Regulatory Peptides, 37 (1992) 101-109 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0167-0115/92/$05.00
101
REGPEP 01128
Galanin is more c o m m o n than N P Y in vascular sympathetic neurons of the brush-tailed possum J.L. Morris, I.L. G i b b i n s a n d S. H o l m g r e n * Department of Anatomy and Histology, and Centrefor Neuroscience, School of Medicine, Flinders University, Bedford Park (Australia)
(Received 13 March 1991; revised version received 12 July 1991; accepted 6 October 1991) Key words: Vasoconstrictor neuron; Perivascular axon; Immunohistochemistry
Summary The distribution of galanin (Gal) in sympathetic vascular neurons of adult and juvenile brush-tailed possums (Trichosurus vulpecula), was examined using doublelabelling immunohistochemistry. This was compared with the distribution of neuropeptide Y (NPY) in the same tissues. Immunoreactivity (IR) to galanin was present in the majority (64-99~o) of nerve cell bodies in paravertebral sympathetic ganglia, where it mostly co-existed with IR to the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH). Gal-IR also was present in most, if not all, T H - I R perivascular axons supplying systemic arteries and veins. N P Y - I R was less common than Gal-IR in all sympathetic ganglia and perivascular axons examined. Some sympathetic, T H - I R axons supplying the abdominal aorta and renal artery contained both GaI-IR and NPY-IR, while T H - I R axons supplying cephalic and thoracic vessels contained Gal-IR but not NPY-IR. Limited observations on sympathetic neurons in two species of wallabies indicated that Gal-IR also was more common than N P Y - I R in other marsupial species, but the incidence of N P Y - I R was higher in these wallabies than in the brush-tailed possum. Together with previous studies, this work suggests that the coexistence of galanin and NPY may be the primitive condition for sympathetic neurons in tetrapods. The differential expression of these peptides in specific populations of sympathetic neurons may have important functional consequences in the autonomic control of the circulation.
* Permanent Address: Department of Zoophysiology0 Zoological Institute, University of GOteborg, $40031 Gtiteborg, Sweden. Correspondence: J.L. Morris, Department of Anatomy and Histology, Flinders Medical Centre, Bedford Park, S.A. 5042, Australia.
102 Introduction
In placental mammals, neuropeptide Y (NPY) often is co-localized with noradrenaline in perivascular sympathetic axons [1-5]. However, there are regional and species differences in the degree of co-existence of catecholamine and NPY in perivascular neurons. For example, sympathetic neurons containing noradrenaline, but lacking NPY, supply venous sinusoids in cat nasal mucosa [2], and small arterioles and arterio-venous anastomoses in non-hairy skin of guinea-pigs [6]. Furthermore, we have reported previously that the majority of sympathetic vascular neurons in an Australian marsupial, the brush-tailed possum (Trichosurus vulpecula), lack NPY [7]. Amongst non-mammalian species, NPY occurs in nearly all sympathetic vascular neurons in the toad Bufo marinus [8] and bullfrog [9], but is absent from sympathetic vascular neurons in some species of Australia lizards [10]. More recently, galanin (Gal) has been localized in sympathetic neurons of some species [10-15], as well as in parasympathetic neurons [13,16], sensory neurons [ 14,17-20 ], enteric neuron s [ 21-24 ] and in the central nervous system [ 24-26 ]. In cats and toads, there is widespread co-existence of Gal and NPY in sympathetic neurons, including those supplying the vasculature [11-13]. Galanin has been shown to be released from sympathetic neurons supplying the dog pancreas, and to inhibit insulin secretion [27]. Although Gal is not found in sympathetic vascular neurons in guineapigs and rats [11], Gal-IR has been located in sensory neurons supplying cerebral arteries in rats [ 19], and in enteric neurons supplying submucosal blood vessels in guinea-pigs [22]. Studies by our colleagues have revealed GaI-IR in prevertebral sympathetic ganglia of the brush-tailed possum (J.B. Furness and R. Padbury, personal communication). Therefore, in the present study we have examined the distribution of Gal in paravertebral sympathetic ganglia and in perivascular axons of the brush-tailed possum, to determine whether Gal occurs in those sympathetic neurons which lack NPY. We have examined both adult and juvenile possums in detail, and have made incidental observations on the distributions of Gal and NPY in vascular neurons in two rarer species of marsupials, the tammar wallaby and the spectacled hare-wallaby.
Materials and Methods
Sympathetic ganglia and systemic arteries were removed from 14 animals after injection of a lethal dose of sodium pentobarbitone. The animals studied were: four adult (1.5-2.5 kg body weight) and five juvenile (20-50 g body weight) brush-tailed possums (Trichosurus vulpecula); four juvenile tammar wallabies (Macropus eugenii, 5-110 g body weight); one adult spectacled hare-wallaby (Lagorchestes conspicillatus, 3 kg body weight). Superior cervical ganglia, stellate ganglia, thoracic sympathetic ganglia, lumbar sympathetic ganglia, common carotid arteries, jugular veins, thoracic aorta and caval veins, coronary arteries, abdominal aorta and vena cava, and renal arteries and veins, were removed from the adult possums. Ganglia and a smaller range of blood vessels were removed from juvenile possums and wallabies, but only thoracic sympathetic ganglia were available from the spectacled hare-wallaby. The tissues were fixed by
103 immersion in a solution of picric acid (15%) and formaldehyde (2 ~o) in phosphatebuffered saline, for 16-24 h at 4 °C. Ganglia and arteries were then processed for cryostat sectioning and double-labelling immunohistochemistry as described previously [13]. The primary antisera used were: anti-NPY RMJ 263 raised in a rabbit, supplied by Drs. C. Maccarrone and B. Jarrott; anti-Gal 8504-8 raised in a rabbit, obtained from Peninsula Laboratories; anti-tyrosine hydroxylase (TH) 10968620-01, a mouse monoclonal antibody, obtained from Boehringer-Mannheim. Secondary antibodies were sheep anti-rabbit IgG coupled to fluorescein isothiocyanate (FITC; Wellcome), together with biotinylated horse anti-mouse IgG (Vector) visualized with streptavidin-conjugated Texas Red (Amersham). Double-labelled sections were mounted in carbonate-buffered glycerol (pH 8.6) and were examined with a Leitz epifluorescence microscope fitted with dichroic filter blocks L3 for FITC fluorescence and N2 for Texas Red fluorescence. Absorption tests for Gal-IR were performed by incubating anti-Gal 8504-8 with synthetic porcine Gal (Peninsula, 10-6-10 -5 M) for 24 h before application of the antiserum to sections of thoracic sympathetic ganglia from adult possums. All Gal-IR was abolished after pre-absorption with 10-6 M Gal. Absorption tests for NPY-IR obtained with anti-NPY RMJ 263 in renal arteries from adult brush-tailed possums has been performed and reported previously [7]. Proportions of sympathetic neurons double-labelled for TH-IR and Gal-IR, or for TH-IR and NPY-IR, were determined quantitatively in ganglia from one animal from each species and age group examined. Between 500-1500 neurons in three to ten non-serial sections from each ganglion were scored for the presence of IR to one, both or neither antigen in the two double-labelling combinations. In all cases, these quantitative results supported the qualitative assessments of ganglia from additional animals.
Results Adult possums
Previously, we reported that no neurons with TH-like immunoreactivity (TH-IR) in thoracic sympathetic ganglia of adult possums contained NPY-IR, whilst 2-3 ~o of TH-IR neurons in lumbar sympathetic ganglia contained NPY-IR [7]. In the present study we confirmed these results qualitatively, and also found that NPY-IR was absent from nerve cell bodies in the superior cervical ganglion (Table I). This differential occurrence of NPY in nerve cell bodies in sympathetic ganglia from different levels matched the differential distribution of NPY-IR in perivascular TH-IR nerve terminals. There was a complete absence of NPY-IR from sympathetic axons supplying the cephalic and thoracic vessels, but NPY-IR occurred in a subpopulation of TH-IR axons supplying the abdominal aorta and the renal artery (Table I; Ref. 7). In contrast to this scarcity of NPY-IR in sympathetic neurons, in the present study we have detected Gal-IR in 64-100 ~o of TH-IR sympathetic nerve cell bodies of adult possums (Table I). Nearly all (98~'o) of the Gal-IR neurons showed TH-IR (Fig. 3). Gal-IR also was prominent in TH-IR axons in the adventitia of systemic blood vessels (Table I). The common carotid artery and renal artery both possessed a particularly
104 TABLE 1 Summary of relative frequencies of TH-IR sympathetic nerve cell bodies and perivascular axons which also have IR to Gal (Gal/TH), or to NPY (NPY/TH) Adult possums
Juvenile possums
Gal/TH
NPY/TH
Gal/TH
NPY/TH
Sympathetic ganglia Superior cervical Thoracic Lumbar
+++ ++++ +++
0 0 +
++++ ++++ ++++
+ + +
Perivascular axons Common carotid artery Internal jugular vein Thoracic aorta Superior caval veins Coronary arteries Abdominal aorta Abdominal caval vein Renal artery Renal vein
+ + + + + + + + +
0 0 0 0 0 + 0 ++ 0
++ + ++ -
0 0 +
++ + +
++
-
Ganglia: 0, no cells with peptide-IR detected; +, 1-20~o of TH-IR cells; + +, 21-50~o of TH-IR cells; + + +, 51-80~o of TH-IR cells; + + + +, >80~o of TH-IR cells. Perivascular axons: 0, no IR axons detected; + to + + +, estimates of relative density of IR axons, from sparse to very dense; - , not examined.
d e n s e p l e x u s o f p e r i v a s c u l a r a x o n s w i t h G a l - I R a n d T H - I R . P r o b a b l y all T H - I R a x o n s c o n t a i n e d G a l - I R . T h e r e f o r e , at l e a s t s o m e o f t h e f i b r e s a r o u n d t h e r e n a l a r t e r y m u s t have contained TH-IR, Gal-IR, and NPY-IR.
Juvenile possums NPY-IR pouch. NPY-IR
w a s also s c a r c e in s y m p a t h e t i c n e u r o n s o f i m m a t u r e p o s s u m s still in t h e
Only
1-6%
o f s y m p a t h e t i c n e r v e cell b o d i e s c o n t a i n e d b o t h T H - I R
(Fig. 1; T a b l e I), a n d less t h a n 1 ~
of neurons contained NPY-IR
and
without
TH-IR. However, 86-90~o of sympathetic neurons contained both TH-IR and Gal-IR (Fig. 2; T a b l e I). A s in a d u l t p o s s u m s , t h e c o m m o n c a r o t i d a r t e r y o f j u v e n i l e s l a c k e d NPY-IR
axons, but had a moderately-dense plexus of axons with Gal-IR and TH-IR
(Fig. 4). I n t h e r e n a l a r t e r y , b o t h G a I - I R a n d N P Y - I R n o t all, o f t h e T H - I R
a x o n s w e r e c o m m o n , M o s t , if
a x o n s s u p p l y i n g t h e r e n a l a r t e r y c o n t a i n e d G a l - I R (Fig. 5), a n d
a subpopulation of these axons also must have contained NPY-IR.
Other species NPY-IR
n e u r o n s w e r e r a r e (1 ~o t o t a l n e u r o n s ) in t h e s u p e r i o r c e r v i c a l g a n g l i o n o f
p o u c h - y o u n g t a m m a r w a l l a b i e s , b u t i n c r e a s e d in f r e q u e n c y in t h e m o r e c a u d a l s y m p a t h e t i c g a n g l i a . 10~o o f n e u r o n s in t h e stellate g a n g l i a c o n t a i n e d b o t h N P Y - I R
and
105
gp
Figs. 1-3. Sections of sympathetic ganglia double-labelled for TH-IR and NPY-IR, or for TH-IR and GaI-IR. Figs. 1 and 2. Superior cervical ganglion from juvenile brush-tailed possum showing a rare clump of nerve cell bodies with NPY-IR and TH-IR (Fig. 1). The NPY-IR is very faint, and is concentrated in the perinuclear region of the cells. Nearly all nerve cell bodies in the same ganglion have both TH-IR and Gal-IR (Fig. 2). Arrows in each pair of micrographs (a,b) show the same double-labelled neurons. Scale bar = 50 #m. Fig. 3. Lumbar sympathetic ganglion from adult possum showing that nearly all TH-IR nerve cell bodies show GaI-IR (white arrows). Open arrows in each pair of micrographs (a,b) show rare nerve cell bodies which contain IR to TH or to Gal, but which lack IR to the other antigen. Note larger size of ganglion cells in adults compared with juveniles (c.f. Figs. 1, 2). Scale bar = 50 #m.
T H - I R , a n d as m a n y as 22 ~o o f n e u r o n s in l u m b a r s y m p a t h e t i c ganglia c o n t a i n e d b o t h N P Y - I R a n d T H - I R . Again, G a I - I R w a s p r e s e n t in a m u c h larger p r o p o r t i o n o f s y m p a t h e t i c n e u r o n s ( 6 8 - 8 5 ~o) t h a n w a s N P Y - I R . All o f the G a I - I R n e u r o n s c o n t a i n e d T H - I R . T h e d e n s e plexus o f T H - I R a x o n s s u r r o u n d i n g the c o m m o n c a r o t i d artery
106
Figs. 4 and 5. GaI-IR and TH-IR in double-labelled sections of the carotid artery (Fig. 4) and renal artery (Fig. 5) from juvenile possums. Most perivascular nerve fibres at adventitio-medial junction contain IR to both TH and Gal (white arrows). Note nerve bundles (b) with intense TH-IR and Gal-IR in the adventitia of the renal artery. Clumps of nerve cell bodies with TH-IR and Gal-IR were sometimes seen associated with these nerve bundles. Scale bars = 25 #m.
c o n t a i n e d strong G a l - I R , but only a few axons contained N P Y - I R . In contrast, the majority o f T H - I R axons supplying the renal artery c o n t a i n e d both strong G a l - I R and weak NPY-IR. Only thoracic sympathetic ganglia from one adult spectacled hare-wallaby were available for i m m u n o h i s t o c h e m i c a l examination. These ganglia contained a much higher p r o p o r t i o n of N P Y - I R neurons (82~o of 692 cells counted) than we have detected in any sympathetic ganglia from the other two marsupial species examined. Nearly all (98~o) o f the N P Y - I R neurons contained T H - I R . Furthermore, 95°~ of all neurons c o n t a i n e d both G a l - I R and T H - I R . Therefore, at least 75~o o f these sympathetic neurons must have contained T H - I R , G a l - I R and N P Y - I R .
Discussion These results d e m o n s t r a t e that galanin is w i d e s p r e a d in postganglionic sympathetic noradrenergic neurons of the brush-tailed possum. G a l a n i n occurs in m a n y m o r e sympathetic ganglion cells than does N P Y , and it is present in p r o b a b l y all sympathetic nerve terminals associated with systemic arteries o f the possums. In particular, galanin
107 is present in those vascular sympathetic neurons which lack NPY [7]. This observation further demonstrates that NPY is not a universal marker for noradrenergic sympathetic neurons innervating blood vessels. Similarly, although galanin is found in sympathetic perivascular neurons in species as diverse as toads [13], possums and cats [11], it is not uniquely associated with this class of neurons. For example, it is not present in sympathetic neurons of guinea-pigs and rats [ 11 ], but it is found in several different classes of parasympathetic neurons in toads [ 13], as well as in enteric and sensory neurons of several species [14,17-24]. The lack of NPY-immunoreactivity in most sympathetic neurons of the brush-tailed possum is not likely to be due to the inability of our antibodies to detect this peptide in marsupials (see Ref. 7). First, NPY co-existed with galanin in sympathetic neurons innervating the renal arteries. Second, NPY-IR, although still rare, was more prominent in sympathetic ganglion cells from immature animals than from adults. These observations strongly suggest that the primitive condition for these neurons is to contain both NPY and galanin, and that synthesis of NPY is down-regulated during development in all but a specialised population of neurons. This conclusion is supported by the widespread co-existence of NPY-IR with Gal-IR in the one specimen of the spectacled hare-wallaby we were able to examine. Furthermore, the co-existence of galanin and NPY in homologous neurons in other distantly related species such as toads and cats is consistent with the hypothesis that this pattern of peptide expression is the primitive one for sympathetic vasomotor neurons in tetrapods. More comparative studies will be required specifically to test this idea. There is now good evidence that NPY can act as a neurotransmitter from sympathetic vasoconstrictor neurons in certain mammalian blood vessels [2,28-31 ]. However, there is less information available regarding the roles of galanin in vascular neurons. Exogenous galanin is a potent pressor agent in Bufo marinus [32], where it is localized in most sympathetic vascular neurons [ 13]. On the other hand, galanin is contained in vasodilator nerve fibres supplying the submucosal arterioles in guinea-pigs [22], and exogenous galanin dilates these vessels [33]. It is necessary to obtain information on the vascular effects of both galanin and NPY in a wide range of blood vessels in many other species, before we can understand the functional significance of the differential distributions of these peptides in sympathetic neurons. Nevertheless, it seems possible that the patterns of expression ofgalanin and NPY in sympathetic vascular neurons may be related to the specific requirements of autonomic regulation of well-defined regions of the peripheral circulation.
Acknowledgements This work was supported by grants from the National Health and Medical Research Council of Australia, and the Swedish Natural Science Research Council. We are grateful to Drs. R.V. Baudinette, R. Padbury, G. Sacconne and R. Baker for supplying wallaby and possum tissue for this study, and to Drs. C. Maccarrone and B. Jarrott for supplying the NPY antibody. Fiona Renton, Pat Vilimas and Sue Matthew provided excellent technical assistance.
108 References 1 Ekblad, E., Edvinsson, L., Wahlestedt, C., Uddman, R., H~tkanson, R. and Sundler, F., Neuropeptide Y co-exists and co-operates with noradrenaline in perivascular nerve fibres, Regul. Pept., 8 (1984) 225-235. 2 Lundberg, J.M. and H6kfelt, T., Multiple co-existence of peptides and classical transmitters in peripheral autonomic and sensory neurons - functional and pharmacological implications. In T. HOkfelt, K. Fuxe and B. Pernow (Eds.), Progress in Brain Research. Vol. 68, Elsevier, Amsterdam, 1986, pp. 241-262. 3 Morris, J. L., The cardiovascular system. In S. Holmgren (Ed.), The Comparative Physiology of Regulatory Peptides, Chapman & Hall, London, 1989, pp. 272-307. 4 Potter, E. K., Neuropeptide Y as an autonomic neurotransmitter, Pharmacol. Ther., 37 (1988) 251-274. 5 Gibbins, I.L., Morris, J.L., Furness, J. B. and Costa, M., Innervation of systemic blood vessels. In G. Burnstock and S. Griffith (Eds.), Non-Adrenergic Innervation of Blood Vessels, CRC Press, Boca Raton, 1988, pp. 1-36. 6 Gibbins, I.L., and Morris, J.L., Sympathetic noradrenergic neurons containing dynorphin but not neuropeptide Y innervate small cutaneous blood vessels of guinea-pigs, J. Autonom. Nerv. Syst., 29 (1990) 137-150. 7 Morris, J. L., Gibbins I.L. and Murphy, R., Neuropeptide Y-like immunoreactivity is absent from most perivascular noradrenergic axons in a marsupial, the brush-tailed possum, Neurosci. Lett., 71 (1986) 264-270. 8 Morris, J. L., Gibbins, I. L., Campbell, G., Murphy, R., Furness, J.B. and Costa, M., Innervation of the large arteries and the heart of the toad (Bufo marinus) by adrenergic and peptide-containing neurons, Cell Tissue Res., 243 (1986) 171-184. 9 Horn, J.P., Stofer, W.D. and Fatherazi, S., Neuropeptide Y-like immunoreactivity in bullfrog sympathetic ganglia is restricted to C-cells, J. Neurosci., 7 (1987) 1717-1727. 10 Gibbins, I.L., Morris, J.L. and Holmgren, S., Distribution of neuropeptide Y and galanin in cardiovascular sympathetic neurons of Australian lizards, Proc. Int. Union Physiol. Sci., 17 (1989) 62. 11 Kummer, W., Galanin- and neuropeptide Y-like immunoreactivities coexist in paravertebral sympathetic neurons of the cat, Neurosci. Lett., 78 (1987) 127-131. 12 Lindh, B., Lundberg, J. M. and H6kfelt, T., NPY-, galanin-, VIP/PHI, CGRP- and substance P-immunoreactive neuronal subpopulations in cat autonomic and sensory ganglia and their projections, Cell Tissue Res., 256 (1989) 256-273. 13 Morris, J.L., Gibbins, I.L. and Osborne, P.B., Galanin-like immunoreactivity in sympathetic and parasympathetic neurons of the toad Bufo marinus, Neurosci. Lett., 102 (1989) 142-148. 14 Str6mberg, I., Bjtirklund, H., Melander, T., R6kaeus, A.., H6kfelt, T. and Olson, L., Galaninimmunoreactive nerves in the rat iris: alterations induced by denervations, Cell Tissue Res., 250 (1987) 267-275. 15 Ahr6n, B., B6ttcher, G., Kowalyk, S., Dunning, B.E., Sundler, F. and Taborsky, G.J., Galanin is co-localized with noradrenaline and neuropeptide Y in dog pancreas and celiac ganglion, Cell Tissue Res., 261 (1990) 49-58. 16 Luts, A., Uddman, R. and Sundler, F., Neuronal galanin is widely distributed in the chicken respiratory tract and co-exists with multiple neuropeptides, Cell Tissue Res., 256 (1989) 95-103. 17 Skofitsch, G. and Jacobowitz, D.M., Galanin-like immunoreactivity in capsaicin sensitive sensory neurons and ganglia, Brain Res. Bull., 15 (1985) 191-195. 18 Grunditz, T., H~ikanson, R., Sundler, F. and Uddman, R., Neuronal pathways to the rat thyroid revealed by retrograde tracing and immunocytochemistry, Neuroscience, 24 (1988) 321-335. 19 Suzuki, N., Hardebo, J.E., K~thrstr6m, J. and Owman, C., Galanin-positive nerves of trigeminal origin innervate rat cerebral vessels, Neurosci. Lett., 100 (1989) 123-129. 20 Philippe, C., Cuber, J.C., Bosshard, A., Rampin, O., Laplace, J.P. and Chayvialle, J.A., Galanin in porcine vagal sensory nerves: immunohistochemical and immunochemical study, Peptides, 11 (1990) 989-993. 21 Ekblad, E., R6kaeus, ,~., H~kanson, R. and Sundler, F., Galanin nerve fibres in the rat gut: distribution origin and projections, Neuroscience, 16 (1985) 355-363.
109 22 Furness, J.B., Costa, M., R6kaeus, A., McDonald, T.J. and Brooks, B., Galanin-immunoreactive neurons in the guinea-pig small intestine: their projections and relationships to other enteric neurons, Cell Tissue Res., 250 (1987) 607-615. 23 Melander, T., H6kfelt, T., R6kaeus, A., Fahrenkrug, J., Tatemoto, K. and Mutt, V., Distribution of galanin-like immunoreactivity in the gastro-intestinal tract of several mammalian species, Cell Tissue Res., 239 (1985) 253-270. 24 R6kaeus, A., Melander, T., HOkfelt, T., Lundberg, J.M., Tatemoto, K., Carlquist, M. and Mutt, V., A galanin-like peptide in the central nervous system and intestine of the rat, Neurosci. Lett., 47 (1984) 161-166. 25 Skofitsch, G. and Jacobowitz, D.M., Immunohistochemical mapping of galanin-like neurons in the rat central nervous system, Peptides, 6 (1985) 509-546. 26 Ch'ng, J.L.C., Christofides, N.D., Anand, P., Gibson, S.J., Allen, Y.S., Su, H.C., Tatemoto, K., Morrison, J. B. F., Polak, J. M. and Bloom, S. R., Distribution of galanin immunoreactivity in the central nervous system and the responses of galanin-containing neuronal pathways to injury, Neuroscience, 16 (1985) 343-354. 27 Dunning, B.E. and Taborsky, G.J., Galanin release during pancreatic nerve stimulation is sufficient to influence islet function, Am. J. Physiol., 256 (1989) E191-E198. 28 Lundberg, J. M., Rudehill, A. Sollevi, A., Theordorsson-Norheim, E. and Hamberger, B., Frequency and reserpine-dependent chemical coding of sympathetic transmission: differential release of noradrenaline and neuropeptide Y from pig spleen, Neurosci. Lett., 93 (1986) 96-100. 29 Morris, J.L., Role of neuropeptide Y and noradrenaline in sympathetic neurotransmission to the thoracic vena cava and aorta of guinea-pigs, Regul. Pept., 32 (1991) 297-310. 30 Morris, J. L., and Murphy, R., Evidence that neuropeptide Y released from noradrenergic axons causes prolonged contraction of the guinea-pig uterine artery, J. Autonom. Nerv. Syst., 24 (1988) 241-249. 31 ()hl6n, A., Persson, M.G., Lindbom, L., Gustafsson, L.E. and Hedqvist, P., Nerve-induced nonadrenergic vasoconstriction and vasodilation in skeletal muscle, Am. J. Physiol., 258 (1990) H1334-H1338. 32 Courtice, G. P., Effect ofneuropeptide Y and galanin on autonomic control of heart rate in the toad, Bufo marinus, J. Autonom. Nerv. Syst., 33 (1991) 231-238. 33 Kotecha, N. and Neild, T. O., Effects ofvasodilator peptides on arterioles of the small intestine, In M. A. Perry and D.G. Garlick (Eds.), Progress in Microcirculation Research: Proc. 6th Australian and New Zealand Symposium, Univ. N.S.W., 1991, pp. 78-80.
101
REGPEP 01128
Galanin is more c o m m o n than N P Y in vascular sympathetic neurons of the brush-tailed possum J.L. Morris, I.L. G i b b i n s a n d S. H o l m g r e n * Department of Anatomy and Histology, and Centrefor Neuroscience, School of Medicine, Flinders University, Bedford Park (Australia)
(Received 13 March 1991; revised version received 12 July 1991; accepted 6 October 1991) Key words: Vasoconstrictor neuron; Perivascular axon; Immunohistochemistry
Summary The distribution of galanin (Gal) in sympathetic vascular neurons of adult and juvenile brush-tailed possums (Trichosurus vulpecula), was examined using doublelabelling immunohistochemistry. This was compared with the distribution of neuropeptide Y (NPY) in the same tissues. Immunoreactivity (IR) to galanin was present in the majority (64-99~o) of nerve cell bodies in paravertebral sympathetic ganglia, where it mostly co-existed with IR to the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH). Gal-IR also was present in most, if not all, T H - I R perivascular axons supplying systemic arteries and veins. N P Y - I R was less common than Gal-IR in all sympathetic ganglia and perivascular axons examined. Some sympathetic, T H - I R axons supplying the abdominal aorta and renal artery contained both GaI-IR and NPY-IR, while T H - I R axons supplying cephalic and thoracic vessels contained Gal-IR but not NPY-IR. Limited observations on sympathetic neurons in two species of wallabies indicated that Gal-IR also was more common than N P Y - I R in other marsupial species, but the incidence of N P Y - I R was higher in these wallabies than in the brush-tailed possum. Together with previous studies, this work suggests that the coexistence of galanin and NPY may be the primitive condition for sympathetic neurons in tetrapods. The differential expression of these peptides in specific populations of sympathetic neurons may have important functional consequences in the autonomic control of the circulation.
* Permanent Address: Department of Zoophysiology0 Zoological Institute, University of GOteborg, $40031 Gtiteborg, Sweden. Correspondence: J.L. Morris, Department of Anatomy and Histology, Flinders Medical Centre, Bedford Park, S.A. 5042, Australia.
102 Introduction
In placental mammals, neuropeptide Y (NPY) often is co-localized with noradrenaline in perivascular sympathetic axons [1-5]. However, there are regional and species differences in the degree of co-existence of catecholamine and NPY in perivascular neurons. For example, sympathetic neurons containing noradrenaline, but lacking NPY, supply venous sinusoids in cat nasal mucosa [2], and small arterioles and arterio-venous anastomoses in non-hairy skin of guinea-pigs [6]. Furthermore, we have reported previously that the majority of sympathetic vascular neurons in an Australian marsupial, the brush-tailed possum (Trichosurus vulpecula), lack NPY [7]. Amongst non-mammalian species, NPY occurs in nearly all sympathetic vascular neurons in the toad Bufo marinus [8] and bullfrog [9], but is absent from sympathetic vascular neurons in some species of Australia lizards [10]. More recently, galanin (Gal) has been localized in sympathetic neurons of some species [10-15], as well as in parasympathetic neurons [13,16], sensory neurons [ 14,17-20 ], enteric neuron s [ 21-24 ] and in the central nervous system [ 24-26 ]. In cats and toads, there is widespread co-existence of Gal and NPY in sympathetic neurons, including those supplying the vasculature [11-13]. Galanin has been shown to be released from sympathetic neurons supplying the dog pancreas, and to inhibit insulin secretion [27]. Although Gal is not found in sympathetic vascular neurons in guineapigs and rats [11], Gal-IR has been located in sensory neurons supplying cerebral arteries in rats [ 19], and in enteric neurons supplying submucosal blood vessels in guinea-pigs [22]. Studies by our colleagues have revealed GaI-IR in prevertebral sympathetic ganglia of the brush-tailed possum (J.B. Furness and R. Padbury, personal communication). Therefore, in the present study we have examined the distribution of Gal in paravertebral sympathetic ganglia and in perivascular axons of the brush-tailed possum, to determine whether Gal occurs in those sympathetic neurons which lack NPY. We have examined both adult and juvenile possums in detail, and have made incidental observations on the distributions of Gal and NPY in vascular neurons in two rarer species of marsupials, the tammar wallaby and the spectacled hare-wallaby.
Materials and Methods
Sympathetic ganglia and systemic arteries were removed from 14 animals after injection of a lethal dose of sodium pentobarbitone. The animals studied were: four adult (1.5-2.5 kg body weight) and five juvenile (20-50 g body weight) brush-tailed possums (Trichosurus vulpecula); four juvenile tammar wallabies (Macropus eugenii, 5-110 g body weight); one adult spectacled hare-wallaby (Lagorchestes conspicillatus, 3 kg body weight). Superior cervical ganglia, stellate ganglia, thoracic sympathetic ganglia, lumbar sympathetic ganglia, common carotid arteries, jugular veins, thoracic aorta and caval veins, coronary arteries, abdominal aorta and vena cava, and renal arteries and veins, were removed from the adult possums. Ganglia and a smaller range of blood vessels were removed from juvenile possums and wallabies, but only thoracic sympathetic ganglia were available from the spectacled hare-wallaby. The tissues were fixed by
103 immersion in a solution of picric acid (15%) and formaldehyde (2 ~o) in phosphatebuffered saline, for 16-24 h at 4 °C. Ganglia and arteries were then processed for cryostat sectioning and double-labelling immunohistochemistry as described previously [13]. The primary antisera used were: anti-NPY RMJ 263 raised in a rabbit, supplied by Drs. C. Maccarrone and B. Jarrott; anti-Gal 8504-8 raised in a rabbit, obtained from Peninsula Laboratories; anti-tyrosine hydroxylase (TH) 10968620-01, a mouse monoclonal antibody, obtained from Boehringer-Mannheim. Secondary antibodies were sheep anti-rabbit IgG coupled to fluorescein isothiocyanate (FITC; Wellcome), together with biotinylated horse anti-mouse IgG (Vector) visualized with streptavidin-conjugated Texas Red (Amersham). Double-labelled sections were mounted in carbonate-buffered glycerol (pH 8.6) and were examined with a Leitz epifluorescence microscope fitted with dichroic filter blocks L3 for FITC fluorescence and N2 for Texas Red fluorescence. Absorption tests for Gal-IR were performed by incubating anti-Gal 8504-8 with synthetic porcine Gal (Peninsula, 10-6-10 -5 M) for 24 h before application of the antiserum to sections of thoracic sympathetic ganglia from adult possums. All Gal-IR was abolished after pre-absorption with 10-6 M Gal. Absorption tests for NPY-IR obtained with anti-NPY RMJ 263 in renal arteries from adult brush-tailed possums has been performed and reported previously [7]. Proportions of sympathetic neurons double-labelled for TH-IR and Gal-IR, or for TH-IR and NPY-IR, were determined quantitatively in ganglia from one animal from each species and age group examined. Between 500-1500 neurons in three to ten non-serial sections from each ganglion were scored for the presence of IR to one, both or neither antigen in the two double-labelling combinations. In all cases, these quantitative results supported the qualitative assessments of ganglia from additional animals.
Results Adult possums
Previously, we reported that no neurons with TH-like immunoreactivity (TH-IR) in thoracic sympathetic ganglia of adult possums contained NPY-IR, whilst 2-3 ~o of TH-IR neurons in lumbar sympathetic ganglia contained NPY-IR [7]. In the present study we confirmed these results qualitatively, and also found that NPY-IR was absent from nerve cell bodies in the superior cervical ganglion (Table I). This differential occurrence of NPY in nerve cell bodies in sympathetic ganglia from different levels matched the differential distribution of NPY-IR in perivascular TH-IR nerve terminals. There was a complete absence of NPY-IR from sympathetic axons supplying the cephalic and thoracic vessels, but NPY-IR occurred in a subpopulation of TH-IR axons supplying the abdominal aorta and the renal artery (Table I; Ref. 7). In contrast to this scarcity of NPY-IR in sympathetic neurons, in the present study we have detected Gal-IR in 64-100 ~o of TH-IR sympathetic nerve cell bodies of adult possums (Table I). Nearly all (98~'o) of the Gal-IR neurons showed TH-IR (Fig. 3). Gal-IR also was prominent in TH-IR axons in the adventitia of systemic blood vessels (Table I). The common carotid artery and renal artery both possessed a particularly
104 TABLE 1 Summary of relative frequencies of TH-IR sympathetic nerve cell bodies and perivascular axons which also have IR to Gal (Gal/TH), or to NPY (NPY/TH) Adult possums
Juvenile possums
Gal/TH
NPY/TH
Gal/TH
NPY/TH
Sympathetic ganglia Superior cervical Thoracic Lumbar
+++ ++++ +++
0 0 +
++++ ++++ ++++
+ + +
Perivascular axons Common carotid artery Internal jugular vein Thoracic aorta Superior caval veins Coronary arteries Abdominal aorta Abdominal caval vein Renal artery Renal vein
+ + + + + + + + +
0 0 0 0 0 + 0 ++ 0
++ + ++ -
0 0 +
++ + +
++
-
Ganglia: 0, no cells with peptide-IR detected; +, 1-20~o of TH-IR cells; + +, 21-50~o of TH-IR cells; + + +, 51-80~o of TH-IR cells; + + + +, >80~o of TH-IR cells. Perivascular axons: 0, no IR axons detected; + to + + +, estimates of relative density of IR axons, from sparse to very dense; - , not examined.
d e n s e p l e x u s o f p e r i v a s c u l a r a x o n s w i t h G a l - I R a n d T H - I R . P r o b a b l y all T H - I R a x o n s c o n t a i n e d G a l - I R . T h e r e f o r e , at l e a s t s o m e o f t h e f i b r e s a r o u n d t h e r e n a l a r t e r y m u s t have contained TH-IR, Gal-IR, and NPY-IR.
Juvenile possums NPY-IR pouch. NPY-IR
w a s also s c a r c e in s y m p a t h e t i c n e u r o n s o f i m m a t u r e p o s s u m s still in t h e
Only
1-6%
o f s y m p a t h e t i c n e r v e cell b o d i e s c o n t a i n e d b o t h T H - I R
(Fig. 1; T a b l e I), a n d less t h a n 1 ~
of neurons contained NPY-IR
and
without
TH-IR. However, 86-90~o of sympathetic neurons contained both TH-IR and Gal-IR (Fig. 2; T a b l e I). A s in a d u l t p o s s u m s , t h e c o m m o n c a r o t i d a r t e r y o f j u v e n i l e s l a c k e d NPY-IR
axons, but had a moderately-dense plexus of axons with Gal-IR and TH-IR
(Fig. 4). I n t h e r e n a l a r t e r y , b o t h G a I - I R a n d N P Y - I R n o t all, o f t h e T H - I R
a x o n s w e r e c o m m o n , M o s t , if
a x o n s s u p p l y i n g t h e r e n a l a r t e r y c o n t a i n e d G a l - I R (Fig. 5), a n d
a subpopulation of these axons also must have contained NPY-IR.
Other species NPY-IR
n e u r o n s w e r e r a r e (1 ~o t o t a l n e u r o n s ) in t h e s u p e r i o r c e r v i c a l g a n g l i o n o f
p o u c h - y o u n g t a m m a r w a l l a b i e s , b u t i n c r e a s e d in f r e q u e n c y in t h e m o r e c a u d a l s y m p a t h e t i c g a n g l i a . 10~o o f n e u r o n s in t h e stellate g a n g l i a c o n t a i n e d b o t h N P Y - I R
and
105
gp
Figs. 1-3. Sections of sympathetic ganglia double-labelled for TH-IR and NPY-IR, or for TH-IR and GaI-IR. Figs. 1 and 2. Superior cervical ganglion from juvenile brush-tailed possum showing a rare clump of nerve cell bodies with NPY-IR and TH-IR (Fig. 1). The NPY-IR is very faint, and is concentrated in the perinuclear region of the cells. Nearly all nerve cell bodies in the same ganglion have both TH-IR and Gal-IR (Fig. 2). Arrows in each pair of micrographs (a,b) show the same double-labelled neurons. Scale bar = 50 #m. Fig. 3. Lumbar sympathetic ganglion from adult possum showing that nearly all TH-IR nerve cell bodies show GaI-IR (white arrows). Open arrows in each pair of micrographs (a,b) show rare nerve cell bodies which contain IR to TH or to Gal, but which lack IR to the other antigen. Note larger size of ganglion cells in adults compared with juveniles (c.f. Figs. 1, 2). Scale bar = 50 #m.
T H - I R , a n d as m a n y as 22 ~o o f n e u r o n s in l u m b a r s y m p a t h e t i c ganglia c o n t a i n e d b o t h N P Y - I R a n d T H - I R . Again, G a I - I R w a s p r e s e n t in a m u c h larger p r o p o r t i o n o f s y m p a t h e t i c n e u r o n s ( 6 8 - 8 5 ~o) t h a n w a s N P Y - I R . All o f the G a I - I R n e u r o n s c o n t a i n e d T H - I R . T h e d e n s e plexus o f T H - I R a x o n s s u r r o u n d i n g the c o m m o n c a r o t i d artery
106
Figs. 4 and 5. GaI-IR and TH-IR in double-labelled sections of the carotid artery (Fig. 4) and renal artery (Fig. 5) from juvenile possums. Most perivascular nerve fibres at adventitio-medial junction contain IR to both TH and Gal (white arrows). Note nerve bundles (b) with intense TH-IR and Gal-IR in the adventitia of the renal artery. Clumps of nerve cell bodies with TH-IR and Gal-IR were sometimes seen associated with these nerve bundles. Scale bars = 25 #m.
c o n t a i n e d strong G a l - I R , but only a few axons contained N P Y - I R . In contrast, the majority o f T H - I R axons supplying the renal artery c o n t a i n e d both strong G a l - I R and weak NPY-IR. Only thoracic sympathetic ganglia from one adult spectacled hare-wallaby were available for i m m u n o h i s t o c h e m i c a l examination. These ganglia contained a much higher p r o p o r t i o n of N P Y - I R neurons (82~o of 692 cells counted) than we have detected in any sympathetic ganglia from the other two marsupial species examined. Nearly all (98~o) o f the N P Y - I R neurons contained T H - I R . Furthermore, 95°~ of all neurons c o n t a i n e d both G a l - I R and T H - I R . Therefore, at least 75~o o f these sympathetic neurons must have contained T H - I R , G a l - I R and N P Y - I R .
Discussion These results d e m o n s t r a t e that galanin is w i d e s p r e a d in postganglionic sympathetic noradrenergic neurons of the brush-tailed possum. G a l a n i n occurs in m a n y m o r e sympathetic ganglion cells than does N P Y , and it is present in p r o b a b l y all sympathetic nerve terminals associated with systemic arteries o f the possums. In particular, galanin
107 is present in those vascular sympathetic neurons which lack NPY [7]. This observation further demonstrates that NPY is not a universal marker for noradrenergic sympathetic neurons innervating blood vessels. Similarly, although galanin is found in sympathetic perivascular neurons in species as diverse as toads [13], possums and cats [11], it is not uniquely associated with this class of neurons. For example, it is not present in sympathetic neurons of guinea-pigs and rats [ 11 ], but it is found in several different classes of parasympathetic neurons in toads [ 13], as well as in enteric and sensory neurons of several species [14,17-24]. The lack of NPY-immunoreactivity in most sympathetic neurons of the brush-tailed possum is not likely to be due to the inability of our antibodies to detect this peptide in marsupials (see Ref. 7). First, NPY co-existed with galanin in sympathetic neurons innervating the renal arteries. Second, NPY-IR, although still rare, was more prominent in sympathetic ganglion cells from immature animals than from adults. These observations strongly suggest that the primitive condition for these neurons is to contain both NPY and galanin, and that synthesis of NPY is down-regulated during development in all but a specialised population of neurons. This conclusion is supported by the widespread co-existence of NPY-IR with Gal-IR in the one specimen of the spectacled hare-wallaby we were able to examine. Furthermore, the co-existence of galanin and NPY in homologous neurons in other distantly related species such as toads and cats is consistent with the hypothesis that this pattern of peptide expression is the primitive one for sympathetic vasomotor neurons in tetrapods. More comparative studies will be required specifically to test this idea. There is now good evidence that NPY can act as a neurotransmitter from sympathetic vasoconstrictor neurons in certain mammalian blood vessels [2,28-31 ]. However, there is less information available regarding the roles of galanin in vascular neurons. Exogenous galanin is a potent pressor agent in Bufo marinus [32], where it is localized in most sympathetic vascular neurons [ 13]. On the other hand, galanin is contained in vasodilator nerve fibres supplying the submucosal arterioles in guinea-pigs [22], and exogenous galanin dilates these vessels [33]. It is necessary to obtain information on the vascular effects of both galanin and NPY in a wide range of blood vessels in many other species, before we can understand the functional significance of the differential distributions of these peptides in sympathetic neurons. Nevertheless, it seems possible that the patterns of expression ofgalanin and NPY in sympathetic vascular neurons may be related to the specific requirements of autonomic regulation of well-defined regions of the peripheral circulation.
Acknowledgements This work was supported by grants from the National Health and Medical Research Council of Australia, and the Swedish Natural Science Research Council. We are grateful to Drs. R.V. Baudinette, R. Padbury, G. Sacconne and R. Baker for supplying wallaby and possum tissue for this study, and to Drs. C. Maccarrone and B. Jarrott for supplying the NPY antibody. Fiona Renton, Pat Vilimas and Sue Matthew provided excellent technical assistance.
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