et al. / Autonomic Neuroscience: Basic and Clinical 106 (2003) 84–90 Table 2 Mean values of the basic morphometric parameters of NADPH-d-positive neurons in the SG of kittens of different ages (n = 100 for each age group) Age
Surface area (Am2)
Maximal diameter (Am)
Optical density
10-day-old 20-day-old 1-month-old 2-month-old 4-month-old 6-month-old
162.4 F 15.31* 243.5 F 24.81w 391.7 F 29.32x 443.2 F 35.63r 428.7 F 32.25r 414.3 F 27.36r
16.7 F 0.86* 22.2 F 0.88w 33.1 F 1.05x 29.0 F 0.95r 28.1 F 0.83r 27.2 F 1.04r
29.8 F 0.89* 28.5 F 0.86* 18.6 F 0.72w 14.2 F 0.82x 15.3 F 0.85x 14.9 F 0.94x
*, w, x, r statistically significant differences ( P < 0.05). Mean values with the same symbol in the same column are not statistically different from each other.
neurons/mm3 was multiplied by the ganglion volume, yielding the total number of neurons. The ganglion volume was calculated by multiplying the mean cross-sectional surface area by the thickness of the section. Statistical methods include calculation of the mean, standard error of the mean. The significance of differences was evaluated by t-test. Differences were considered statistically significant if P < 0.05.
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3.1. AChe-positive neurons The staining intensity of AChE-positive neurons ranged from weak to intense, with intensely stained AChE-positive neurons found in the SG of all animals. AChE-positive neurons had a round or oval shape. The mean cross-sectional surface area and maximal diameter increased postnatally (Table 1, Fig. 1). No differences in size were observed between neurons in 10- and 20-day-old kittens. Soma size, however, started to increase from 20 days onwards up to 2 months of life, after which the parameters under study did not change further. The optical density of neurons was virtually identical in all animals under study. In newborn kittens, AChE-positive neurons were located in the entire ganglionic area, except at places where the SG anastomosed with the vagosympathetic trunk (Fig. 3). This pattern was found in newborn, 10- and 20-day-old kittens. Subsequently, the distribution area of the neurons decreased to reach its minimal level as from 2 months of life (Table 3), with neurons now restricted to the lateral part of the ganglion. The number of AChE-positive neurons in the SG was not constant but increased from birth onwards, reaching its peak in 20-day-old kittens (Table 4), and then decreased in 1- and 2-month-old animals.
3. Results 3.2. NADPH-d-positive neurons The optical density of both populations (AChE-positive and NADPH-d-positive neurons) varied from 0.18 to 0.63. Neurons were divided into three groups according to staining intensity (weak, medium and intense); only cells with intense staining (0.18 – 0.33) were included in our study.
NADPH-d-positive cells were observed in newborn kittens but their number was still very small at this stage of development and they displayed only very weak staining (Fig. 2). Intensely stained, NADPH-d-positive neurons were first detected in 10-day-old animals. The size of NADPH-d-
Fig. 3. Schematic drawing of zones (dotted), where AChE-(1 – 4) and NADPH-d-positive (5 – 8) neurons in the SG are located in (1, 5) newborn, (2, 6) 10-dayold, (3, 7) 20-day-old, (4, 8) 2-month-old kittens. (T1) and (T2): communicating branches with ThI and ThII spinal nerves.
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Table 3 Areas occupied by AChE- and NADPH-d-positive neurons in the SG of kittens of different ages (%) (n = 3 for each age group) Age
AChE-positive neurons
NADPH-d-positive neurons
Newborn 10-day-old 20-day-old 1-month-old 2-month-old 4-month-old 6-month-old
79.4 F 3.89* 81.8 F 5.13* 85.3 F 4.45* 67.1 F 3.38w 54.3 F 3.45x 51.5 F 2.88x 55.1 F 4.08x
– 30.3 F 3.28* 75.8 F 4.56w 62.4 F 3.15x 53.1 F 3.63r 48.7 F 4.67r 45.5 F 3.81r
*, w, x, r statistically significant differences ( P < 0.05). Mean values with the same symbol in the same column are not statistically different from each other.
positive neurons increased from 10 days to 2 months of life (Table 2). The number of these neurons also increased gradually up to the second month of life (Table 4). No differences in shape were found between AChE- and NADPH-d-positive neurons. The optical density of NADPH-d-positive neurons decreased postnatally from 20 days to 2 months of life. The occurrence of NADPH-d-positive neurons in 10day-old kittens was restricted to the lateral part of the ganglion (Fig. 3, Table 3). The surface area was highest in 20-day-old animals and then decreased until the second month of life. No further changes were observed afterwards.
4. Discussion The findings of this study demonstrate that the development of the AChE- and NADPH-d phenotypes in cat SG neurons does not take place simultaneously. Intensely stained AChE-positive neurons are already present at the moment of birth. Their number increases up to 20 days of age and then declines. A similar pattern was previously found in SG neurons sending their axons to different target organs (Masliukov et al., 2000). The reason for this decrease is not clear. Some neurons may die during ontogenesis due to apoptosis. However, we found that the total number of neurons (Table 4) in the cat SG remains constant from 20 days of life onwards (Masliukov, 2001). It could be speculated that neurons might change their transmitter content, as also shown for cholinergic neurons that were initially noradrenergic in nature prior to establishing synaptic contacts with sweat glands (Ernsberger and Rohrer, 1999). In contrast, in rat sympathetic ganglia, AChE activity already starts during the first week after birth (Ernsberger and Rohrer, 1999). These discrepancies could reflect species differences. Intensely stained NADPH-d-positive neurons were first observed in 10-day-old animals and their number increased gradually. Since only intensely stained NADPH-d-positive
neurons are involved in NOS production (Hope et al., 1991; Santer and Symons, 1993), it could be suggested that NOSpositive cells in the SG are absent in newborn animals but only appear later. Both neuronal populations are located in the same area, i.e., the lateral part of the SG. Our previous results indicate that neurons located in this area send their axons to the forelimb and spinal ganglia (Fateev et al., 2000; Masliukov et al., 2000). It is generally accepted that cholinergic neurons innervate sweat glands and blood vessels in cats. The maximal number of AChE-positive neurons was observed at the time when the animal is able to stand. AChE-positive neurons declined in number after the first month of life when motor activity of the animal is fully developed. Differences in the number of nitroxidergic neurons could be explained by possible other functions. NO-containing neurons may also influence heart activity (Choate and Paterson, 1999). The constant number of AChE- and NADPH-d-positive neurons observed after the second month of life is in accordance with data obtained by Haddad and Armour (1991), who found that the dog SG functionally matures also during this stage. AChE- and NADPH-d-positive neurons are differently sized. The cross-sectional area and maximal diameter of AChE-positive neurons were larger than those of the general methylene blue-stained population of SG neurons in all studied animals. NADPH-d-positive cells, on the other hand, were smaller in comparison with this basic population of SG neurons in newborn and older kittens (Masliukov, 2001). In conclusion, AChE- and NADPH-d-positive cells developed in different ways of development. Nevertheless, both neuronal populations have completed maturation by the second month of life. The presence of AChe-positive cells even at the moment of birth confirm our previous observations that the SG in newborn have heterogeneous populations of neurons (Masliukov et al., 2000). Thus, the SG of newborn kittens may not only regulate the heart work but presumably, control
Table 4 Number of neurons with different histochemical patterns in the SG (n = 3 for each age group) Age
AChE-positive neurons
NADPH-d-positive neurons
Newborn 10-day-old 20-day-old 1-month-old 2-month-old 4-month-old 6-month-old
6260 F 748* 7627 F 825* 9109 F 624w 4225 F 513x 1698 F 223r 1515 F 321r 1711 F 264r
– 15 F 4* 312 F 29w 2195 F 381x 8368 F 629r 7954 F 543r 8384 F 429r
*, w, x, r statistically significant differences ( P < 0.05). Mean values with the same symbol in the same column are not statistically different from each other.
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vasodilatation. However, this control at this stage is not mature.
Acknowledgements M.P.M. is recipient of an INTAS Fellowship grant for Young Scientists (Fellowship Reference YSF 2002-0020). We are grateful to Drs. Rumyantseva and Telushkin (Yaroslavl State Medical Academy) for their help in studying the methods used in this work.
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Histochemical features of neurons in the cat stellate ganglion during postnatal ontogenesis Petr M. Masliukov a,*, Valentin V. Shilkin b, Alexandr D. Nozdrachev c, Jean-Pierre Timmermans d a
Department of Normal Physiology and Biophysics, Yaroslavl State Medical Academy, Revoliucionnaya 5, Yaroslavl 150000, Russia b Department of Human Anatomy, Yaroslavl State Medical Academy, Yaroslavl, Russia c Department of Physiology, St. Petersburg State University, St. Petersburg, Russia d Laboratory of Cell Biology and Histology, Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium Received 15 February 2003; received in revised form 6 April 2003; accepted 7 April 2003
Abstract Changes in the distribution of NADPH-diaphorase (NADPH-d) and acetylcholinesterase (AChE) were studied in neurons of the stellate ganglion in newborn, 10-, 20-day-old, 1-, 2-, 4- and 6-month-old kittens. AChE-positive neurons were revealed in the stellate ganglion (SG) from birth onwards. The number of these neurons increased until 20 days of postnatal life and then declined in 1- and 2-month-old kittens. A small number of weakly stained, NADPH-d-positive cells were found in newborn kittens, while intensely stained neurons first appeared in 10-day-old animals and increased in number up to the second month of life. The size of AChE-positive neurons was larger in comparison with NADPH-d-positive cells in the stellate ganglion of all animals under study. We suggest that putative vasodilator neurons or cells innervating sweat glands exhibit different development patterns from the moment of birth. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Sympathetic nervous system; Sympathetic ganglia; Stellate ganglion; NADPH diaphorase activity; Acetylcholinesterase activity; Postnatal ontogenesis
1. Introduction Postganglionic sympathetic neurons can be distinguished by their characteristic palette of cotransmitters and by morphological features, such as soma size and dendritic morphology (Gibbins, 1992, 1995; Elfvin et al., 1993; Klimaschewski et al., 1996; Gibbins et al., 2000). Vasoconstrictor and vasodilator neurons are known to have different sizes (Gibbins and Matthew, 1996). Histochemical and immunohistochemical techniques revealed that the large majority of principal sympathetic ganglionic neurons contain noradrenaline as their main neurotransmitter (von Euler, 1946; Hartman, 1972). Certain subpopulations of sympathetic neurons are cholinergic. These neurons display particularly high acetylcholinesterase (AChE) activity (Sjo¨qvist, 1963) and contain VIP and calcitonin gene-related peptide (CGRP) (Lundberg et al., 1979; Heym et al., 1993), and, in the cat, additionally substance P (Lindth et al., 1990). Further substances found * Corresponding author. Tel.: +7-852-305763; fax: +7-852-305013. E-mail address: [email protected] (P.M. Masliukov).
in these populations are choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) (Buckley et al., 1967; Weihe et al., 1996; Guidry and Landis, 1998). Some neurons express nitric oxide synthase (NOS) (Bredt et al., 1990; Morris et al., 1995; Klimaschewski et al., 1996). NO may colocalize with acetylcholine and one or more neuropeptides. In the cat, NOS was detected in 99% of presumably sudomotor neurons exhibiting CGRP and VIP immunoreactivity and in 70% of presumably muscle vasodilatator neurons containing VIP but not CGRP (Anderson et al., 1995). Nevertheless, the neurotransmitter features in sympathetic neurons are subject to change during development. It is generally accepted that the catecholaminergic transmitter phenotype in sympathetic neurons develops during embryogenesis (Cochard et al., 1979; Ernsberger and Rohrer, 1996). Some studies describe changes in neuropeptide content in sympathetic ganglionic neurons during maturation (Baetge et al., 1990; Roudenok, 2000; Anderson et al., 2001; Roudenok and Kuhnel, 2001). Sympathetic AChE-positive neurons innervating sweat glands in rats are initially noradrenergic but, after birth, change their neuro-
1566-0702/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1566-0702(03)00051-1
P.M. Masliukov et al. / Autonomic Neuroscience: Basic and Clinical 106 (2003) 84–90 Table 1 Mean values of the basic morphometric parameters of AChE-positive neurons in the SG of kittens of different ages (n = 100 for each age group) Age
Surface area (Am2)
Maximal diameter (Am)
Optical density
Newborn 10-day-old 20-day-old 1-month-old 2-month-old 4-month-old 6-month-old
295.1 F 16.95* 471.1 F 43.18w 453.3 F 18.63w 584.2 F 32.14x 663.2 F 31.44r 651.8 F 25.75r 683.5 F 24.86r
24.7 F 0.58* 30.4 F 0.85w 30.3 F 0.66w 33.1 F 1.05x 35.2 F 0.53r 35.1 F 0.64r 35.3 F 0.81r
14.7 F 0.86 16.4 F 0.75 16.2 F 0.91 14.6 F 0.63 15.9 F 0.56 14.5 F 0.82 16.1 F 0.63
*, w, x, r statistically significant differences ( P < 0.05). Mean values with the same symbol in the same column are not statistically different from each other.
transmitter phenotype from noradrenergic to cholinergic (Ernsberger and Rohrer, 1999). Other data, however, indicate the presence of AChE-positive neurons in the rat stellate ganglion (SG) in newborn puppies (Rumjanceva, 2000). In addition, the way in which neurons with an NADPH-diaphorase (NADPH-d)-positive phenotype develop is still unknown. The majority of studies dealing with the postnatal ontogenesis of cholinergic transmission so far were performed
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using a histochemical method for AChE detection (Sjo¨qvist, 1963; Lundberg et al., 1979; Lindth et al., 1989; Ernsberger and Rohrer, 1999), although it should be kept in mind that nowadays more specific immunocytochemical markers are available. Intense AChE and NADPH-d activity correlates highly with the activity of enzymes involving in acetylcholine metabolism (Sjo¨qvist, 1963) and NOS (Hope et al., 1991; Santer and Symons, 1993) in neurons. The purpose of this study was to investigate the development of AChE- and NADPH-d-positive neurons in the SG of kittens aged 0 –180 days. The SG is known as important center of sympathetic innervation of organs of the thoracic cavity, neck and upper extremity (Gabella, 1976; Nozdrachev, 1983; Phillips et al., 1986). There are some data indicating that sympathetic ganglion consists of different populations of neurons. Neurons in the ganglion can differ by their function and neurotransmitter compound (Gibbins, 1992; Morris et al., 1998). Our previous study indicated that neurons in the cat SG innervating different targets exhibit a clear topography (Masliukov et al., 2000). In the present study, we aimed at correlating the size and neurochemical features of these neurons with their specific topography.
Fig. 1. Microphotographs of AChE-positive neurons in the SG in (1) newborn, (2) 10-day-old, (3) 20-day-old, (4) 2-month-old kittens. Bar, 30 Am.
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2. Materials and methods
buffer for 1 h at 37 jC, after which they were rinsed for 10 min in distilled water.
2.1. Anesthesia and tissue handling 2.3. NADPH diaphorase histochemistry Six newborn (weight 80 –120 g), six 10-day-old (weight 150 – 170 g), six 20-day-old (200 –250 g), six 1-month-old (300 –350 g), six 2-month-old (400 –500 g), six 4-monthold (700 – 1000 g) and six 6-month-old (1.5 –3.0 kg) kittens were used in this study. All animals were killed with a lethal dose of sodium pentobarbital (Nembutal, St. Petersburg, Russia, 300 mg/kg, i.p.), after which the SG were removed from each side in all animals. Ganglia were fixed for 2 h with 4% paraformaldehyde in 0.1 M phosphate buffer (PB) (pH 7.4) and washed twice in 0.1 M PB. For NADPH-d staining, ganglia were transferred to a 0.1 M PB solution containing 15% sucrose (pH 7.4) and stored overnight at 4 jC. Serial sections (30 Am) were prepared with a cryomicrotome. 2.2. AChe histochemistry AChE histochemistry was performed according to the method of Crevier and Belanger (1955). Sections were placed in a reaction medium containing 0.3 mM lead nitrate, 16 mM thioacetic acid in 0.1 M sodium citrate (pH 6.2)
NADPH-d reactivity was detected histochemically (Hope and Vincent, 1989). Frozen sections were washed in 0.1 M phosphate buffered saline for 20 min, 0.05 M Tris buffer (pH 7.6) for 10 min and then incubated for 60 min in the dark at 37 jC in 0.05 M Tris buffer, containing 1 mM hNADPH and 0.5 mM nitroblue tetrazolium and 0.2% Triton X-100 (all purchased from Sigma, St. Louis, USA). The reaction was stopped by adding excess 0.05 mM Tris buffer. 2.4. Data analysis Morphometric analysis (cross-sectional surface area, maximal diameter, optical (from 0 (dark) to 1 (light)) and distribution density) was performed with BIOSCAN (Konako, Minsk) software running on a base IBM PC. The number of neurons in the SG was determined using the formula: N = P/D, where N was the number of neurons/ mm3, P the distribution density in sections of the SG and D the diameter of the cross-sectional area (hypothetically conceived as a circle) (Avtandilov, 1990). The number of
Fig. 2. Microphotographs of NADPH-d-positive neurons in the SG in (1) newborn, (2) 10-day-old, (3) 20-day-old, (4) 2-month-old kittens. Bar, 30 Am.
P.M. Masliukov et al. / Autonomic Neuroscience: Basic and Clinical 106 (2003) 84–90 Table 2 Mean values of the basic morphometric parameters of NADPH-d-positive neurons in the SG of kittens of different ages (n = 100 for each age group) Age
Surface area (Am2)
Maximal diameter (Am)
Optical density
10-day-old 20-day-old 1-month-old 2-month-old 4-month-old 6-month-old
162.4 F 15.31* 243.5 F 24.81w 391.7 F 29.32x 443.2 F 35.63r 428.7 F 32.25r 414.3 F 27.36r
16.7 F 0.86* 22.2 F 0.88w 33.1 F 1.05x 29.0 F 0.95r 28.1 F 0.83r 27.2 F 1.04r
29.8 F 0.89* 28.5 F 0.86* 18.6 F 0.72w 14.2 F 0.82x 15.3 F 0.85x 14.9 F 0.94x
*, w, x, r statistically significant differences ( P < 0.05). Mean values with the same symbol in the same column are not statistically different from each other.
neurons/mm3 was multiplied by the ganglion volume, yielding the total number of neurons. The ganglion volume was calculated by multiplying the mean cross-sectional surface area by the thickness of the section. Statistical methods include calculation of the mean, standard error of the mean. The significance of differences was evaluated by t-test. Differences were considered statistically significant if P < 0.05.
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3.1. AChe-positive neurons The staining intensity of AChE-positive neurons ranged from weak to intense, with intensely stained AChE-positive neurons found in the SG of all animals. AChE-positive neurons had a round or oval shape. The mean cross-sectional surface area and maximal diameter increased postnatally (Table 1, Fig. 1). No differences in size were observed between neurons in 10- and 20-day-old kittens. Soma size, however, started to increase from 20 days onwards up to 2 months of life, after which the parameters under study did not change further. The optical density of neurons was virtually identical in all animals under study. In newborn kittens, AChE-positive neurons were located in the entire ganglionic area, except at places where the SG anastomosed with the vagosympathetic trunk (Fig. 3). This pattern was found in newborn, 10- and 20-day-old kittens. Subsequently, the distribution area of the neurons decreased to reach its minimal level as from 2 months of life (Table 3), with neurons now restricted to the lateral part of the ganglion. The number of AChE-positive neurons in the SG was not constant but increased from birth onwards, reaching its peak in 20-day-old kittens (Table 4), and then decreased in 1- and 2-month-old animals.
3. Results 3.2. NADPH-d-positive neurons The optical density of both populations (AChE-positive and NADPH-d-positive neurons) varied from 0.18 to 0.63. Neurons were divided into three groups according to staining intensity (weak, medium and intense); only cells with intense staining (0.18 – 0.33) were included in our study.
NADPH-d-positive cells were observed in newborn kittens but their number was still very small at this stage of development and they displayed only very weak staining (Fig. 2). Intensely stained, NADPH-d-positive neurons were first detected in 10-day-old animals. The size of NADPH-d-
Fig. 3. Schematic drawing of zones (dotted), where AChE-(1 – 4) and NADPH-d-positive (5 – 8) neurons in the SG are located in (1, 5) newborn, (2, 6) 10-dayold, (3, 7) 20-day-old, (4, 8) 2-month-old kittens. (T1) and (T2): communicating branches with ThI and ThII spinal nerves.
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Table 3 Areas occupied by AChE- and NADPH-d-positive neurons in the SG of kittens of different ages (%) (n = 3 for each age group) Age
AChE-positive neurons
NADPH-d-positive neurons
Newborn 10-day-old 20-day-old 1-month-old 2-month-old 4-month-old 6-month-old
79.4 F 3.89* 81.8 F 5.13* 85.3 F 4.45* 67.1 F 3.38w 54.3 F 3.45x 51.5 F 2.88x 55.1 F 4.08x
– 30.3 F 3.28* 75.8 F 4.56w 62.4 F 3.15x 53.1 F 3.63r 48.7 F 4.67r 45.5 F 3.81r
*, w, x, r statistically significant differences ( P < 0.05). Mean values with the same symbol in the same column are not statistically different from each other.
positive neurons increased from 10 days to 2 months of life (Table 2). The number of these neurons also increased gradually up to the second month of life (Table 4). No differences in shape were found between AChE- and NADPH-d-positive neurons. The optical density of NADPH-d-positive neurons decreased postnatally from 20 days to 2 months of life. The occurrence of NADPH-d-positive neurons in 10day-old kittens was restricted to the lateral part of the ganglion (Fig. 3, Table 3). The surface area was highest in 20-day-old animals and then decreased until the second month of life. No further changes were observed afterwards.
4. Discussion The findings of this study demonstrate that the development of the AChE- and NADPH-d phenotypes in cat SG neurons does not take place simultaneously. Intensely stained AChE-positive neurons are already present at the moment of birth. Their number increases up to 20 days of age and then declines. A similar pattern was previously found in SG neurons sending their axons to different target organs (Masliukov et al., 2000). The reason for this decrease is not clear. Some neurons may die during ontogenesis due to apoptosis. However, we found that the total number of neurons (Table 4) in the cat SG remains constant from 20 days of life onwards (Masliukov, 2001). It could be speculated that neurons might change their transmitter content, as also shown for cholinergic neurons that were initially noradrenergic in nature prior to establishing synaptic contacts with sweat glands (Ernsberger and Rohrer, 1999). In contrast, in rat sympathetic ganglia, AChE activity already starts during the first week after birth (Ernsberger and Rohrer, 1999). These discrepancies could reflect species differences. Intensely stained NADPH-d-positive neurons were first observed in 10-day-old animals and their number increased gradually. Since only intensely stained NADPH-d-positive
neurons are involved in NOS production (Hope et al., 1991; Santer and Symons, 1993), it could be suggested that NOSpositive cells in the SG are absent in newborn animals but only appear later. Both neuronal populations are located in the same area, i.e., the lateral part of the SG. Our previous results indicate that neurons located in this area send their axons to the forelimb and spinal ganglia (Fateev et al., 2000; Masliukov et al., 2000). It is generally accepted that cholinergic neurons innervate sweat glands and blood vessels in cats. The maximal number of AChE-positive neurons was observed at the time when the animal is able to stand. AChE-positive neurons declined in number after the first month of life when motor activity of the animal is fully developed. Differences in the number of nitroxidergic neurons could be explained by possible other functions. NO-containing neurons may also influence heart activity (Choate and Paterson, 1999). The constant number of AChE- and NADPH-d-positive neurons observed after the second month of life is in accordance with data obtained by Haddad and Armour (1991), who found that the dog SG functionally matures also during this stage. AChE- and NADPH-d-positive neurons are differently sized. The cross-sectional area and maximal diameter of AChE-positive neurons were larger than those of the general methylene blue-stained population of SG neurons in all studied animals. NADPH-d-positive cells, on the other hand, were smaller in comparison with this basic population of SG neurons in newborn and older kittens (Masliukov, 2001). In conclusion, AChE- and NADPH-d-positive cells developed in different ways of development. Nevertheless, both neuronal populations have completed maturation by the second month of life. The presence of AChe-positive cells even at the moment of birth confirm our previous observations that the SG in newborn have heterogeneous populations of neurons (Masliukov et al., 2000). Thus, the SG of newborn kittens may not only regulate the heart work but presumably, control
Table 4 Number of neurons with different histochemical patterns in the SG (n = 3 for each age group) Age
AChE-positive neurons
NADPH-d-positive neurons
Newborn 10-day-old 20-day-old 1-month-old 2-month-old 4-month-old 6-month-old
6260 F 748* 7627 F 825* 9109 F 624w 4225 F 513x 1698 F 223r 1515 F 321r 1711 F 264r
– 15 F 4* 312 F 29w 2195 F 381x 8368 F 629r 7954 F 543r 8384 F 429r
*, w, x, r statistically significant differences ( P < 0.05). Mean values with the same symbol in the same column are not statistically different from each other.
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vasodilatation. However, this control at this stage is not mature.
Acknowledgements M.P.M. is recipient of an INTAS Fellowship grant for Young Scientists (Fellowship Reference YSF 2002-0020). We are grateful to Drs. Rumyantseva and Telushkin (Yaroslavl State Medical Academy) for their help in studying the methods used in this work.
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