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Histochemical properties of axons associated with the salivary apparatus of the cockroach, Nauphoeta cinerea
0040-8166/80/00520703 $02.00
TISSUE & CELL 12(4)703%711 (("1980 Longman Group Ltd
DAVID
J. MAXWELL
HISTOCHEMICAL PROPERTIES OF AXONS ASSOCIATED WITH THE SALIVARY APPARATUS OF THE COCKROACH, NAUPHOETA CINEREA ABSTRACT. A variety of techniques have indicated that dopamine is probably the neurotransmitter at the salivary gland of the cockroach, Nauphoeta cinerea (Olivier). It is known from a previous ultrastructural study that two types of axon are associated with the gland but it is not known which of these axons contain catecholamines. The present study, using permanganate fixation or incubation in 5-hydroxydopamine or h-hydroxydopamine, shows that only one category of axon contains catecholamines.
Introduction
differentiate between these two types of axon and to demonstrate the presence of catecholamines within them. Potassium permanganate fixative has been used to demonstrate the presence of catecholamines in autonomic nerve endings (Richardson, 1966). In vitro tests with permanganate fixative and various compounds suggested that it reacts specifically with biogenic monoamines, forming an electrondense precipitate (HGkfelt and Jonsson, 1968). Permanganate fixative has been used to demonstrate the presence of monoamines in axons investing the salivary gland of the moth, Manduca sexta (Robertson, 1974). S-Hydroxydopamine is believed to be taken up by catecholaminergic boutons, displacing the natural transmitter, and resulting in the appearance of granular vesicles within them (Tranzer and Thoenen, 1967). It has been suggested that high concentrations of 5-hydroxydopamine will also enter 5-hydroxytryptamine endings (Ajika and Hiikfelt, 1973) indicating that this method is specific for monoamines rather than catecholamines. There is a considerable body of evidence to support the idea that 6-hydroxydopamine selectively destroys catecholamine containing 1968; neurones (Tranzer and Thoenen, Thoenen and Tranzer, 1968; Malmfors and Thoenen, 1971). However like permanganate
studies (BowserRiley and House, 1976) and secretion studies (Smith, 1977; Smith and House, 1977) have indicated that the neurotransmitter at the cockroach salivary gland is probably dopamine (for reviews see House, 1977; House and Ginsborg, 1979). Fluorescence histochemistry of the axons on the surface of the gland has shown that they contain a catecholamine (Bland et al., 1973). However, a previous electron microscopa1 study has revealed that two types of axon are associated with the salivary apparatus of the cockroach (Maxwell, 1978). These two types of axon were designated type A and type B according to their vesicular contents and are illustrated in Figs. 1 and 2. The main characteristics of the two axons are shown in Fig. 3. Type A axons contain large granular vesicles and small agranular vesicles whereas type B axons contain only large granular vesicles which are considerably larger than those of type A. Three methods were used in an attempt to ELECTROPHYSIOLOGICAL
University of Edinburgh, Department of Veterinary Physiology. Royal (Dick) School of Veterinary Studies. Summerhall. Edinburgh EH9 IQH. Scotland. Received I3 March 1980. Revised 24 July 1980. 703
MAXWELL
Abbreviations A : BM Gl GV LGV P
for figurer
type A axon type B axon central cell of salivary gland basement membrane glial cell granular vesicle of type PI axon large granular vesicle of type B axon peripheral cell of salivary gland
Fig. I. A type A axon. Note the small elliptical granular vesicles. x 40,000. Fig, 2. A type B awn
that is crammed
agranular
with large granular
vesicles and the larger vesicles.
x 60,000.
HISTOCHEMISTRY
OF
COCKROACH
TYPE
0
0
705
AXONS
A
TYPE
B
40+15t32nm 91t21.47 nm
0
134f2389nm
Fig. 3. A diagram representing the salient features of type A and B axons v+ith their vesicular dimensions. The dark objects represent electron-dense granules; elliptical objects represent the agranular ve;iclex of type A axons.
and 5-hydroxydopamine it seems probable that 5-hydroxytryptamine systems are also affected by 6-hydroxydopamine as Berry et al. (1974) found that it abolished paraformaldehyde induced 5-hydroxytryptamine Auorescence in the central nervous system of the water snail Planorbis corneus. Materials and Methods The entire salivary apparatuses of adult cockroaches, Nauphoeta cinerea (Olivier), were removed under saline (Smith and House, 1977). Potassium permanganate fixation was performed by placing the tissue in an ice cold solution of 3% KMn04 in 0.1 M phosphate buffer at pH 7.0 for 3045 min (Richardson, 1966). The tissue was then washed in phosphate buffer, dehydrated in graded alcohol solutions, cleared in propylene oxide and embedded in Araldite. Thin sections were stained with uranyl acetate and lead citrate (Reynolds, 1963). Treatment with 5-hydroxydopamine or 6-hydroxydopamine was performed by incubating fresh tissue in 0.1 mg/ml of 5hydroxydopamine (Sigma) in cockroach saline for 1 hr or in 1 mg/ml 6-hydroxydopamine (Sigma) in saline for 4 hr. Control tissues were incubated in cockroach saline alone for equal periods of time. Incubated glands were then fixed i hr in 4% glutaraldehyde in 0.05 M sodium cacodylate buffer
with 2.8% sucrose added. This solution had a tonicity of 370 mOsm, a pH of 7.2 and was used at 4°C. The tissue was then processed conventionally for electron microscopy (Maxwell, 1978) except that some sections of tissue that was incubated in 5-hydroxydopamine were not stained with uranyl acetate and lead citrate. This procedure facilitated discovery of electron-dense deposits of 5-hydroxydopamine. Data from micrographs were processed and stored in the files of a PDP-12 computer. Results Potassium permanganate fixation Figs. 4 and 5 illustrate the typical product of potassium permanganate fixation of the tissue of the cockroach salivary gland. In keeping with the observations of other authors (e.g. Hokfelt, 1968) mitochondria were found to be swollen and the general state of preservation of the tissue was not as good as that obtained with osmium and gldtaraldehyde fixation. Type A axons exhibited a positive reaction to permanganate fixation; i.e. they were noted to contain small granular vesicles of a circular profile. These vesicles have a mean diameter of 46~ 9 nm. The large granular vesicles of type A axons had a more opaque appearance (Fig. 4) than those after glutaraldehyde and osmium fixation (Fig. I). Type B axons did not react strongly with
706
MAXWELL
permanganate fixative. Their large opaque vesicles were found to have a mean diameter of 139 f 21 nm which is not statistically different from populations of large granular vesicles in type B axons observed after glutaraldehyde and osmium fixation (Fig. 2). These results indicate that the small vesicles of type A axons contain a biogenic monamine. Incubation in S-hydroxydopamine After incubation for 1 hr in 5hydroxydopamine type A axons were seen to contain some small flattened vesicles that contained dense material (Figs. 6, 7). These vesicles had a mean dimension of 40 nm. Electron-dense deposits of 5-hydroxydopamine could readily be seen in sections of type A axons that had not been stained with lead and uranium salts (Fig. 8). In addition to the small vesicles the large granular vesicles were also observed and did not seem to have been altered by the 5-hydroxydopamine. Type B axons did not exhibit any changes after incubation in 5hydroxydopamine (Fig. 8) and tissue that was incubated cockroach
saline did not display any dense material within the small vesicles of type A axons. Incubation it? 6-hydroxydopamine After 4 hr of incubation in 6-hydroxydopamine virtually every type A axon observed showed some signs of degeneration (Figs. 9, 10, 1I). Mitochondria within nerves were often seen to be damaged and vesicles appeared to aggregate. According to Smith et al. (1966) this aggregation is a sign of axonal degeneration. On the other hand type B axons had a completely normal appearance (Fig. 12). Tissue that was incubated in cockroach saline for 4 hr showed no signs of axonal degeneration. Vesicle shapes During the course of this study it was noted that the small vesicles of type A axons displayed different shaped profiles, depending upon the type of fixative employed. A previous study has shown that, after glutaraldehyde and osmium fixation, the small agranular vesicles of type A axons come from
Fig. 4. A type A axon that was fixed in potassium small granular vesicles (arrow) and the more opaque Fig. 5. Tissue that was fixed in potassium shown. x 30,000.
permanganate fixative. Note the larger vesicles. x 45,000.
permanganate.
Fig. 6. The effect of incubation in 5-hydroxydopamine. vesicles of type A axons are now granular in appearance. Fig. 7. A type A axon after incubation
Type A and B axons are Note that many of the small x 30,000.
in Shydroxydopamine.
x 60,000.
Fig. 8. An unstained section of tissue that was incubated in Shydroxydopamine. Electron-dense deposits of 5-hydroxydopamine may be seen within the small vesicles of a type A axon, whereas a type B axon has not been affected by the incubation. x 60,000. Fig. 9. A type A axon after incubation in 6-hydroxydopamine. Note the aggregation of the vesicles (arrows) and a damaged mitochondrion (asterisk). x 40,000. Fig. IO. Incubation in 6-hydroxydopamine; in a state of degeneration (arrows). x 22,500.
two type A axons are shown which are
Fig. 1 I. Two type A axons (arrows) in a state of degeneration h-hydroxydopamine. The axons are situated between the central gland. x 28,000.
after incubation in cells of the salivary
Fig. 12. Incubation in 6-hydroxydopamine: type A axons are seen to be in a state of degeneration (arrows) whereas type B axons have a normal appearance. x 21,000.
i
08
HISTOCHEMISTRY
OF
COCKROACH
709
AXONS
a population of prolate rotary ellipsoids (Maxwell, 1978). In this study the shapes of vesicles were compared by plotting the logarithm of the maximum diameter divided by the minimum diameter (measured at right
a *or
I :
angles to each other) of each vesicle as a histogram (Fig. f3). This method shows that vesicles from a glutaraldehyde and osmium fixed population (Fig. 13a) and those fixed by the same method after incubation in 5-hydroxydopamine (Fig. 13~) have a similar distribution, whereas those fixed in potassium permanganate (Fig. 13b) have a narrower distribution clustering around zero. This indicates that permanganate fixed tissue has predominantly vesicles of a circular profile (i.e. probably spheres in three dimensions), whereas those fixed by glutaraldehyde and osmium tend to be elliptical. Discussion
b
Mf. 1. t. *(I t*
*
tt
t
*.
*
,o_::;;.
. . . . .,
. .. ..
! ***’ Lt..
**.*
*
If.”
.
.
I”‘:“‘. ~;
0’
z
1
c
10r
I.....;
. . .._........_.._........_....
Log
Ratio max/min
Fig. 13. Histograms of the logarithm of the maximum diameter divided by the minimum diameter of the small agranular vesicles observed in type A axons. (a) is a plot of vesicle dimensions taken from several axons that were fixed in glutaraldehyde and osmium fixative. (b) represents similar data collected after permanganate fixation. (c) is data collected from axons that were incubated in Shydroxydopamine prior to glutaraldehyde and osmium fixation. The wider distribution in graphs (a) and (c) suggests that these vesicles tend to be elliptical whereas the narrower distribution, around zero, in graph (b) is indicative of spherical vesicles.
Bland et al. (1973) have shown by microspectrofluorimetry that the fluorescence exhibited by axons on the surface of the gland after treatment by the method of Falck et al. (1962) has a spectrum consistent with the presence of catecholamines. There was no evidence to suggest the presence of S-hydroxytryptamine. Paraformaldehyde-induced fluorescence has been abolished in the nerves of the gland after incubation in 6-hydroxydopamine (Maxwell and Laszlo, unpublished observations). Pharmacological experiments, using the antagonist phentolamine, also suggest that 5-hydroxytryptamine is not the natural transmitter at the neuroglandular junction: both secretory (House and Smith, 1978) and electrophysiological (BowserRiley et al., 1978) experiments show that phentolamine can distinguish between receptors for 5-hydroxytryptamine and those for dopamine, noradrenaline and the natural transmitter. Fry et al. (1974) have shown by a radiochemical assay that homogenized salivary glands of this cockroach contain dopamine but not noradrenaline. Evidence, obtained via the three methods reported in this study conclusively demonstrates the presence of monoamines within the small vesicles of type A axons. If this evidence is considered in conjunction with these previous findings, then it is reasonable to suggest that type A axons are dopaminergic. The function and contents of type B axons are not known. It was previously suggested that they may be neurosecretory cells (Maxwell, 1978). The significance of different vesicular shapes in type A axons after potassium per-
710
MAXWELL
manganate fixation or fixation with glutaraldehyde and osmium is also not understood. It has been suggested that the osmolality of the fixative buffer may be important (Valdivia, 1970); buffers of an osmolality greater than 1715 mOsm were found to produce flattened vesicles in certain cells in the mammalian cerebellum. This suggestion is not applicable to the present study as the total tonicity of the fixative did not exceed 370 mOsm. It has also been claimed that aldehyde fixatives produce flattened vesicles. Thus Walberg (1966) found 40 % more flattened vesicles after fixation of the terminal boutons of the
mammalian central nervous system with glutaraldehyde and osmium than with osmium fixation alone. In view of these findings it is unwise to classify vesicles on the basis of their shapes. Acknowledgements I wish to thank Drs K. P. Bland and C. R. House for their useful criticism of this manuscript and also Mr C. M. Warwick for assistance in the production of the photographic plates. Financial support was provided by the Science Research Council.
References AJIKA, K. and HGKFELT, T. 1973. Ultrastructural identification of catecholamine neurones in the hypothalamic periventricular-arcuate nucleus-median eminence complex with special reference to quantitative aspects. Bruin Res., 57, 97-117. BERRY, M. S., PENTREATH,V. W., TURNER, J. D. and COTTRELL, G. A. 1974. Effects of 6-hydroxydopamine on an identified dopamine containing neuron in the central nervous system of Planorbis corneus. Brain Res., 76, 304-324.
BLAND, K. P., HOUSE, C. R., GINSBORG, B. L. and LASZLO, I. 1973. Catecholamine transmitter for salivary secretion in the cockroach. Nature New Biol., 244, 26-27. BOWSER-RILEY, F. and HOUSE, C. R. 1976. The actions of some putative neurotransmitters on the cockroach salivary gland. J. exp. Biol., 64, 665-676. BOWSER-RILEY, F., HOUSE, C. R. and SMITH, R. K. 1978. Competitive antagonism by phentolamine of responses to biogenic amines and the transmitter at a neuroglandular junction. J. Physiol., 279,473-489. FALCK, B., HILLARP, N.-A., THIEME, G. and TORP, A. 1962. Fluorescence of catecholamines and related compounds condensed with formaldehyde. J. Histochem. Cytochem., 10, 348-354. FRY, J. P., HOUSE, C. R. and SHARMAN,D. F. 1974. An analysis of the catecholamine content of the salivary gland of the cockroach. Br. J. Phnmac., 51, 116p-117~. H&FELT, T. 1968. In vitro studies on central and peripheral monamine neurons at the ultrastructural level. Z. Zellforsch. mikrosk. Amt., 91, l-74. HBKFELT, T. and JONSSON, G. 1968. Studies on reaction and binding of monoamines after fixation and processing for electron microscopy with special reference to fixation with potassium permanganate. Histochemie,
16, 45-67.
HOUSE, C. R. 1977. Cockroach Transport
Academic Press, London. HOUSE, C. R. and GINSBORG, Physiol.,
salivary
gland: a secretory epithelium with a dopaminergic (eds. B. J. Gupta, R. B. Moreton, J. L. Oschman
of Ions and Water in Animals
63C,
B. L. 1979. Pharmacology
of cockroach
salivary
secretion.
innervation. In and B. J. Wall). Camp.
Biochem.
1-6.
HOUSE, C. R. and SMITH, R. K. 1978. On the receptors involved in the nervous control of salivary secretion by Nauphoeta cinerea (Olivier). J. Physiol., 279, 457-47 I. MALMFORS, T. and THOENEN, H. 1971. 6-Hydroxydopamine and Catecholamine Neurons. North-Holland, Amsterdam, London. MAXWELL, D. J. 1978. Fine structure of axons associated with the salivary apparatus of the cockroach, Nauphoeta
cinerea.
Tissue & Cell, IO, 699-706.
REYNOLDS, E. S. 1963. The use of lead citrate at high pH as electron-opaque stain in electron microscopy. J. Cell Biol., 17, 208-212. RICHARDSON, K. L. 1966. Electron microscopic identification of autonomic nerve endings. Nature, 210, 756. ROBERTSON,H. A. 1974. The innervation of the salivary gland of the moth Manduca sexta. Ceii Tiss. Res., 148, 237-245. SMITH, K. R., HUDGENS, R. W. and O’LEARY, J. L. 1966. An electron microscopic study of degenerative changes in the cat cerebellum after intrinsic lesions. J. camp. Neural., 126, 15-36.
HISTOCHEMISTRY
OF
COCKROACH
AXONS
711
SMITH, R. K. 1977. Catecholamine receptors mediating cockroach salivary secretion. Biochem. SW. Tran,\., 5, 173-174. SMITH, R. K. and HOUSE, C. R. 1977. Fluid secretion by isolated cockroach salivary glands. Expermtia, 33. 1182-1183. THOENEN. H. and TRANZER, J. P. 1968. Chemical sympathectomy by selective destruction of adrenergic nerve endings with 6-hydroxydopamine. Naunyn-Schmiedebergs Arch. Pharmak. exp. Path., 261, 271-288.
TRANZER, J. P. and THOENEN, H. 1967. Electron microscopic localisation of 5-hydroxydopamine (3,4,5trihydroxyphenyl-ethylamine), a new ‘false’ sympathetic transmitter. Experentia, 23, 743-745. TRANZER, J. P. and THOENEN, H. 1968. An electron microscopic study of selective, acute degeneration of sympathetic nerve terminals after administration of 6-hydroxydopamine. Experentia. 24, 155-156. VALDIVIA, 0. 1970. Methods of fixation and the morphology of synaptic vesicles. Amt. Rec., 166, 392. WALBERG, F. 1966. Elongated vesicles in terminal boutons of the central nervous system: a result of aldehyde fixarion. Acta amt., 65, 224-235.
TISSUE & CELL 12(4)703%711 (("1980 Longman Group Ltd
DAVID
J. MAXWELL
HISTOCHEMICAL PROPERTIES OF AXONS ASSOCIATED WITH THE SALIVARY APPARATUS OF THE COCKROACH, NAUPHOETA CINEREA ABSTRACT. A variety of techniques have indicated that dopamine is probably the neurotransmitter at the salivary gland of the cockroach, Nauphoeta cinerea (Olivier). It is known from a previous ultrastructural study that two types of axon are associated with the gland but it is not known which of these axons contain catecholamines. The present study, using permanganate fixation or incubation in 5-hydroxydopamine or h-hydroxydopamine, shows that only one category of axon contains catecholamines.
Introduction
differentiate between these two types of axon and to demonstrate the presence of catecholamines within them. Potassium permanganate fixative has been used to demonstrate the presence of catecholamines in autonomic nerve endings (Richardson, 1966). In vitro tests with permanganate fixative and various compounds suggested that it reacts specifically with biogenic monoamines, forming an electrondense precipitate (HGkfelt and Jonsson, 1968). Permanganate fixative has been used to demonstrate the presence of monoamines in axons investing the salivary gland of the moth, Manduca sexta (Robertson, 1974). S-Hydroxydopamine is believed to be taken up by catecholaminergic boutons, displacing the natural transmitter, and resulting in the appearance of granular vesicles within them (Tranzer and Thoenen, 1967). It has been suggested that high concentrations of 5-hydroxydopamine will also enter 5-hydroxytryptamine endings (Ajika and Hiikfelt, 1973) indicating that this method is specific for monoamines rather than catecholamines. There is a considerable body of evidence to support the idea that 6-hydroxydopamine selectively destroys catecholamine containing 1968; neurones (Tranzer and Thoenen, Thoenen and Tranzer, 1968; Malmfors and Thoenen, 1971). However like permanganate
studies (BowserRiley and House, 1976) and secretion studies (Smith, 1977; Smith and House, 1977) have indicated that the neurotransmitter at the cockroach salivary gland is probably dopamine (for reviews see House, 1977; House and Ginsborg, 1979). Fluorescence histochemistry of the axons on the surface of the gland has shown that they contain a catecholamine (Bland et al., 1973). However, a previous electron microscopa1 study has revealed that two types of axon are associated with the salivary apparatus of the cockroach (Maxwell, 1978). These two types of axon were designated type A and type B according to their vesicular contents and are illustrated in Figs. 1 and 2. The main characteristics of the two axons are shown in Fig. 3. Type A axons contain large granular vesicles and small agranular vesicles whereas type B axons contain only large granular vesicles which are considerably larger than those of type A. Three methods were used in an attempt to ELECTROPHYSIOLOGICAL
University of Edinburgh, Department of Veterinary Physiology. Royal (Dick) School of Veterinary Studies. Summerhall. Edinburgh EH9 IQH. Scotland. Received I3 March 1980. Revised 24 July 1980. 703
MAXWELL
Abbreviations A : BM Gl GV LGV P
for figurer
type A axon type B axon central cell of salivary gland basement membrane glial cell granular vesicle of type PI axon large granular vesicle of type B axon peripheral cell of salivary gland
Fig. I. A type A axon. Note the small elliptical granular vesicles. x 40,000. Fig, 2. A type B awn
that is crammed
agranular
with large granular
vesicles and the larger vesicles.
x 60,000.
HISTOCHEMISTRY
OF
COCKROACH
TYPE
0
0
705
AXONS
A
TYPE
B
40+15t32nm 91t21.47 nm
0
134f2389nm
Fig. 3. A diagram representing the salient features of type A and B axons v+ith their vesicular dimensions. The dark objects represent electron-dense granules; elliptical objects represent the agranular ve;iclex of type A axons.
and 5-hydroxydopamine it seems probable that 5-hydroxytryptamine systems are also affected by 6-hydroxydopamine as Berry et al. (1974) found that it abolished paraformaldehyde induced 5-hydroxytryptamine Auorescence in the central nervous system of the water snail Planorbis corneus. Materials and Methods The entire salivary apparatuses of adult cockroaches, Nauphoeta cinerea (Olivier), were removed under saline (Smith and House, 1977). Potassium permanganate fixation was performed by placing the tissue in an ice cold solution of 3% KMn04 in 0.1 M phosphate buffer at pH 7.0 for 3045 min (Richardson, 1966). The tissue was then washed in phosphate buffer, dehydrated in graded alcohol solutions, cleared in propylene oxide and embedded in Araldite. Thin sections were stained with uranyl acetate and lead citrate (Reynolds, 1963). Treatment with 5-hydroxydopamine or 6-hydroxydopamine was performed by incubating fresh tissue in 0.1 mg/ml of 5hydroxydopamine (Sigma) in cockroach saline for 1 hr or in 1 mg/ml 6-hydroxydopamine (Sigma) in saline for 4 hr. Control tissues were incubated in cockroach saline alone for equal periods of time. Incubated glands were then fixed i hr in 4% glutaraldehyde in 0.05 M sodium cacodylate buffer
with 2.8% sucrose added. This solution had a tonicity of 370 mOsm, a pH of 7.2 and was used at 4°C. The tissue was then processed conventionally for electron microscopy (Maxwell, 1978) except that some sections of tissue that was incubated in 5-hydroxydopamine were not stained with uranyl acetate and lead citrate. This procedure facilitated discovery of electron-dense deposits of 5-hydroxydopamine. Data from micrographs were processed and stored in the files of a PDP-12 computer. Results Potassium permanganate fixation Figs. 4 and 5 illustrate the typical product of potassium permanganate fixation of the tissue of the cockroach salivary gland. In keeping with the observations of other authors (e.g. Hokfelt, 1968) mitochondria were found to be swollen and the general state of preservation of the tissue was not as good as that obtained with osmium and gldtaraldehyde fixation. Type A axons exhibited a positive reaction to permanganate fixation; i.e. they were noted to contain small granular vesicles of a circular profile. These vesicles have a mean diameter of 46~ 9 nm. The large granular vesicles of type A axons had a more opaque appearance (Fig. 4) than those after glutaraldehyde and osmium fixation (Fig. I). Type B axons did not react strongly with
706
MAXWELL
permanganate fixative. Their large opaque vesicles were found to have a mean diameter of 139 f 21 nm which is not statistically different from populations of large granular vesicles in type B axons observed after glutaraldehyde and osmium fixation (Fig. 2). These results indicate that the small vesicles of type A axons contain a biogenic monamine. Incubation in S-hydroxydopamine After incubation for 1 hr in 5hydroxydopamine type A axons were seen to contain some small flattened vesicles that contained dense material (Figs. 6, 7). These vesicles had a mean dimension of 40 nm. Electron-dense deposits of 5-hydroxydopamine could readily be seen in sections of type A axons that had not been stained with lead and uranium salts (Fig. 8). In addition to the small vesicles the large granular vesicles were also observed and did not seem to have been altered by the 5-hydroxydopamine. Type B axons did not exhibit any changes after incubation in 5hydroxydopamine (Fig. 8) and tissue that was incubated cockroach
saline did not display any dense material within the small vesicles of type A axons. Incubation it? 6-hydroxydopamine After 4 hr of incubation in 6-hydroxydopamine virtually every type A axon observed showed some signs of degeneration (Figs. 9, 10, 1I). Mitochondria within nerves were often seen to be damaged and vesicles appeared to aggregate. According to Smith et al. (1966) this aggregation is a sign of axonal degeneration. On the other hand type B axons had a completely normal appearance (Fig. 12). Tissue that was incubated in cockroach saline for 4 hr showed no signs of axonal degeneration. Vesicle shapes During the course of this study it was noted that the small vesicles of type A axons displayed different shaped profiles, depending upon the type of fixative employed. A previous study has shown that, after glutaraldehyde and osmium fixation, the small agranular vesicles of type A axons come from
Fig. 4. A type A axon that was fixed in potassium small granular vesicles (arrow) and the more opaque Fig. 5. Tissue that was fixed in potassium shown. x 30,000.
permanganate fixative. Note the larger vesicles. x 45,000.
permanganate.
Fig. 6. The effect of incubation in 5-hydroxydopamine. vesicles of type A axons are now granular in appearance. Fig. 7. A type A axon after incubation
Type A and B axons are Note that many of the small x 30,000.
in Shydroxydopamine.
x 60,000.
Fig. 8. An unstained section of tissue that was incubated in Shydroxydopamine. Electron-dense deposits of 5-hydroxydopamine may be seen within the small vesicles of a type A axon, whereas a type B axon has not been affected by the incubation. x 60,000. Fig. 9. A type A axon after incubation in 6-hydroxydopamine. Note the aggregation of the vesicles (arrows) and a damaged mitochondrion (asterisk). x 40,000. Fig. IO. Incubation in 6-hydroxydopamine; in a state of degeneration (arrows). x 22,500.
two type A axons are shown which are
Fig. 1 I. Two type A axons (arrows) in a state of degeneration h-hydroxydopamine. The axons are situated between the central gland. x 28,000.
after incubation in cells of the salivary
Fig. 12. Incubation in 6-hydroxydopamine: type A axons are seen to be in a state of degeneration (arrows) whereas type B axons have a normal appearance. x 21,000.
i
08
HISTOCHEMISTRY
OF
COCKROACH
709
AXONS
a population of prolate rotary ellipsoids (Maxwell, 1978). In this study the shapes of vesicles were compared by plotting the logarithm of the maximum diameter divided by the minimum diameter (measured at right
a *or
I :
angles to each other) of each vesicle as a histogram (Fig. f3). This method shows that vesicles from a glutaraldehyde and osmium fixed population (Fig. 13a) and those fixed by the same method after incubation in 5-hydroxydopamine (Fig. 13~) have a similar distribution, whereas those fixed in potassium permanganate (Fig. 13b) have a narrower distribution clustering around zero. This indicates that permanganate fixed tissue has predominantly vesicles of a circular profile (i.e. probably spheres in three dimensions), whereas those fixed by glutaraldehyde and osmium tend to be elliptical. Discussion
b
Mf. 1. t. *(I t*
*
tt
t
*.
*
,o_::;;.
. . . . .,
. .. ..
! ***’ Lt..
**.*
*
If.”
.
.
I”‘:“‘. ~;
0’
z
1
c
10r
I.....;
. . .._........_.._........_....
Log
Ratio max/min
Fig. 13. Histograms of the logarithm of the maximum diameter divided by the minimum diameter of the small agranular vesicles observed in type A axons. (a) is a plot of vesicle dimensions taken from several axons that were fixed in glutaraldehyde and osmium fixative. (b) represents similar data collected after permanganate fixation. (c) is data collected from axons that were incubated in Shydroxydopamine prior to glutaraldehyde and osmium fixation. The wider distribution in graphs (a) and (c) suggests that these vesicles tend to be elliptical whereas the narrower distribution, around zero, in graph (b) is indicative of spherical vesicles.
Bland et al. (1973) have shown by microspectrofluorimetry that the fluorescence exhibited by axons on the surface of the gland after treatment by the method of Falck et al. (1962) has a spectrum consistent with the presence of catecholamines. There was no evidence to suggest the presence of S-hydroxytryptamine. Paraformaldehyde-induced fluorescence has been abolished in the nerves of the gland after incubation in 6-hydroxydopamine (Maxwell and Laszlo, unpublished observations). Pharmacological experiments, using the antagonist phentolamine, also suggest that 5-hydroxytryptamine is not the natural transmitter at the neuroglandular junction: both secretory (House and Smith, 1978) and electrophysiological (BowserRiley et al., 1978) experiments show that phentolamine can distinguish between receptors for 5-hydroxytryptamine and those for dopamine, noradrenaline and the natural transmitter. Fry et al. (1974) have shown by a radiochemical assay that homogenized salivary glands of this cockroach contain dopamine but not noradrenaline. Evidence, obtained via the three methods reported in this study conclusively demonstrates the presence of monoamines within the small vesicles of type A axons. If this evidence is considered in conjunction with these previous findings, then it is reasonable to suggest that type A axons are dopaminergic. The function and contents of type B axons are not known. It was previously suggested that they may be neurosecretory cells (Maxwell, 1978). The significance of different vesicular shapes in type A axons after potassium per-
710
MAXWELL
manganate fixation or fixation with glutaraldehyde and osmium is also not understood. It has been suggested that the osmolality of the fixative buffer may be important (Valdivia, 1970); buffers of an osmolality greater than 1715 mOsm were found to produce flattened vesicles in certain cells in the mammalian cerebellum. This suggestion is not applicable to the present study as the total tonicity of the fixative did not exceed 370 mOsm. It has also been claimed that aldehyde fixatives produce flattened vesicles. Thus Walberg (1966) found 40 % more flattened vesicles after fixation of the terminal boutons of the
mammalian central nervous system with glutaraldehyde and osmium than with osmium fixation alone. In view of these findings it is unwise to classify vesicles on the basis of their shapes. Acknowledgements I wish to thank Drs K. P. Bland and C. R. House for their useful criticism of this manuscript and also Mr C. M. Warwick for assistance in the production of the photographic plates. Financial support was provided by the Science Research Council.
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