Brain Research, 128 (1977) 361-368
361
© Elsevier/North-HollandBiomedicalPress
Catecholamine fluorescence in the brains of chemically sympathectomized adult rats: increased fluorescence in bulbospinal neurons
H. P. L O R E Z
Pharmaceutical Research Department, F. Hoffmann-La Roche and Co. Ltd., Basel (Switzerland)
(Accepted February 17th, 1977)
The adrenergic neuron blocking drugs guanacline2s,~7 and guanethidine2,15,23 deplete and, if chronically administered in high doses, destroy peripheral sympathetic adrenergic neurons in adult rats. In contrast, the catecholamine (CA) content of the whole brain or large parts of the brain showed little or no decrease after treatment with guanacline27 or guanethidineS,15,22. However these findings do not exclude possible major changes in small groups of central monoamine neurons e.g. those outside the blood-brain barrier which might be depleted or damaged, or the A1 and A2 CA neurons in the myelencephalon9, which are probably synaptically connected with the preganglionic sympathetic neurons10, 30 and which might react to the destruction of peripheral sympathetic neurons. On the basis of this reasoning, central monoamine neurons in adult rats chemically sympathectomized with guanacline were investigated using the Falck-Hillarp fluorescence histochemical technique. Guanacline sulphate • 2H20 (Leron®, cyclazenine, N-(2-guanidinoethyl)-4methyl-l,2,3,6-tetrahydropyridine) was given p.o. to I 1 male albino Fiillinsdorf rats (Wistar origin, 100-110 g, about 40 days old) daily for 21 days in the following doses: days 14, 1500 mg/kg; days 5-10, 1000 m g/kg; days 11-21, 1500 mg/kg. The animals were decapitated 26 h after the last drug application. Three of the above rats were injected i.v. with 10 mg/kg a-methyl-noradrenaline (a-MNA, Corbasil®, base) 15 min and phentolamine (Regitin®) 16 min before decapitation. Three control rats received the same phentolamine and a-MNA injections after reserpine (Serpasil®, 10 mg/kg i.p. 16 h). The monoamines were demonstrated in air-dried stretch preparations of the irides and in freeze-dried brains according to Falck et al. 14. After embedding in paraffin the brains were serially sectioned at 8 #m. The superior cervical and coeliac ganglia were fixed in Susa-solution, sectioned at 5/~m and stained with hematoxylin-eosin. Nine animals survived the guanacline treatment with a mean, final weight of 166 g, compared with 240 g in the controls. The following observations indicated an effective sympathectomy. (1) Adrenergic sympathetic nerves. The irides contained far fewer noradrenaline (NA)-containing axons, with decreased CA fluorescence, but some swollen axon endings with intense specific fluorescence, suggesting degeneration (Fig. 1). The NA content of
362 w i]6 ¢
e-
j
Fig. I. CA axons in the iris of a control rat (a) and of a rat chronically treated with guanacline (b). In the latter both fewer axons and swollen axon endings showing strong CA fluorescence indicate neuronal damage, x 580 (88 x 10). Fig. 2. Superior cervical ganglion of a guanacline-treated rat. Note the very marked loss of ganglion cells and the infiltration of small rounded cells. The few remaining ganglion cells (l~) show signs of degeneration. × 205 (16 × 10).
the heart was reduced to less than 2 ~ o f controls (Thoenen, personal c o m m u n i c a t i o n ) . Rats, which received guanacline and a - M N A , displayed a similar reduction in axonal n u m b e r with some increase in fluorescence intensity o f the r e m a i n i n g axons. This also indicated n o t only a N A depletion, but also a destruction o f N A axons. In s u p p o r t o f these findings electron microscopic e x a m i n a t i o n o f the vasa deferentia (Tranzer, p e r s o n a l c o m m u n i c a t i o n ) showed a partial d i s a p p e a r a n c e (75-90 ~ ) o f the varicosities with dense-cored vesicles, i.e. adrenergic varicosities. (2) Sympathetic ganglion cells. The n u m b e r o f ganglion cells in the superior cervical a n d coeliac ganglia was reduced to 2 0 4 0 ~ o f controls (Fig. 2). The r e m a i n i n g ganglion cells showed p r o n o u n c e d chromatolysis, vacuolisation a n d h o m o g e n o u s eosinophilia. The ganglia were enlarged a n d infiltrated with r o u n d e d cells, m o s t l y small
363
P
Fig. 3. a: CA fluorescence of median eminence (m, mostly dopamine nerve terminals), arcuate and periventricular nuclei (a and p respectively, mostly NA nerve terminals) in a control rat, frontal section, v, third ventricle, b: after guanacline treatment the CA fluorescence has almost disappeared. c: injection of a-MNA restored the CA fluorescence in the median emioence of guanacline-treated rats, just as in the median eminence of reserpine-treated controls injected with a-MNA (d). In the latter rat some A 12 cells are visible. × 80 (8 × 10).
lymphocytes. Accordingly in the stellate ganglia the protein content was increased by 30 % (cellular infiltration), but the dopamine-fl-hydroxylase content was reduced to less than 5 % o f controls (Thoenen, personal communication). The brain C A fluorescence was distributed as previously described 9,16. However guanacline reduced the fluorescence intensity and n u m b e r of axons in two regions. (1) The median eminence including adjacent regions (Fig. 3a and b) e.g. retrochiasmatic area, periventricular nucleus, arcuate nucleus and mammillary complex. (2) The area postrema including adjacent parts o f the nucleus tractus solitariJ, nucleus commissuralis and nucleus motorius dorsalis nervi vagi (Fig. 4). Guanacline might have reached these regions which are outside (median eminence, area postrema), or partially outside, the b l o o d - b r a i n barrier and depleted the N A stores as in peripheral sympathetic nerves 27. Similarly guanethidine, another guanidine derivative, also depletes central C A
364
Fig. 4. Frontal section through area postrema and adjacent regions of a control (a) and a guanaclinetreated rat (b). After guanacline treatment the axonal CA fluorescence is greatly diminished in the area postrema (a), nucleus tractus solitarti (t) and nucleus motorius dorsalis nervi vagi (m). Note the three A2 cell bodies showing increased CA fluorescence, c, canalis centralis, x 80 (8 x 10). Fig. 5. a : small cells exhibiting moderate CA fluorescence in the area postrema of a control rat treated with reserpine and a-MNA. In guanacline-treated rats injected with a-MNA (b), the area postrema shows a similar diffuse CA fluorescence, but almost no fluorescent cells are visible, x 300 (28 x 10). n e u r o n s w h e n it r e a c h e s t h e m b y - p a s s i n g the b l o o d - b r a i n b a r r i e r a f t e r i n t r a c e r e b r o v e n t r i c u l a r s or i n t r a c e r e b r a l i n j e c t i o n s 12,1~. N o signs o f n e u r o n a l d e g e n e r a t i o n , e.g. f l u o r e s c e n c e a c c u m u l a t i o n s were o b s e r v e d . T h i s was also t r u e a f t e r i n t r a c e r e b r a l g u a n e t h i d i n e injections12, is. A d d i t i o n a l l y , f l u o r e s c e n t C A n e r v e t e r m i n a l s h a d v i r t u a l l y d i s a p p e a r e d in the pineal g l a n d a n d a r o u n d t h e m e n i n g e a l a n d c h o r o i d a l vessels. In o r d e r to find o u t w h e t h e r the r e d u c e d n u m b e r o f specifically f l u o r e s c e n t a x o n s
365 in regions outside the blood-brain barrier, mainly median eminence and area postrema, was due to neuronal degeneration, the brains were examined after i.v. injection of a-MNA. To compare the amine accumulation in intact neurons, three control rats received 10 mg/kg reserpine i.p. 16 h before a-MNA. After reserpine alone the fluorescence of monoamine axons had completely disappeared. The median eminence of both groups, guanacline- and reserpine-treated rats, showed a similar intense CA fluorescence after injection of a-MNA (Fig. 3c and d). It was not possible to determine the intra- and extraneuronal localization of fluorescence. However in the area postrema a difference in fluorescence was observed (Fig. 5). In guanacline- and a-MNA-treated rats this area showed a homogenous moderate CA fluorescence with only a few small cells exhibiting moderate CA fluorescence. Such cells were frequent in controls injected with reserpine and a-MNA. These are probably the small neuronal cells of the area postrema, which contain and accumulate monoaminesg, 16, and which were occasionally visible with weak CA fluorescence in controls not receiving a-MNA. That they were almost absent after guanacline treatment in spite of the large dose of a-MNA indicates cell destruction. Around the meningeal and choroidal vessels, intense CA fluorescence reappeared after a-MNA in reserpine-treated controls, but almost none in guanaclinetreated rats, indicating an effective sympathectomy of the vasculature. The yellow formaldehyde-induced indolealkylamine fluorescence in the brains appeared unchanged, but this fluorescence is more difficult to judge than the bluegreen CA fluorescence, due to the former's low intensity and rapid fading. In contrast to the decreased fluorescence of some CA systems in the brain described above, the CA fluorescence of the A1 and A2 cell bodies in the myelencephalon (nomenclature of Dahlstr6m and Fuxe 9) was considerably increased after guanacline treatment (Figs. 6 and 7). The number of highly fluorescent cells was increased, with many cells showing brighter fluorescence than in controls. CA fluorescence was not increased in the other CA cell groups, A4-A141, 9. It must be stressed that these qualitative studies only show changes in the CA content of the neurons, when these changes are considerable, and also that the sensitivity of the method is not the same for all CA systems 25. The A1 and A2 cell groups comprise NA 9 and probably adrenaline containing cells 2°,~6,3z. Based on the distribution of phenylethanolamine-N-methyltransferase containing cells ~0 it seems that NA containing cells showed increased fluorescence in the A2 group, and also in the A1 group - - in the caudal part at least. The fluorescence increase probably reflected a change in neuronal activity. The neuronal activity, inside the blood-brain barrier, was probably not directly altered by guanacline; it was also probably not linked to the changes in the brain regions outside the blood-brain barrier since increased CA fluorescence of A1 and A2 cells was also observed in rats killed 24 h after a p.o. guanacline treatment with 300 mg/kg for 28 days, in spite of normal CA fluorescence in the regions outside the blood-brain barrier; the peripheral sympathetic neurons were markedly destroyed in these rats (unpublished observations). It seems more likely that the change in neuronal activity of the A1 and A2 cells is secondary to physiological changes resulting from the sympathectomy, and indicates central compensatory mechanisms.
366
Figs. 6 and 7. CA cells of the cell groups A i (Fig. 6) and A2 (Fig. 7). Figs. 6a and 7a, control rat. After guanacline treatment (Figs. 6b and 7b) most cells show increased CA fluorescence and some appear swollen. Note the binucleated cell ( ~ ) . Fig. 6, × 250; Fig. 7, × 300 (28 x 10).
367 Studies on central C A cells showed a positive relationship between C A fluorescence intensity and neuronal activity 28. It is clear that the bulbospinal neurons A1 and A2 send their axons into the spinal cord to synapse inter alia with the sympathetic preganglionic neurons or the interneurons that surround them x°,a°. However it is still a matter o f controversy whether they exert an excitatorya,4,19,29, al,32 or inhibitory~-7, H, is,z1,24 effect on these sympathetic neurons. Thus, two compensatory mechanisms leading to enhanced activity of the A l and A2 neurons may be tentatively hypothesized. lfthey excite the preganglionic sympathetic neurons or facilitate excitatory transmission then perhaps they might have reacted to enhance the level of postsynaptic sympathetic activity reduced by the guanacline treatment. It is known that increased activity o f spinal C A neurons results from loss of tonic arterial baroreceptor activity 4. If they are directly inhibitory or inhibit the transmission o f sympatho-excitatory impulses to preganglionic sympathetic neurons they might have been involved in a negative feedback mechanism to control the preganglionic sympathetic neurons activated by other mechanisms. To summarize, adult rats were chemically sympathectomized by chronic guanacline treatment. The formaldehyde-induced C A fluorescence in the brain was diminished in regions outside the b l o o d - b r a i n barrier, probably due to a direct depleting effect on these C A neurons. Additionally, m o n o a m i n e neurons were destroyed in the area postrema. In contrast, the bulbospinal C A neurons, A1 and A2 showed an increased C A fluorescence inside the b l o o d - b r a i n barrier. It is assumed that the fluorescence increase indicates a feed-back induced activation of these cells and, therefore, that the findings provide morphological support for an integral or m o d u l a t o r y role o f the A1 and A2 neurons in sympathetic reflex mechanisms. I would like to thank Dr. G. H~iusler for valuable criticism of the manuscript, and Mr. R. Reese for technical assistance.
1 Bj6rklund, A. and Nobin, A., Fluorescence histochemical and microspectrofluorometric mapping of dopamine and noradrenaline cell groups in the rat diencephalon, Brain Research, 51 (1973) 193-205. 2 Burnstock, G., Evans, B., Gannon, B. J., Heath, J. W. and James, V., A new method of destroying adrenergic nerves in adult animals using guanethidine, Brit. J. PharmacoL, 43 (1971) 295-301. 3 Chalmers, J. P., Brain amines and models of experimental hypertension, Circulat. Res., 36 (1975) 469-480. 4 Chalmers, J. P. and Wurtman, R. J., Participation of central noradrenergic neurons in arterial baroreceptor reflexes in the rabbit, Cireulat. Res., 28 (1971) 480-491. 5 Coote, J. H. and Macleod, V. H., The possibility that noradrenaline is a sympatho-inhibitory transmitter in the spinal cord, J. Physiol. (Lond.), 225 (1972) 44P-46P. 6 Coote, J. H. and Macleod, V. H., The influence of bulbospinal monoaminergic pathways on sympathetic nerve activity, J. Physiol. (Lond.), 241 (1974) 453-475. 7 Coote, J. H. and Macleod, V. H., The spinal route of sympatho-inhibitory pathways descending from the medulla oblongata, Pfliigers Arch. ges. Physiol., 359 (1975) 335-347. 8 Cox, R. H. and Maickel, R. P., Effects of guanethidine on rat brain serotonin and norepinephrine, Life Sei., 8 (1969) 1319-1324. 9 Dahlstr6m, A., and Fuxe, K., Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta physiol, scand., 62, (Suppl. 232) (1964) 1-55.
368 10 Dahlstr6m, A. and Fuxe, K., Evidence for the existence of monoamine neurons in the central nervous system. II. Experimentally induced changes in the intraneuronal amine levels of bulbospinal neuron systems, Actaphysiol. scand., 64, Suppl. 247 (1965) 1-36. 11 De Groat, W. C. and Ryall, R. W., An excitatory action of 5-hydroxytryptamine on sympathetic preganglionic neurones, Exp. Braiu Res., 3 (1967) 299-305. 12 Evans, B. K., Armstrong, S., Singer, G., Cook, R. D. and Burnstock, G., lntracranial injection of drugs: comparison of diffusion of 6-OHDA and guanethidine, Pharmacol. Biochem. Behav., 3 (1975) 205-217. 13 Evans, B. K., Singer, G., Armstrong, S., Saunders, P. E. and Burnstock, G., Effects of chronic intracranial injection of low and high concentrations of guanethidine in the rat, Pharmacol. Biochem. Behav., 3 (1975) 219-228. 14 Falck, B., Hillarp, N.-~., Thieme, G. and Torp, A., Fluorescence of catecholamines and related compounds condensed with formaldehyde, J. Histochem. Cytochem., 10 (1962) 348-354. 15 Furst, C. I., The biochemistry of guanethidine, Advanc. Drug Res., 4 (1967) 133-161. 16 Fuxe, K., Evidence for the existence of monoamine neurons in the central nervous system. IV. The distribution of monoamine terminals in the central nervous system, Acta physioL scand., 64, Suppl. 247 (1965) 37-85. 17 Fuxe, K. and Owman, C., Cellular localization of monoamines in the area postrema of certain mammals, J. comp. NeuraL, 125 (1965) 337-354. 18 Haeusler, G., Inhibition of the preganglionic sympathetic neurone through spinal a-adrenoceptors, Arch. PharmacoL, 293, Suppl. (1976) R15. 19 Hare, B. D., Neumayr, R. J. and Franz, D. N., Opposite effects of L-DOPA and 5-HTP on spinal sympathetic reflexes, Nature (Lond.), 239 (1972) 336-337. 20 H6kfelt, T., Fuxe, K., Goldstein, M. and Johansson, O., lmmunohistochemical evidence for the existence of adrenaline neurons in the rat brain, Brain Research, 66 (1974) 235 251. 21 Illert, M., Influence of monoaminergic pathways on the spinal somato-sympathetic reflex transmission, Europ. J. PhysioL, 339, Suppl. (1973) R66. 22 Johnson, E. M., O'Brien, F. and Werbitt, R., Modification and characterization of the permanent sympathectomy produced by the administration of guanethidine to newborn rats, Europ. J. Pharmacol., 37 (1976) 45-54. 23 Juul, P., Effects of various antihypertensive guanidine derivatives on the adult rat superior cervical ganglion: histology, ultrastructure, and cholinesterase histochemistry, dcta pharmacol. (Kbh.), 32 (1973) 500-512. 24 Kirchner, F. and Polosa, C., The effect of L-Dopa on the spinal sympathetic reflex of chronic spinal cats, Europ. J. PhysioL, 362, Suppl. (1976) R41. 25 Kopin, I. J., Palkovits, M., Kobayashi, R. M. and Jacobowitz, D. M., Quantitative relationship of catecholamine content and histofluorescence in brain of rats, Brain Research, 80 (1974) 229-235. 26 Koslow, S. H. and Schlumpf, M., Quantitation of adrenaline in rat brain nuclei and areas by mass fragmentography, Nature (Lond.), 251 (1974) 530-531. 27 Kroneberg, G., Schlossmann, K. und Stoepel, K., Zur Pharmakologie von N-(2-Guanidino~ithyl)4-methyl-l,2,3,6-tetrahydropyridin, einer neuen antihypertensiv wirkenden Verbindung, ArzneimitteI-Forsch., 17 (1967) 199-207. 28 Lichtensteiger, W., Felix, D., Lienhart, R. and Hefti, F., A quantitative correlation between single unit activity and fluorescence intensity of dopamine neurones in zona compacta of substantia nigra, as demonstrated under the influence of nicotine and physostigmine, Brain Research, 117 (1976) 85-103. 29 Neumayr, R. J., Hare, B. D. and Franz, D. N., Evidence for bulbospinal control of sympathetic preganglionic neurons by monoaminergic pathways, Life Sci., 14 (1974) 793-806. 30 R6thelyi, M., Cell and neuropil architecture of the intermediolateral (sympathetic) nucleus of cat spinal cord, Brain Research, 46 (1972) 203-213. 31 Taylor, D. G., Spinal adrenergic fibers and vasoconstrictor outflow to cat hindlimbs, Fed. Proc., 34 (1975) 420. 32 Taylor, D. G. and Brody, M. J., Spinal adrenergic mechanisms regulating sympathetic outflow to blood vessels, Circulat. Res., 38, Suppl. 2 (1976) 10-20. 33 Van der Gugten, J., Palkovits, M., Wijnen, H. L. J. M. and Versteeg, D. H. G., Regional distribution of adrenaline in rat brain, Brain Research, 107 (1976) 171-175.
361
© Elsevier/North-HollandBiomedicalPress
Catecholamine fluorescence in the brains of chemically sympathectomized adult rats: increased fluorescence in bulbospinal neurons
H. P. L O R E Z
Pharmaceutical Research Department, F. Hoffmann-La Roche and Co. Ltd., Basel (Switzerland)
(Accepted February 17th, 1977)
The adrenergic neuron blocking drugs guanacline2s,~7 and guanethidine2,15,23 deplete and, if chronically administered in high doses, destroy peripheral sympathetic adrenergic neurons in adult rats. In contrast, the catecholamine (CA) content of the whole brain or large parts of the brain showed little or no decrease after treatment with guanacline27 or guanethidineS,15,22. However these findings do not exclude possible major changes in small groups of central monoamine neurons e.g. those outside the blood-brain barrier which might be depleted or damaged, or the A1 and A2 CA neurons in the myelencephalon9, which are probably synaptically connected with the preganglionic sympathetic neurons10, 30 and which might react to the destruction of peripheral sympathetic neurons. On the basis of this reasoning, central monoamine neurons in adult rats chemically sympathectomized with guanacline were investigated using the Falck-Hillarp fluorescence histochemical technique. Guanacline sulphate • 2H20 (Leron®, cyclazenine, N-(2-guanidinoethyl)-4methyl-l,2,3,6-tetrahydropyridine) was given p.o. to I 1 male albino Fiillinsdorf rats (Wistar origin, 100-110 g, about 40 days old) daily for 21 days in the following doses: days 14, 1500 mg/kg; days 5-10, 1000 m g/kg; days 11-21, 1500 mg/kg. The animals were decapitated 26 h after the last drug application. Three of the above rats were injected i.v. with 10 mg/kg a-methyl-noradrenaline (a-MNA, Corbasil®, base) 15 min and phentolamine (Regitin®) 16 min before decapitation. Three control rats received the same phentolamine and a-MNA injections after reserpine (Serpasil®, 10 mg/kg i.p. 16 h). The monoamines were demonstrated in air-dried stretch preparations of the irides and in freeze-dried brains according to Falck et al. 14. After embedding in paraffin the brains were serially sectioned at 8 #m. The superior cervical and coeliac ganglia were fixed in Susa-solution, sectioned at 5/~m and stained with hematoxylin-eosin. Nine animals survived the guanacline treatment with a mean, final weight of 166 g, compared with 240 g in the controls. The following observations indicated an effective sympathectomy. (1) Adrenergic sympathetic nerves. The irides contained far fewer noradrenaline (NA)-containing axons, with decreased CA fluorescence, but some swollen axon endings with intense specific fluorescence, suggesting degeneration (Fig. 1). The NA content of
362 w i]6 ¢
e-
j
Fig. I. CA axons in the iris of a control rat (a) and of a rat chronically treated with guanacline (b). In the latter both fewer axons and swollen axon endings showing strong CA fluorescence indicate neuronal damage, x 580 (88 x 10). Fig. 2. Superior cervical ganglion of a guanacline-treated rat. Note the very marked loss of ganglion cells and the infiltration of small rounded cells. The few remaining ganglion cells (l~) show signs of degeneration. × 205 (16 × 10).
the heart was reduced to less than 2 ~ o f controls (Thoenen, personal c o m m u n i c a t i o n ) . Rats, which received guanacline and a - M N A , displayed a similar reduction in axonal n u m b e r with some increase in fluorescence intensity o f the r e m a i n i n g axons. This also indicated n o t only a N A depletion, but also a destruction o f N A axons. In s u p p o r t o f these findings electron microscopic e x a m i n a t i o n o f the vasa deferentia (Tranzer, p e r s o n a l c o m m u n i c a t i o n ) showed a partial d i s a p p e a r a n c e (75-90 ~ ) o f the varicosities with dense-cored vesicles, i.e. adrenergic varicosities. (2) Sympathetic ganglion cells. The n u m b e r o f ganglion cells in the superior cervical a n d coeliac ganglia was reduced to 2 0 4 0 ~ o f controls (Fig. 2). The r e m a i n i n g ganglion cells showed p r o n o u n c e d chromatolysis, vacuolisation a n d h o m o g e n o u s eosinophilia. The ganglia were enlarged a n d infiltrated with r o u n d e d cells, m o s t l y small
363
P
Fig. 3. a: CA fluorescence of median eminence (m, mostly dopamine nerve terminals), arcuate and periventricular nuclei (a and p respectively, mostly NA nerve terminals) in a control rat, frontal section, v, third ventricle, b: after guanacline treatment the CA fluorescence has almost disappeared. c: injection of a-MNA restored the CA fluorescence in the median emioence of guanacline-treated rats, just as in the median eminence of reserpine-treated controls injected with a-MNA (d). In the latter rat some A 12 cells are visible. × 80 (8 × 10).
lymphocytes. Accordingly in the stellate ganglia the protein content was increased by 30 % (cellular infiltration), but the dopamine-fl-hydroxylase content was reduced to less than 5 % o f controls (Thoenen, personal communication). The brain C A fluorescence was distributed as previously described 9,16. However guanacline reduced the fluorescence intensity and n u m b e r of axons in two regions. (1) The median eminence including adjacent regions (Fig. 3a and b) e.g. retrochiasmatic area, periventricular nucleus, arcuate nucleus and mammillary complex. (2) The area postrema including adjacent parts o f the nucleus tractus solitariJ, nucleus commissuralis and nucleus motorius dorsalis nervi vagi (Fig. 4). Guanacline might have reached these regions which are outside (median eminence, area postrema), or partially outside, the b l o o d - b r a i n barrier and depleted the N A stores as in peripheral sympathetic nerves 27. Similarly guanethidine, another guanidine derivative, also depletes central C A
364
Fig. 4. Frontal section through area postrema and adjacent regions of a control (a) and a guanaclinetreated rat (b). After guanacline treatment the axonal CA fluorescence is greatly diminished in the area postrema (a), nucleus tractus solitarti (t) and nucleus motorius dorsalis nervi vagi (m). Note the three A2 cell bodies showing increased CA fluorescence, c, canalis centralis, x 80 (8 x 10). Fig. 5. a : small cells exhibiting moderate CA fluorescence in the area postrema of a control rat treated with reserpine and a-MNA. In guanacline-treated rats injected with a-MNA (b), the area postrema shows a similar diffuse CA fluorescence, but almost no fluorescent cells are visible, x 300 (28 x 10). n e u r o n s w h e n it r e a c h e s t h e m b y - p a s s i n g the b l o o d - b r a i n b a r r i e r a f t e r i n t r a c e r e b r o v e n t r i c u l a r s or i n t r a c e r e b r a l i n j e c t i o n s 12,1~. N o signs o f n e u r o n a l d e g e n e r a t i o n , e.g. f l u o r e s c e n c e a c c u m u l a t i o n s were o b s e r v e d . T h i s was also t r u e a f t e r i n t r a c e r e b r a l g u a n e t h i d i n e injections12, is. A d d i t i o n a l l y , f l u o r e s c e n t C A n e r v e t e r m i n a l s h a d v i r t u a l l y d i s a p p e a r e d in the pineal g l a n d a n d a r o u n d t h e m e n i n g e a l a n d c h o r o i d a l vessels. In o r d e r to find o u t w h e t h e r the r e d u c e d n u m b e r o f specifically f l u o r e s c e n t a x o n s
365 in regions outside the blood-brain barrier, mainly median eminence and area postrema, was due to neuronal degeneration, the brains were examined after i.v. injection of a-MNA. To compare the amine accumulation in intact neurons, three control rats received 10 mg/kg reserpine i.p. 16 h before a-MNA. After reserpine alone the fluorescence of monoamine axons had completely disappeared. The median eminence of both groups, guanacline- and reserpine-treated rats, showed a similar intense CA fluorescence after injection of a-MNA (Fig. 3c and d). It was not possible to determine the intra- and extraneuronal localization of fluorescence. However in the area postrema a difference in fluorescence was observed (Fig. 5). In guanacline- and a-MNA-treated rats this area showed a homogenous moderate CA fluorescence with only a few small cells exhibiting moderate CA fluorescence. Such cells were frequent in controls injected with reserpine and a-MNA. These are probably the small neuronal cells of the area postrema, which contain and accumulate monoaminesg, 16, and which were occasionally visible with weak CA fluorescence in controls not receiving a-MNA. That they were almost absent after guanacline treatment in spite of the large dose of a-MNA indicates cell destruction. Around the meningeal and choroidal vessels, intense CA fluorescence reappeared after a-MNA in reserpine-treated controls, but almost none in guanaclinetreated rats, indicating an effective sympathectomy of the vasculature. The yellow formaldehyde-induced indolealkylamine fluorescence in the brains appeared unchanged, but this fluorescence is more difficult to judge than the bluegreen CA fluorescence, due to the former's low intensity and rapid fading. In contrast to the decreased fluorescence of some CA systems in the brain described above, the CA fluorescence of the A1 and A2 cell bodies in the myelencephalon (nomenclature of Dahlstr6m and Fuxe 9) was considerably increased after guanacline treatment (Figs. 6 and 7). The number of highly fluorescent cells was increased, with many cells showing brighter fluorescence than in controls. CA fluorescence was not increased in the other CA cell groups, A4-A141, 9. It must be stressed that these qualitative studies only show changes in the CA content of the neurons, when these changes are considerable, and also that the sensitivity of the method is not the same for all CA systems 25. The A1 and A2 cell groups comprise NA 9 and probably adrenaline containing cells 2°,~6,3z. Based on the distribution of phenylethanolamine-N-methyltransferase containing cells ~0 it seems that NA containing cells showed increased fluorescence in the A2 group, and also in the A1 group - - in the caudal part at least. The fluorescence increase probably reflected a change in neuronal activity. The neuronal activity, inside the blood-brain barrier, was probably not directly altered by guanacline; it was also probably not linked to the changes in the brain regions outside the blood-brain barrier since increased CA fluorescence of A1 and A2 cells was also observed in rats killed 24 h after a p.o. guanacline treatment with 300 mg/kg for 28 days, in spite of normal CA fluorescence in the regions outside the blood-brain barrier; the peripheral sympathetic neurons were markedly destroyed in these rats (unpublished observations). It seems more likely that the change in neuronal activity of the A1 and A2 cells is secondary to physiological changes resulting from the sympathectomy, and indicates central compensatory mechanisms.
366
Figs. 6 and 7. CA cells of the cell groups A i (Fig. 6) and A2 (Fig. 7). Figs. 6a and 7a, control rat. After guanacline treatment (Figs. 6b and 7b) most cells show increased CA fluorescence and some appear swollen. Note the binucleated cell ( ~ ) . Fig. 6, × 250; Fig. 7, × 300 (28 x 10).
367 Studies on central C A cells showed a positive relationship between C A fluorescence intensity and neuronal activity 28. It is clear that the bulbospinal neurons A1 and A2 send their axons into the spinal cord to synapse inter alia with the sympathetic preganglionic neurons or the interneurons that surround them x°,a°. However it is still a matter o f controversy whether they exert an excitatorya,4,19,29, al,32 or inhibitory~-7, H, is,z1,24 effect on these sympathetic neurons. Thus, two compensatory mechanisms leading to enhanced activity of the A l and A2 neurons may be tentatively hypothesized. lfthey excite the preganglionic sympathetic neurons or facilitate excitatory transmission then perhaps they might have reacted to enhance the level of postsynaptic sympathetic activity reduced by the guanacline treatment. It is known that increased activity o f spinal C A neurons results from loss of tonic arterial baroreceptor activity 4. If they are directly inhibitory or inhibit the transmission o f sympatho-excitatory impulses to preganglionic sympathetic neurons they might have been involved in a negative feedback mechanism to control the preganglionic sympathetic neurons activated by other mechanisms. To summarize, adult rats were chemically sympathectomized by chronic guanacline treatment. The formaldehyde-induced C A fluorescence in the brain was diminished in regions outside the b l o o d - b r a i n barrier, probably due to a direct depleting effect on these C A neurons. Additionally, m o n o a m i n e neurons were destroyed in the area postrema. In contrast, the bulbospinal C A neurons, A1 and A2 showed an increased C A fluorescence inside the b l o o d - b r a i n barrier. It is assumed that the fluorescence increase indicates a feed-back induced activation of these cells and, therefore, that the findings provide morphological support for an integral or m o d u l a t o r y role o f the A1 and A2 neurons in sympathetic reflex mechanisms. I would like to thank Dr. G. H~iusler for valuable criticism of the manuscript, and Mr. R. Reese for technical assistance.
1 Bj6rklund, A. and Nobin, A., Fluorescence histochemical and microspectrofluorometric mapping of dopamine and noradrenaline cell groups in the rat diencephalon, Brain Research, 51 (1973) 193-205. 2 Burnstock, G., Evans, B., Gannon, B. J., Heath, J. W. and James, V., A new method of destroying adrenergic nerves in adult animals using guanethidine, Brit. J. PharmacoL, 43 (1971) 295-301. 3 Chalmers, J. P., Brain amines and models of experimental hypertension, Circulat. Res., 36 (1975) 469-480. 4 Chalmers, J. P. and Wurtman, R. J., Participation of central noradrenergic neurons in arterial baroreceptor reflexes in the rabbit, Cireulat. Res., 28 (1971) 480-491. 5 Coote, J. H. and Macleod, V. H., The possibility that noradrenaline is a sympatho-inhibitory transmitter in the spinal cord, J. Physiol. (Lond.), 225 (1972) 44P-46P. 6 Coote, J. H. and Macleod, V. H., The influence of bulbospinal monoaminergic pathways on sympathetic nerve activity, J. Physiol. (Lond.), 241 (1974) 453-475. 7 Coote, J. H. and Macleod, V. H., The spinal route of sympatho-inhibitory pathways descending from the medulla oblongata, Pfliigers Arch. ges. Physiol., 359 (1975) 335-347. 8 Cox, R. H. and Maickel, R. P., Effects of guanethidine on rat brain serotonin and norepinephrine, Life Sei., 8 (1969) 1319-1324. 9 Dahlstr6m, A., and Fuxe, K., Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta physiol, scand., 62, (Suppl. 232) (1964) 1-55.
368 10 Dahlstr6m, A. and Fuxe, K., Evidence for the existence of monoamine neurons in the central nervous system. II. Experimentally induced changes in the intraneuronal amine levels of bulbospinal neuron systems, Actaphysiol. scand., 64, Suppl. 247 (1965) 1-36. 11 De Groat, W. C. and Ryall, R. W., An excitatory action of 5-hydroxytryptamine on sympathetic preganglionic neurones, Exp. Braiu Res., 3 (1967) 299-305. 12 Evans, B. K., Armstrong, S., Singer, G., Cook, R. D. and Burnstock, G., lntracranial injection of drugs: comparison of diffusion of 6-OHDA and guanethidine, Pharmacol. Biochem. Behav., 3 (1975) 205-217. 13 Evans, B. K., Singer, G., Armstrong, S., Saunders, P. E. and Burnstock, G., Effects of chronic intracranial injection of low and high concentrations of guanethidine in the rat, Pharmacol. Biochem. Behav., 3 (1975) 219-228. 14 Falck, B., Hillarp, N.-~., Thieme, G. and Torp, A., Fluorescence of catecholamines and related compounds condensed with formaldehyde, J. Histochem. Cytochem., 10 (1962) 348-354. 15 Furst, C. I., The biochemistry of guanethidine, Advanc. Drug Res., 4 (1967) 133-161. 16 Fuxe, K., Evidence for the existence of monoamine neurons in the central nervous system. IV. The distribution of monoamine terminals in the central nervous system, Acta physioL scand., 64, Suppl. 247 (1965) 37-85. 17 Fuxe, K. and Owman, C., Cellular localization of monoamines in the area postrema of certain mammals, J. comp. NeuraL, 125 (1965) 337-354. 18 Haeusler, G., Inhibition of the preganglionic sympathetic neurone through spinal a-adrenoceptors, Arch. PharmacoL, 293, Suppl. (1976) R15. 19 Hare, B. D., Neumayr, R. J. and Franz, D. N., Opposite effects of L-DOPA and 5-HTP on spinal sympathetic reflexes, Nature (Lond.), 239 (1972) 336-337. 20 H6kfelt, T., Fuxe, K., Goldstein, M. and Johansson, O., lmmunohistochemical evidence for the existence of adrenaline neurons in the rat brain, Brain Research, 66 (1974) 235 251. 21 Illert, M., Influence of monoaminergic pathways on the spinal somato-sympathetic reflex transmission, Europ. J. PhysioL, 339, Suppl. (1973) R66. 22 Johnson, E. M., O'Brien, F. and Werbitt, R., Modification and characterization of the permanent sympathectomy produced by the administration of guanethidine to newborn rats, Europ. J. Pharmacol., 37 (1976) 45-54. 23 Juul, P., Effects of various antihypertensive guanidine derivatives on the adult rat superior cervical ganglion: histology, ultrastructure, and cholinesterase histochemistry, dcta pharmacol. (Kbh.), 32 (1973) 500-512. 24 Kirchner, F. and Polosa, C., The effect of L-Dopa on the spinal sympathetic reflex of chronic spinal cats, Europ. J. PhysioL, 362, Suppl. (1976) R41. 25 Kopin, I. J., Palkovits, M., Kobayashi, R. M. and Jacobowitz, D. M., Quantitative relationship of catecholamine content and histofluorescence in brain of rats, Brain Research, 80 (1974) 229-235. 26 Koslow, S. H. and Schlumpf, M., Quantitation of adrenaline in rat brain nuclei and areas by mass fragmentography, Nature (Lond.), 251 (1974) 530-531. 27 Kroneberg, G., Schlossmann, K. und Stoepel, K., Zur Pharmakologie von N-(2-Guanidino~ithyl)4-methyl-l,2,3,6-tetrahydropyridin, einer neuen antihypertensiv wirkenden Verbindung, ArzneimitteI-Forsch., 17 (1967) 199-207. 28 Lichtensteiger, W., Felix, D., Lienhart, R. and Hefti, F., A quantitative correlation between single unit activity and fluorescence intensity of dopamine neurones in zona compacta of substantia nigra, as demonstrated under the influence of nicotine and physostigmine, Brain Research, 117 (1976) 85-103. 29 Neumayr, R. J., Hare, B. D. and Franz, D. N., Evidence for bulbospinal control of sympathetic preganglionic neurons by monoaminergic pathways, Life Sci., 14 (1974) 793-806. 30 R6thelyi, M., Cell and neuropil architecture of the intermediolateral (sympathetic) nucleus of cat spinal cord, Brain Research, 46 (1972) 203-213. 31 Taylor, D. G., Spinal adrenergic fibers and vasoconstrictor outflow to cat hindlimbs, Fed. Proc., 34 (1975) 420. 32 Taylor, D. G. and Brody, M. J., Spinal adrenergic mechanisms regulating sympathetic outflow to blood vessels, Circulat. Res., 38, Suppl. 2 (1976) 10-20. 33 Van der Gugten, J., Palkovits, M., Wijnen, H. L. J. M. and Versteeg, D. H. G., Regional distribution of adrenaline in rat brain, Brain Research, 107 (1976) 171-175.
