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Development of adrenergic nerve terminals: The effects of decentralization
Brain Research, 158 (1978) 259-268 © Elsevier/North-HollandBiomedicalPress
259
DEVELOPMENT OF ADRENERGIC NERVE TERMINALS: THE EFFECTS OF DECENTRALIZATION
CATHERINE MYTILINEOUand IRA B. BLACK Department of Neurology, Mount Sinai School of Medicine, New York, N. Y. 10029 and Department of Neurology, Cornell University Medical College, New York, N.Y. 10021 (U.S.A.)
(Accepted March 30th, 1978)
SUMMARY The effect of decentralization (deafferentation) on the ontogeny of adrenergic nerve terminals was studied in the rat iris. Transection of the cholinergic trunk, which innervates the superior cervical ganglion (SCG), in 2-3-day-old rats, inhibited the developmental increase in iris nerve terminal density, as indicated by fluorescence microscopy. However, terminal varicosities, containing the norepinephrine (NE) storage vesicles, appeared larger and more brightly fluorescent in decentralized irides. Tyrosine hydroxylase, localized to nerve terminal cytoplasm, developed to only 40 ~ of normal in decentralized irides. In contrast, dopamine-fl-hydroxylase, which is localized to NE storage vesicles in varicosities, developed normally. The ability to accumulate and store [SH]NE was examined in control and decentralized terminals in the presence or absence of reserpine. This drug inhibits NE storage capacity without primarily affecting accumulation itself. Decentralization reduced the in vitro uptake of NE to 54 ~o, measured after reserpine pretreatment; in contrast, apparent uptake of transmitter was reduced to only 76 ~o in vehicle-treated rats. These results suggest that decentralization prevents the normal development of nerve terminal membrane without markedly interfering with vesicular storage capacity. This contention was supported by the observation that in vitro retention of [3H]NE over time was actually increased in decentralized irides. Our results suggest that the ontogeny of different subcellular structures is affected differently by deafferentation. The transsynaptic regulation of nerve terminal maturation appears to be most critical in younger rats, since decentralization exerts most marked effects when performed during the perinatal period.
INTRODUCTION The normal growth and maturation of peripheral sympathetic neurons requires
260 intact preganglionic innervation2-4,19. Decentralization (denervation) of the superior cervical ganglion (SCG) shortly after birth, or treatment with ganglionic blocking agents, prevents the normal ontogenetic increase in the activities of tyrosine hydroxylase (T-OH) and dopamine-fl-hydroxylase (DBH) in adrenergic neurons2,4,5,19. These enzymes are involved in the biosynthesis of norepinephrine (NE) 17, and are localized to adrenergic perikarya of the SCG 17. We have recently demonstrated that decentralization of the SCG prevents normal development of the adrenergic nerve terminals in target organs s, as well as interfering with maturation of ganglion cell bodies. The normal growth of the adrenergic nerve terminals in the iris was prevented by ganglion decentralization in 2-3-day-old rats s. T-OH activity, localized to adrenergic terminals of the iris, remained depressed by 60 in irides innervated by decentralized ganglia s . Moreover, fluorescence microscopy demonstrated that the number of adrenergic nerve terminals present in the iris and their degree of branching was markedly reduced by ganglion decentralization. In contrast, the in vitro uptake of [SH]NE, a quantitative measure of functional nerve terminal membrane 14, was depressed by only 20 ~ in irides innervated by decentralized ganglia s. Observations made by fluorescence microscopy also suggested that fibers emanating from decentralized ganglia were qualitatively altered. The present report describes the neurochemical properties of decentralized adrenergic nerve terminals in greater detail. Our studies confirm the observation that decentralization prevents the normal ontogenetic increase in the number of nerve terminals innervating the iris. However, although ganglion decentralization prevents the normal development of iris T-OH activity, iris DBH develops normally. Furthermore, the relative capacity of the nerve fibers to store [SH]NE actually increases in the decentralized iris. Lastly, we report that the effect of decentralization on nerve terminal development is critically related to the age of the animal at the time of surgery. METHODS
Enzyme assays T-OH and DBH activities were assayed in irides as previously described1,13.
[SH] Norepinephrine uptake Irides were removed under a dissecting microscope and placed in ice-cold KrebsRinger phosphate buffer containing per ml: glucose, 1.0 mg; EDTA, 0.5 mg; ascorbate, 0.2 rag; pargyline, 0.16 mg. After a 10 min equilibration at 37 °C [3H]NE was added to a final concentration of 10 7 M and the tissues were incubated with shaking for 15 min. Then the tissues were washed in fresh buffer for 15 more minutes and transferred to vials containing 3 ml 100 ~o ethanol and heated at 60 °C for 1 h. After addition of 10 ml Bray's solution the vials were counted for radioactivity in a liquid scintillation spectrometer (Packard Tri-Carb 2450).
Spontaneous release of [3H] NE Irides innervated by intact and decentralized ganglia were removed and incubated
261 with [3H]NE (10-7 M) for 30 min in the absence of pargyline. After a 15 rain wash, individual irides were transferred to the wells of a tissue culture plate which contained 1 ml of fresh buffer at 37 °C. At 5 min intervals the irides were transferred to the next well. The plate contained 4 rows of 6 wells each so that the spontaneous release of [3H]NE was studied, using 4 irides each for 30 min period. At the end of the 30 min the irides were transferred to vials containing 3 ml of 100 ~ ethanol for determination of radioactivity as described above. In addition, 0.5 ml buffer was collected from each well, added to a vial containing 10 ml Bray's solution, and subjected to scintillation spectroscopy.
Fluorescence histochemistr y The irides were isolated and prepared for uptake of a-methyl-NE as described above, except that pargyline was not added to the buffer. The final concentration of a-methyl-NE in the medium was 10-5 M, and the incubation period was 30 min. After a wash the irides were stretched on glass slides and left to dry overnight in a dessicator containing P~Os. They were then exposed to formaldehyde vapor (70 ~o relative humidity) for 1 h at 80 °C 16. They were observed and photographed by a Leitz Ortholux fluorescence photomicroscope. Statistical analyses Grouped data were analyzed using Student's t-test and paired data were analyzed with the paired t-test. RESULTS
Effect of ganglion decentralization on the morphological development of iris innervation Fluorescence microscopy of irides was performed to evaluate the effect of neonatal ganglion decentralization on the development of adrenergic nerve terminals. To aid in the visualization of all adrenergic axon terminals, irides were preincubated with a-methyl-NE. This compound is concentrated in adrenergic neurons by the same uptake process as NE, but is resistant to metabolism by monoamine oxidase. Consequently, virtually all adrenergic terminals, regardless of endogenous amine stores, are rendered visible, allowing more detailed morphologic observations. Irides of 8-week-old rats innervated by ganglia decentralized at 3 days of age exhibited marked reductions in the density of the adrenergic ground plexus, in agreement with previous results3. In addition, qualitative differences in the pattern of innervation were evident at higher magnification. In the decentralized iris the nerve plexus consisted predominantly of single nerve fibers, whereas fibers tended to run in pairs in normal irides (Fig. la, arrows). Moreover, the terminal varicosities appeared larger and more brightly fluorescent in decentralized irides (Fig. lb, arrows). However, it was not possible by fluorescence microscopy to determine whether the varicosities were in fact larger, or simply appeared larger due to increased fluorescence intensity. Effect of decentralization on the neurochemical development of iris terminals To determine whether the development of decentralized adrenergic terminals
262
Fig. 1. Stretch preparations of irides from a rat 8 weeks after SCG decentralization at 3 days of age. The irides were incubated in 10 5 M a-methyl norepinephrine prior to treatment for catecbolamine histofluorescence; a: normal iris. A rich adrenergic plexus is present with many fibers running in bundles of 2 or more (arrows). b: decentralized iris. The nerve fibers are fewer with less branching. Some large brightly fluorescent varicosities can be seen (arrows). × 300.
exhibited neurochemical as well as morphologic abnormalities, catecholamine biosynthetic enzymes were examined. In agreement with previous results 3, T-OH activity, which is localized to the supernatant fraction of nerve terminals 17, was 40 ~ of normal in irides 3 weeks after decentralization (Fig. 2). In contrast, the activity of DBH, which is localized to noradrenergic storage vesicles 17, was normal in decentralized irides (Fig. 2). Since these observations suggested that the development of vesicles and nerve terminal density may be affected differently by decentralization, we studied the uptake and storage of NE.
Uptake and storage of norepinephrine The in vitro uptake of [3H]NE by normal and contralateral decentralized irides was studied 3 weeks after neonatal ganglion decentralization. To define the functions of membrane uptake and vesicular storage separately, one group of animals was pretreated with reserpine, and littermates received vehicle. Reserpine interferes with vesicular storage (retention) of N E without altering the high-affinity uptake process itself 12,17. The experimental protocol resulted in 4 sets of irides: (a) vehicle, intact; (b) vehicle, decentralized; (c) reserpine, intact; (d) reserpine, decentralized. In vehicletreated rats, decentralization reduced [3H]NE uptake to 76 ~o of that in contralateral control, intact irides (Fig. 3). However, after reserpine treatment, which removed the
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Fig. 2. Effect of ganglion decentralization on iris enzyme activities. Eight rats aged 3 days were subjected to unilateral decentralization of the superior cervical ganglion; 3 weeks postoperatively ipsilateral decentralized irides and contralateral control irides were removed and enzyme activities were assayed. Tyrosine hydroxylase activity is expressed as pmole/iris/h 4- S.E.M. (vertical bars) and dopamine-flhydroxylase as nmole/iris/h. * Differs from respective control at P < 0.001. The dopamine-fl-hydroxylase groups do not differ significantly (P > 0.05). vesicular storage component of the apparent uptake, decentralized irides accumulated only 54 ~o of the [aH]NE in contralateral controls (Fig. 3). Reserpinization reduced the uptake of [3H]NE by 20 ~ in the control irides and by 44 ~ in the decentralized irides. Thus, decentralized iris terminals apparently have reduced capacity to take up transmitter, but a relatively higher storage capacity. Terminal storage capacity was also evaluated by examining in vitro retention of exogenous [aH]NE. Decentralized terminals retained more of the previously taken up amine at all times examined (Fig. 4). To determine whether the [aH]NE was metabolized differently in control and decentralized iris terminals, non-catechol metabolites were measure&. There were no significant differences between the groups, non-catechol metabolites constituting 2.4 :~ 0.09 ~ of total radioactivity in the controls, and 2.6 40.22 ~ in the decentralized irides (P > 0.05).
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Fig. 3. Effect of reserpine administration on the uptake of [3H]norepinephrine by normal and decentralized rat irides. Animals were decentralized at 2-3 days of age and the accumulation of [3H]norepinephrine was measured as indicated under Methods; 3 weeks postoperatively one group of 6 animals received 10 mg/kg reserpine i.p. 20 h before the experiment. The controls (6 animals) received an equal volume of vehicle. Each value represents mean counts/min × 10 3/iris ± S.E.M. * Differs from control at P < 0 . 0 0 1 .
Effect of ganglion decentralization at different ages Fluorescence microscopy. To determine whether there is a critical period during which transsynaptic factors regulate terminal development, ganglia were decentralized in rats of different ages. Control and contralateral decentralized irides were examined by fluorescence microscopy after incubation with a-methyl-NE, as described above. Decentralization at 3 days of age resulted in abnormal development of the adrenergic ground plexus (Fig. 1). Similar abnormalities were apparent in irides of animals undergoing surgery at 10 days of age. However, decentralization in older rats resulted in no discernible alteration of terminal development as observed by fluorescence microscopy.
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Fig. 4. Effect of decentralization on the retention of previously accumulated [aH]NE by the iris. The irides from 8 rats, 7 weeks after unilateral SCG decentralization performed at 3 days of age, were processed for [aH]NE uptake as described under Methods. The NE retained by the decentralized and control irides over a 30 min wash period is expressed as per cent of the radioactivity (CPM/iris -t- S.E.M.) present in each iris at the beginning of the experiment. * Differs from respective control, P < 0.05 ; • * Differs from respective control, P < 0.025; *=* Differs from respective control, P < 0.005.
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Fig. 5. Effect of ganglion decentralization at different ages. Groups of 6-8 rats were subjected to unilateral ganglion decentralization at the indicated ages. Tyrosine hydroxylase activity was assayed in the ipsilateral iris and the contralateral control iris in each animal 21 days postoperatively. Enzyme activity is expressed as pmole/iris/h 4- S.E.M. (vertical bars). * Differs from respective control by paired t-test at P < 0.01.
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Fig. 6. Effect of decentralization at different ages on the uptake of [3H]NE by the irides. SCG from groups of 12 animals were unilaterally decentralized at various postnatal ages and the [aH]NE uptake by the normal and decentralized iris was determined 3 weeks after the operation as described under Methods. 6 animals from each age group received 10 mg/kg reserpine i.p. 20 h before the experiment. Points represent CPM/iris 4- S.E.M. expressed as per cent of the contralatelal control iris. * Differs from the respective control at P < 0.05.
266
Tyrosine hydroxylase Ganglion decentralization had a more pronounced effect on development of iris T-OH activity when performed in younger animals (Fig. 5). Surgery was performed in rats of varying ages and irides were examined 3 weeks thereafter. Decentralization during the first postnatal weeks inhibited iris T-OH development by 60 ~, whereas decentralization at 55 days of age resulted in 32 ~o inhibition (Fig. 5).
Norepinephrine uptake The development of [3H]NE uptake was examined in irides of rats subjected to surgery at different ages. As previously defined, reserpine pretreatment was used to examine uptake and storage functions separately. Decentralization exerted greatest effects when performed in younger animals. In fact, in the absence of reserpine treatment, only decentralization at 3 days of age significantly inhibited development of [3H]NE uptake. However, reserpine treatment, by blocking vesicular storage, unmasked significant inhibition of the development of [3H]NE uptake at every age examined. Nevertheless, the effect was greatest when surgery was performed in younger animals. Decentralization at 3 days reduced [3H]NE uptake by 47 ~o, whereas surgery at 20 days of age or later reduced uptake by only 17 ~o. DISCUSSION The normal maturation of sympathetic neurons is dependent on a number of extracellular factors. Target organs regulate the survival and development of innervating sympathetic nerves 8-1°, and considerable evidence suggests that this influence is mediated, at least in part, by nerve growth factor1°,11,1~. Additionally, anterograde transsynaptic mechanisms also regulate sympathetic ontogeny. Transection of the preganglionic cholinergic fibers innervating the SCG prevents the normal development of adrenergic perikarya within the ganglion as well as terminals within target structures 2-5, 19. For example, ganglion decentralization prevents the developmental accumulation of T-OH molecules in neuronal cell bodies ~ and the ontogenetic increase in nerve terminal numbers and T-OH activity in target organs 3. Our present studies focus on transsynaptic regulation of axon terminal maturation, and demonstrate that different subcellular structures within terminals are affected differently by decentralization. Decentralization inhibited the developmental increase in nerve terminal density within the iris by approximately 50 ~ (Fig. 1). However, the nerve terminal varicosities, which contain the noradrenergic storage vesicles, actually appeared larger and more brightly fluorescent in decentralized terminals by fluorescence microscopy (Fig. 1). To more definitively determine whether decentralization affected varicosities and the remainder of the axon terminals differently, we compared T-OH and DBH activities. T-OH is a supernatant enzyme, present throughout the nerve terminal cytoplasm. By contrast, DBH is bound to the noradrenergic storage vesicle membrane, and thus constitutes a vesicle and varicosity marker. Decentralization prevented the developmental rise of T-OH activity, remaining at approximately 40 ~ of normal. DBH activity developed normally, consistent with our morphologic observations, suggesting that
267 vesicular development was normal. Since nerve terminal numbers were markedly reduced by decentralization, these observations suggest that the vesicle population was increased in each decentralized nerve terminal. More generally, our results suggest that different portions of nerve terminals are regulated differently by transsynaptic factors. The functional significance of these developmental alterations was studied by examining the uptake and storage of exogenous neurotransmitters. NE is concentrated by the high-affinity uptake process of axon terminal membranes, and is stored within vesicles12,17. The two processes, uptake and storage, were examined independently after decentralization by using reserpine as described under Results. Apparent NE uptake was reduced by 76 ~ of normal in decentralized iris terminals. However, this measure is a summation of both uptake and storage. Elimination of the storage component by reserpine treatment indicated that high-affinity uptake was actually reduced to 54 ~o of normal in decentralized terminals (Fig. 3). The magnitude of this effect is entirely consistent with the degree of inhibition of ground plexus ramification (Fig. 1) and of inhibition of T-OH development (Fig. 2), suggesting that axon terminal membrane was reduced by approximately half after decentralization. Conversely, these results suggest that vesicular storage function is increased in decentralized terminals. This impression was confirmed by noting that in vitro retention of exogenous NE was actually increased in decentralized terminals (Fig. 4). It may be inferred that transsynaptic factors exert a profound effect on nerve terminal development and relatively little effect on vesicle development. Alternatively, reduced impulse traffic consequent to decentralization may radically alter the kinetics of vesicle turnover, favoring vesicle accumulation in terminals. Recent studies in adult rats suggest that manipulations which alter nerve impulse frequency affect different vesicle populations differently18. However, additional studies are required to determine whether decentralization actually alters vesicle numbers, or simply alters DBH activity and storage capacity within the same number of vesicles. Regardless of underlying mechanisms, our observations suggest that development of different subcellular structures within axon terminals are subject to different regulatory controls. Lastly, our studies indicate that transsynaptic regulation of terminal maturation is most critical in the perinatal period. Decentralization profoundly inhibits the ontogeny ofT-OH and [3H]NE uptake when performed during the first few postnatal days, whereas surgery at 50-60 days has a significant, but smaller effect. Consequently, there does appear to be a critical period during which transsynaptic regulation is necessary for normal terminal maturation. ACKNOWLEDGEMENTS We thank Dorothy Dembiec for providing the aluminum hydroxide analyses and Ms. Maria C. Papaconstantinou, Ms. Dahna Boyer and Ms. Elise Grossman for excellent technical assistance. This work was supported by NIH Grants NS 11631, 10259 and aided by a grant from the Dysautonomia Foundation, Inc. I.B.B. is the recipient of the Irma T. Hirschl Career Scientist Award.
268 REFERENCES 1 Black, I. B., Increased tyrosine hydroxylase activity in frontal cortex and cerebellum after reserpine, Brain Research, 95 (1975) 170-176. 2 Black, 1. B. and Geen, S. C., Inhibition of the biochemical and morphological maturation of adrenergic neurons by nicotinic receptor blockade, J. Neurochem., 22 (1974) 301-306. 3 Black, I. B. and Mytilineou, C., Trans-synaptic regulation of the development of end organ innervation by sympathetic neurons, Brain Research, 101 (1976) 503-52l. 4 Black, I. B., Hendry, I. A. and Iversen, L. L., Trans-synaptic regulation of growth and development of ad~energic neurons in a mouse sympathetic ganglion, Brain Research, 34 (1971) 229-240. 5 Black, I. B., Hendry, I. A. and Iversen, L. L., Effects of surgical decentralization and nerve growth factor on the maturation of adrenergic neurons in a mouse sympathetic ganglion, J. Neurochem., 19 (1972) 1367-1377. 6 Black, I. B., Joh, T. H. and Reis, D. J., Accumulation of tyrosine hydroxylase molecules during growth and development of the superior cervical ganglion, Brain Research, 75 (1974) 133-144. 7 Dembiec, D. and Cohen, G., The effect ofcarbonyl-binding agents on the potassium-evoked release of [3H]catecholamines from brain and peripheral tissues, J. Neurochem., 28 (1977) 1125-1128. 8 Dibner, M. D. and Black, 1. B., The effect of target organ removal on the development of sympathetic neurons, Brain Research, 103 (1976) 93-102. 9 Dibner, M. D., Mytilineou, C. and Black, I. B., Target organ regulation of sympathetic neuron development, Brain Research, 123 (1977) 301-310. 10 Hendry, I.A. and lversen, L. L., Changes in tissue and plasma concentrations of nerve growth factor following removal of the submaxillary glands in adult mice and their effects on the sympathetic nervous system, Nature (Lond.), 243 (1973) 500-504. 11 Hendry, I. A., Stbckel, K., Thoenen, H. and lversen, L. L., The retrograde axonal transport ofnerve growth factor, Brain Research, 68 (1974) 103-121. 12 Iversen, L. L., The Uptake and Storage of Noradrenaline in Sympathetic Nerves, Cambridge Univ. Press, London, 1967. 13 Joh, T. H., Ross, R. A. and Reis, D. J., A simple and sensitive assay for dopamine-fl-hydroxylase, Analyt. Biochem., 62 (1974) 248-254. 14 Jonsson, G., Hamberger, B., Malmfors, T. and Sachs C., Uptake and accumulation of [3H]noradrenaline in adrenergic nerves of rat iris. Effect of reserpine, monoamine oxidase and tyrosine hydroxylase inhibition, Europ. J. Pharmacol., 8 (1969) 58-72. 15 Levi-Montalcini, R. and Angeletti, P. U., Nerve growth factor, Physiol. Rev., 48 (1968) 534-569. 16 Malmfors, T., Studies on adrenergic nerves: the use of rat and mouse iris for direct observations on their physiology and pharmacology at cellular and subcellular levels, Acta physiol, scand., 64, Suppl. 248 (1965) 1-93. 17 Molinoff, P. B. and Axelrod, J., Biochemistry of catecholamines, Ann. Rev. Biochem., 40 (1971) 465-500. 18 Nelson, D. L. and Molinoff, P. B., Differential effects of nerve impulses on adrenergic storage vesicles in rat heart, J. Pharmacol. exp. Ther., 198 (1976) 112-122. 19 Thoenen, H., Saner, A., Kettler, R. and Angeletti, P. U., Nerve growth factor and preganglionic cholinergic nerves; their relative importance to the development of the terminal adrenergic neurone, Brain Research, 44 (1972) 593-602.
259
DEVELOPMENT OF ADRENERGIC NERVE TERMINALS: THE EFFECTS OF DECENTRALIZATION
CATHERINE MYTILINEOUand IRA B. BLACK Department of Neurology, Mount Sinai School of Medicine, New York, N. Y. 10029 and Department of Neurology, Cornell University Medical College, New York, N.Y. 10021 (U.S.A.)
(Accepted March 30th, 1978)
SUMMARY The effect of decentralization (deafferentation) on the ontogeny of adrenergic nerve terminals was studied in the rat iris. Transection of the cholinergic trunk, which innervates the superior cervical ganglion (SCG), in 2-3-day-old rats, inhibited the developmental increase in iris nerve terminal density, as indicated by fluorescence microscopy. However, terminal varicosities, containing the norepinephrine (NE) storage vesicles, appeared larger and more brightly fluorescent in decentralized irides. Tyrosine hydroxylase, localized to nerve terminal cytoplasm, developed to only 40 ~ of normal in decentralized irides. In contrast, dopamine-fl-hydroxylase, which is localized to NE storage vesicles in varicosities, developed normally. The ability to accumulate and store [SH]NE was examined in control and decentralized terminals in the presence or absence of reserpine. This drug inhibits NE storage capacity without primarily affecting accumulation itself. Decentralization reduced the in vitro uptake of NE to 54 ~o, measured after reserpine pretreatment; in contrast, apparent uptake of transmitter was reduced to only 76 ~o in vehicle-treated rats. These results suggest that decentralization prevents the normal development of nerve terminal membrane without markedly interfering with vesicular storage capacity. This contention was supported by the observation that in vitro retention of [3H]NE over time was actually increased in decentralized irides. Our results suggest that the ontogeny of different subcellular structures is affected differently by deafferentation. The transsynaptic regulation of nerve terminal maturation appears to be most critical in younger rats, since decentralization exerts most marked effects when performed during the perinatal period.
INTRODUCTION The normal growth and maturation of peripheral sympathetic neurons requires
260 intact preganglionic innervation2-4,19. Decentralization (denervation) of the superior cervical ganglion (SCG) shortly after birth, or treatment with ganglionic blocking agents, prevents the normal ontogenetic increase in the activities of tyrosine hydroxylase (T-OH) and dopamine-fl-hydroxylase (DBH) in adrenergic neurons2,4,5,19. These enzymes are involved in the biosynthesis of norepinephrine (NE) 17, and are localized to adrenergic perikarya of the SCG 17. We have recently demonstrated that decentralization of the SCG prevents normal development of the adrenergic nerve terminals in target organs s, as well as interfering with maturation of ganglion cell bodies. The normal growth of the adrenergic nerve terminals in the iris was prevented by ganglion decentralization in 2-3-day-old rats s. T-OH activity, localized to adrenergic terminals of the iris, remained depressed by 60 in irides innervated by decentralized ganglia s . Moreover, fluorescence microscopy demonstrated that the number of adrenergic nerve terminals present in the iris and their degree of branching was markedly reduced by ganglion decentralization. In contrast, the in vitro uptake of [SH]NE, a quantitative measure of functional nerve terminal membrane 14, was depressed by only 20 ~ in irides innervated by decentralized ganglia s. Observations made by fluorescence microscopy also suggested that fibers emanating from decentralized ganglia were qualitatively altered. The present report describes the neurochemical properties of decentralized adrenergic nerve terminals in greater detail. Our studies confirm the observation that decentralization prevents the normal ontogenetic increase in the number of nerve terminals innervating the iris. However, although ganglion decentralization prevents the normal development of iris T-OH activity, iris DBH develops normally. Furthermore, the relative capacity of the nerve fibers to store [SH]NE actually increases in the decentralized iris. Lastly, we report that the effect of decentralization on nerve terminal development is critically related to the age of the animal at the time of surgery. METHODS
Enzyme assays T-OH and DBH activities were assayed in irides as previously described1,13.
[SH] Norepinephrine uptake Irides were removed under a dissecting microscope and placed in ice-cold KrebsRinger phosphate buffer containing per ml: glucose, 1.0 mg; EDTA, 0.5 mg; ascorbate, 0.2 rag; pargyline, 0.16 mg. After a 10 min equilibration at 37 °C [3H]NE was added to a final concentration of 10 7 M and the tissues were incubated with shaking for 15 min. Then the tissues were washed in fresh buffer for 15 more minutes and transferred to vials containing 3 ml 100 ~o ethanol and heated at 60 °C for 1 h. After addition of 10 ml Bray's solution the vials were counted for radioactivity in a liquid scintillation spectrometer (Packard Tri-Carb 2450).
Spontaneous release of [3H] NE Irides innervated by intact and decentralized ganglia were removed and incubated
261 with [3H]NE (10-7 M) for 30 min in the absence of pargyline. After a 15 rain wash, individual irides were transferred to the wells of a tissue culture plate which contained 1 ml of fresh buffer at 37 °C. At 5 min intervals the irides were transferred to the next well. The plate contained 4 rows of 6 wells each so that the spontaneous release of [3H]NE was studied, using 4 irides each for 30 min period. At the end of the 30 min the irides were transferred to vials containing 3 ml of 100 ~ ethanol for determination of radioactivity as described above. In addition, 0.5 ml buffer was collected from each well, added to a vial containing 10 ml Bray's solution, and subjected to scintillation spectroscopy.
Fluorescence histochemistr y The irides were isolated and prepared for uptake of a-methyl-NE as described above, except that pargyline was not added to the buffer. The final concentration of a-methyl-NE in the medium was 10-5 M, and the incubation period was 30 min. After a wash the irides were stretched on glass slides and left to dry overnight in a dessicator containing P~Os. They were then exposed to formaldehyde vapor (70 ~o relative humidity) for 1 h at 80 °C 16. They were observed and photographed by a Leitz Ortholux fluorescence photomicroscope. Statistical analyses Grouped data were analyzed using Student's t-test and paired data were analyzed with the paired t-test. RESULTS
Effect of ganglion decentralization on the morphological development of iris innervation Fluorescence microscopy of irides was performed to evaluate the effect of neonatal ganglion decentralization on the development of adrenergic nerve terminals. To aid in the visualization of all adrenergic axon terminals, irides were preincubated with a-methyl-NE. This compound is concentrated in adrenergic neurons by the same uptake process as NE, but is resistant to metabolism by monoamine oxidase. Consequently, virtually all adrenergic terminals, regardless of endogenous amine stores, are rendered visible, allowing more detailed morphologic observations. Irides of 8-week-old rats innervated by ganglia decentralized at 3 days of age exhibited marked reductions in the density of the adrenergic ground plexus, in agreement with previous results3. In addition, qualitative differences in the pattern of innervation were evident at higher magnification. In the decentralized iris the nerve plexus consisted predominantly of single nerve fibers, whereas fibers tended to run in pairs in normal irides (Fig. la, arrows). Moreover, the terminal varicosities appeared larger and more brightly fluorescent in decentralized irides (Fig. lb, arrows). However, it was not possible by fluorescence microscopy to determine whether the varicosities were in fact larger, or simply appeared larger due to increased fluorescence intensity. Effect of decentralization on the neurochemical development of iris terminals To determine whether the development of decentralized adrenergic terminals
262
Fig. 1. Stretch preparations of irides from a rat 8 weeks after SCG decentralization at 3 days of age. The irides were incubated in 10 5 M a-methyl norepinephrine prior to treatment for catecbolamine histofluorescence; a: normal iris. A rich adrenergic plexus is present with many fibers running in bundles of 2 or more (arrows). b: decentralized iris. The nerve fibers are fewer with less branching. Some large brightly fluorescent varicosities can be seen (arrows). × 300.
exhibited neurochemical as well as morphologic abnormalities, catecholamine biosynthetic enzymes were examined. In agreement with previous results 3, T-OH activity, which is localized to the supernatant fraction of nerve terminals 17, was 40 ~ of normal in irides 3 weeks after decentralization (Fig. 2). In contrast, the activity of DBH, which is localized to noradrenergic storage vesicles 17, was normal in decentralized irides (Fig. 2). Since these observations suggested that the development of vesicles and nerve terminal density may be affected differently by decentralization, we studied the uptake and storage of NE.
Uptake and storage of norepinephrine The in vitro uptake of [3H]NE by normal and contralateral decentralized irides was studied 3 weeks after neonatal ganglion decentralization. To define the functions of membrane uptake and vesicular storage separately, one group of animals was pretreated with reserpine, and littermates received vehicle. Reserpine interferes with vesicular storage (retention) of N E without altering the high-affinity uptake process itself 12,17. The experimental protocol resulted in 4 sets of irides: (a) vehicle, intact; (b) vehicle, decentralized; (c) reserpine, intact; (d) reserpine, decentralized. In vehicletreated rats, decentralization reduced [3H]NE uptake to 76 ~o of that in contralateral control, intact irides (Fig. 3). However, after reserpine treatment, which removed the
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Fig. 2. Effect of ganglion decentralization on iris enzyme activities. Eight rats aged 3 days were subjected to unilateral decentralization of the superior cervical ganglion; 3 weeks postoperatively ipsilateral decentralized irides and contralateral control irides were removed and enzyme activities were assayed. Tyrosine hydroxylase activity is expressed as pmole/iris/h 4- S.E.M. (vertical bars) and dopamine-flhydroxylase as nmole/iris/h. * Differs from respective control at P < 0.001. The dopamine-fl-hydroxylase groups do not differ significantly (P > 0.05). vesicular storage component of the apparent uptake, decentralized irides accumulated only 54 ~o of the [aH]NE in contralateral controls (Fig. 3). Reserpinization reduced the uptake of [3H]NE by 20 ~ in the control irides and by 44 ~ in the decentralized irides. Thus, decentralized iris terminals apparently have reduced capacity to take up transmitter, but a relatively higher storage capacity. Terminal storage capacity was also evaluated by examining in vitro retention of exogenous [aH]NE. Decentralized terminals retained more of the previously taken up amine at all times examined (Fig. 4). To determine whether the [aH]NE was metabolized differently in control and decentralized iris terminals, non-catechol metabolites were measure&. There were no significant differences between the groups, non-catechol metabolites constituting 2.4 :~ 0.09 ~ of total radioactivity in the controls, and 2.6 40.22 ~ in the decentralized irides (P > 0.05).
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Fig. 3. Effect of reserpine administration on the uptake of [3H]norepinephrine by normal and decentralized rat irides. Animals were decentralized at 2-3 days of age and the accumulation of [3H]norepinephrine was measured as indicated under Methods; 3 weeks postoperatively one group of 6 animals received 10 mg/kg reserpine i.p. 20 h before the experiment. The controls (6 animals) received an equal volume of vehicle. Each value represents mean counts/min × 10 3/iris ± S.E.M. * Differs from control at P < 0 . 0 0 1 .
Effect of ganglion decentralization at different ages Fluorescence microscopy. To determine whether there is a critical period during which transsynaptic factors regulate terminal development, ganglia were decentralized in rats of different ages. Control and contralateral decentralized irides were examined by fluorescence microscopy after incubation with a-methyl-NE, as described above. Decentralization at 3 days of age resulted in abnormal development of the adrenergic ground plexus (Fig. 1). Similar abnormalities were apparent in irides of animals undergoing surgery at 10 days of age. However, decentralization in older rats resulted in no discernible alteration of terminal development as observed by fluorescence microscopy.
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Fig. 4. Effect of decentralization on the retention of previously accumulated [aH]NE by the iris. The irides from 8 rats, 7 weeks after unilateral SCG decentralization performed at 3 days of age, were processed for [aH]NE uptake as described under Methods. The NE retained by the decentralized and control irides over a 30 min wash period is expressed as per cent of the radioactivity (CPM/iris -t- S.E.M.) present in each iris at the beginning of the experiment. * Differs from respective control, P < 0.05 ; • * Differs from respective control, P < 0.025; *=* Differs from respective control, P < 0.005.
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Fig. 5. Effect of ganglion decentralization at different ages. Groups of 6-8 rats were subjected to unilateral ganglion decentralization at the indicated ages. Tyrosine hydroxylase activity was assayed in the ipsilateral iris and the contralateral control iris in each animal 21 days postoperatively. Enzyme activity is expressed as pmole/iris/h 4- S.E.M. (vertical bars). * Differs from respective control by paired t-test at P < 0.01.
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Fig. 6. Effect of decentralization at different ages on the uptake of [3H]NE by the irides. SCG from groups of 12 animals were unilaterally decentralized at various postnatal ages and the [aH]NE uptake by the normal and decentralized iris was determined 3 weeks after the operation as described under Methods. 6 animals from each age group received 10 mg/kg reserpine i.p. 20 h before the experiment. Points represent CPM/iris 4- S.E.M. expressed as per cent of the contralatelal control iris. * Differs from the respective control at P < 0.05.
266
Tyrosine hydroxylase Ganglion decentralization had a more pronounced effect on development of iris T-OH activity when performed in younger animals (Fig. 5). Surgery was performed in rats of varying ages and irides were examined 3 weeks thereafter. Decentralization during the first postnatal weeks inhibited iris T-OH development by 60 ~, whereas decentralization at 55 days of age resulted in 32 ~o inhibition (Fig. 5).
Norepinephrine uptake The development of [3H]NE uptake was examined in irides of rats subjected to surgery at different ages. As previously defined, reserpine pretreatment was used to examine uptake and storage functions separately. Decentralization exerted greatest effects when performed in younger animals. In fact, in the absence of reserpine treatment, only decentralization at 3 days of age significantly inhibited development of [3H]NE uptake. However, reserpine treatment, by blocking vesicular storage, unmasked significant inhibition of the development of [3H]NE uptake at every age examined. Nevertheless, the effect was greatest when surgery was performed in younger animals. Decentralization at 3 days reduced [3H]NE uptake by 47 ~o, whereas surgery at 20 days of age or later reduced uptake by only 17 ~o. DISCUSSION The normal maturation of sympathetic neurons is dependent on a number of extracellular factors. Target organs regulate the survival and development of innervating sympathetic nerves 8-1°, and considerable evidence suggests that this influence is mediated, at least in part, by nerve growth factor1°,11,1~. Additionally, anterograde transsynaptic mechanisms also regulate sympathetic ontogeny. Transection of the preganglionic cholinergic fibers innervating the SCG prevents the normal development of adrenergic perikarya within the ganglion as well as terminals within target structures 2-5, 19. For example, ganglion decentralization prevents the developmental accumulation of T-OH molecules in neuronal cell bodies ~ and the ontogenetic increase in nerve terminal numbers and T-OH activity in target organs 3. Our present studies focus on transsynaptic regulation of axon terminal maturation, and demonstrate that different subcellular structures within terminals are affected differently by decentralization. Decentralization inhibited the developmental increase in nerve terminal density within the iris by approximately 50 ~ (Fig. 1). However, the nerve terminal varicosities, which contain the noradrenergic storage vesicles, actually appeared larger and more brightly fluorescent in decentralized terminals by fluorescence microscopy (Fig. 1). To more definitively determine whether decentralization affected varicosities and the remainder of the axon terminals differently, we compared T-OH and DBH activities. T-OH is a supernatant enzyme, present throughout the nerve terminal cytoplasm. By contrast, DBH is bound to the noradrenergic storage vesicle membrane, and thus constitutes a vesicle and varicosity marker. Decentralization prevented the developmental rise of T-OH activity, remaining at approximately 40 ~ of normal. DBH activity developed normally, consistent with our morphologic observations, suggesting that
267 vesicular development was normal. Since nerve terminal numbers were markedly reduced by decentralization, these observations suggest that the vesicle population was increased in each decentralized nerve terminal. More generally, our results suggest that different portions of nerve terminals are regulated differently by transsynaptic factors. The functional significance of these developmental alterations was studied by examining the uptake and storage of exogenous neurotransmitters. NE is concentrated by the high-affinity uptake process of axon terminal membranes, and is stored within vesicles12,17. The two processes, uptake and storage, were examined independently after decentralization by using reserpine as described under Results. Apparent NE uptake was reduced by 76 ~ of normal in decentralized iris terminals. However, this measure is a summation of both uptake and storage. Elimination of the storage component by reserpine treatment indicated that high-affinity uptake was actually reduced to 54 ~o of normal in decentralized terminals (Fig. 3). The magnitude of this effect is entirely consistent with the degree of inhibition of ground plexus ramification (Fig. 1) and of inhibition of T-OH development (Fig. 2), suggesting that axon terminal membrane was reduced by approximately half after decentralization. Conversely, these results suggest that vesicular storage function is increased in decentralized terminals. This impression was confirmed by noting that in vitro retention of exogenous NE was actually increased in decentralized terminals (Fig. 4). It may be inferred that transsynaptic factors exert a profound effect on nerve terminal development and relatively little effect on vesicle development. Alternatively, reduced impulse traffic consequent to decentralization may radically alter the kinetics of vesicle turnover, favoring vesicle accumulation in terminals. Recent studies in adult rats suggest that manipulations which alter nerve impulse frequency affect different vesicle populations differently18. However, additional studies are required to determine whether decentralization actually alters vesicle numbers, or simply alters DBH activity and storage capacity within the same number of vesicles. Regardless of underlying mechanisms, our observations suggest that development of different subcellular structures within axon terminals are subject to different regulatory controls. Lastly, our studies indicate that transsynaptic regulation of terminal maturation is most critical in the perinatal period. Decentralization profoundly inhibits the ontogeny ofT-OH and [3H]NE uptake when performed during the first few postnatal days, whereas surgery at 50-60 days has a significant, but smaller effect. Consequently, there does appear to be a critical period during which transsynaptic regulation is necessary for normal terminal maturation. ACKNOWLEDGEMENTS We thank Dorothy Dembiec for providing the aluminum hydroxide analyses and Ms. Maria C. Papaconstantinou, Ms. Dahna Boyer and Ms. Elise Grossman for excellent technical assistance. This work was supported by NIH Grants NS 11631, 10259 and aided by a grant from the Dysautonomia Foundation, Inc. I.B.B. is the recipient of the Irma T. Hirschl Career Scientist Award.
268 REFERENCES 1 Black, I. B., Increased tyrosine hydroxylase activity in frontal cortex and cerebellum after reserpine, Brain Research, 95 (1975) 170-176. 2 Black, 1. B. and Geen, S. C., Inhibition of the biochemical and morphological maturation of adrenergic neurons by nicotinic receptor blockade, J. Neurochem., 22 (1974) 301-306. 3 Black, I. B. and Mytilineou, C., Trans-synaptic regulation of the development of end organ innervation by sympathetic neurons, Brain Research, 101 (1976) 503-52l. 4 Black, I. B., Hendry, I. A. and Iversen, L. L., Trans-synaptic regulation of growth and development of ad~energic neurons in a mouse sympathetic ganglion, Brain Research, 34 (1971) 229-240. 5 Black, I. B., Hendry, I. A. and Iversen, L. L., Effects of surgical decentralization and nerve growth factor on the maturation of adrenergic neurons in a mouse sympathetic ganglion, J. Neurochem., 19 (1972) 1367-1377. 6 Black, I. B., Joh, T. H. and Reis, D. J., Accumulation of tyrosine hydroxylase molecules during growth and development of the superior cervical ganglion, Brain Research, 75 (1974) 133-144. 7 Dembiec, D. and Cohen, G., The effect ofcarbonyl-binding agents on the potassium-evoked release of [3H]catecholamines from brain and peripheral tissues, J. Neurochem., 28 (1977) 1125-1128. 8 Dibner, M. D. and Black, 1. B., The effect of target organ removal on the development of sympathetic neurons, Brain Research, 103 (1976) 93-102. 9 Dibner, M. D., Mytilineou, C. and Black, I. B., Target organ regulation of sympathetic neuron development, Brain Research, 123 (1977) 301-310. 10 Hendry, I.A. and lversen, L. L., Changes in tissue and plasma concentrations of nerve growth factor following removal of the submaxillary glands in adult mice and their effects on the sympathetic nervous system, Nature (Lond.), 243 (1973) 500-504. 11 Hendry, I. A., Stbckel, K., Thoenen, H. and lversen, L. L., The retrograde axonal transport ofnerve growth factor, Brain Research, 68 (1974) 103-121. 12 Iversen, L. L., The Uptake and Storage of Noradrenaline in Sympathetic Nerves, Cambridge Univ. Press, London, 1967. 13 Joh, T. H., Ross, R. A. and Reis, D. J., A simple and sensitive assay for dopamine-fl-hydroxylase, Analyt. Biochem., 62 (1974) 248-254. 14 Jonsson, G., Hamberger, B., Malmfors, T. and Sachs C., Uptake and accumulation of [3H]noradrenaline in adrenergic nerves of rat iris. Effect of reserpine, monoamine oxidase and tyrosine hydroxylase inhibition, Europ. J. Pharmacol., 8 (1969) 58-72. 15 Levi-Montalcini, R. and Angeletti, P. U., Nerve growth factor, Physiol. Rev., 48 (1968) 534-569. 16 Malmfors, T., Studies on adrenergic nerves: the use of rat and mouse iris for direct observations on their physiology and pharmacology at cellular and subcellular levels, Acta physiol, scand., 64, Suppl. 248 (1965) 1-93. 17 Molinoff, P. B. and Axelrod, J., Biochemistry of catecholamines, Ann. Rev. Biochem., 40 (1971) 465-500. 18 Nelson, D. L. and Molinoff, P. B., Differential effects of nerve impulses on adrenergic storage vesicles in rat heart, J. Pharmacol. exp. Ther., 198 (1976) 112-122. 19 Thoenen, H., Saner, A., Kettler, R. and Angeletti, P. U., Nerve growth factor and preganglionic cholinergic nerves; their relative importance to the development of the terminal adrenergic neurone, Brain Research, 44 (1972) 593-602.