Developmental interactions between sweat glands and the sympathetic neurons which innervate them: Effects of delayed innervation on neurotransmitter plasticity and gland maturation

DEVELOPMENTAL

BIOLOGY

130,703-720 (1988)

Developmental Interactions between Sweat Glands and the Sympathetic Neurons Which Innervate Them: Effects of Delayed Innervation on Neurotransmitter Plasticity and Gland Maturation LESLIE M. STEVENS AND STORY C. LANDIS Department of Neurobiobg~, Harvard Medical School, Boston, Massachusetts 02115;and Center for Neurosciences, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106 Accepted August 22, 1988 The neurotransmitter properties of the sympathetic innervation of sweat glands in rat footpads have previously been shown to undergo a striking change during development. When axons first reach the developing glands, they contain catecholamine histofluorescence and immunoreactivity for catecholamine synthetic enzymes. As the glands and their innervation mature, catecbolamines disappear and cholinergic and peptidergic properties appear. Final maturation of the sweat glands, assayed by secretory competence, is correlated temporally with the development of cholinergic function in the innervation. To determine if the neurotransmitter phenotype of sympathetic neurons developing in viva is plastic, if sympathetic targets can play a role in determining neurotransmitter properties of the neurons which innervate them, and if gland maturation is dependent upon its innervation, the normal developmental interaction between sweat glands and their innervation was disrupted. This was accomplished by a single injection of B-hydroxydopamine (6-OHDA) on Postnatal Day 2. Following this treatment, the arrival of noradrenergic sympathetic axons at the developing glands was delayed 7 to 10 days. Like the gland innervation of normal rats, the axons which innervated the sweat glands of 6-OHDA-treated animals acquired cholinergic function and their expression of endogenous catecholamines declined. The change in neurotransmitter properties, however, occurred later in development than in untreated animals and was not always complete. Even in adult animals, some fibers continued to express endogenous catecholamines and many nerve terminals contained a small proportion of small granular vesicles after permanganate fixation. The gland innervation in the 6OHDA-treated animals also differed from that of normal rats in that immunoreactivity for VIP was not expressed in the majority of glands. It seems likely that following treatment with 6-OHDA sweat glands were innervated both by neurons that would normally have done so and by neurons that would normally have innervated other, noradrenergic targets in the footpads, such as blood vessels. Contact with sweat glands, therefore, appears to suppress noradrenergic function and induce cholinergic function not only in the neurons which normally innervate the glands but also in neurons which ordinarily innervate other targets. Effects of delayed innervation were also observed on target development. The appearance of sensitivity to cholinergic agonists by the sweat glands was coupled with the onset of cholinergie transmission. Our results provide evidence for reciprocal developmental interactions between the sweat glands and their innervation which permit the establishment of functional transIt&.SiOn.

Q 1988 Academic

Press, Inc.

in phenotype and allowed a description of several factors that can influence the transition in vitro (for reThe developmental mechanisms which determine the views see Patterson, 19’78;Bunge et al., 1978; Black et al., final neurotransmitter phenotype of peripheral 1984; Potter et al., 1986). neurons are not well understood. The cholinergic symThe neurotransmitter-related properties of the sweat pathetic innervation of rat sweat glands has proven to gland innervation change in a striking manner during be an interesting system in which to study neurotransdevelopment. The developing sweat glands first become mitter choice for several reasons. First, cholinergic innervated on Postnatal Day 4 by catecholamine-conneurons form a minority population of sympathetic taining fibers. At 10 days the fibers exhibit intense catneurons, the majority of which are noradrenergic. Sec- echolamine fluorescence (Landis and Keefe, 1983), ond, the fibers which innervate the glands undergo a strong immunoreactivity for tyrosine hydroxylase, the transition in transmitter status from noradrenergic to rate-limiting enzyme in the synthesis of norepinephrine cholinergic during postnatal development (Landis and (Landis et ah, 1988), and small granular vesicles after Keefe, 1983; Landis et aZ., 1988; Leblanc and Landis, fixation with potassium permanganate indicating ve1986). Finally, previous studies of sympathetic neurons sicular stores of norepinephrine (Landis and Keefe, developing in culture had documented a similar change 1983). By 21 days, however, the gland innervation is INTRODUCTION

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DEVELOPMENTALBIOLOGYV0~~~~130,1988

almost devoid of detectable catecholamine fluorescence and the vesicles in gland-associated terminals are clear after permanganate fixation (Landis and Keefe, 1983). Concurrently, the gland innervation begins to express choline acetyltransferase, the synthetic enzyme for acetylcholine; activity is first detectable at Day 11 and increases fourfold during the third postnatal week (Leblanc and Landis, 1986). The onset of the secretory response, which is cholinergically mediated, occurs in parallel with the development of choline acetyltransferase activity but is slightly delayed (Stevens and Landis, 1987). The close temporal correlation between the appearance of cholinergic properties in the innervation and the onset of secretory responsiveness coupled with the failure of secretory competence to develop in chronically sympathectomized rats has led to the suggestion that the development of cholinergic function in the innervation is critical for the final maturation of the glands. Immunoreactivities for two neuropeptides, vasoactive intestinal peptide (VIP) (Lundberg et aZ., 1979; Yodlowski et aL, 1984; Landis et aZ.,1988) and calcitonin gene-related peptide (CGRP) (Landis and Fredieu, 1986), are present in the gland innervation of mature rats. VIP immunoreactivity becomes detectable at approximately the same time as cholinergic function, while CGRP immunoreactivity appears about 1 week later (Landis et ah, 1988). Examination of the effects of multiple treatments with 6-hydroxydopamine (6OHDA), a toxic catecholamine congener, during the first postnatal week provides evidence that the change in neurotransmitter properties from noradrenergic to cholinergic that occurs in the developing sweat gland innervation is due to changes in a single population of fibers rather than to replacement of early noradrenergic fibers by later arriving cholinergic axons (Yodlowski et ah, 1984; Leblanc and Landis, 1986). A similar transition in neurotransmitter phenotype has been described in sympathetic neurons developing in culture, and several factors that influence which transmitter the neurons will synthesize in culture have been identified. When adrenergic sympathetic neurons from neonatal rats are grown in the absence of other cell types (Mains and Patterson, 1973), in defined medium (Iacovitti et uL, 1982; Wolinsky et al, 1985), or under conditions in which they are chronically depolarized (Walicke et al., 197’7;Walicke and Patterson, 1981; Hefti et aL, 1982; Raynaud et ak, 1987), they continue to differentiate noradrenergically. If, however, the neurons are cultured with certain nonneuronal cells (Patterson and Chun, 1974) or in medium conditioned by these cells (Patterson et aZ,, 1975; Patterson and Chun, 1977a,b; Kessler, 1984, 1985), the neurons lose their noradrenergic properties and differentiate cholinergically. In particular, heart cell conditioned me-

dium contains a protein of molecular weight 45,000 which has recently been purified lOO,OOO-fold and shown to be capable of simultaneously suppressing noradrenergic properties and inducing cholinergic function in cultured sympathetic neurons (Fukada, 1985). Similar activities have been detected in medium conditioned by rat skeletal muscle (Swerts et al., 1983; Weber et uZ., 1985), in human placental serum and chick embryo extract (Johnson et ah, 1980a; Iacovitti et al, 1981), in brain extracts (Kessler et al., 1986), and in rat serum (Wolinsky et al., 1985) where it appears to be developmentally regulated (Wolinsky and Patterson, 1985). More recently, it has been shown that cholinergic differentiation is promoted by growing the neurons in high density cultures (Adler and Black, 1985; Kessler, 1985), an effect which seems to be mediated by a membrane-associated molecule (Adler and Black, 1986; Kessler et ah, 1986; Wong and Kessler, 1987). The finding that the transmitter phenotype of sympathetic neurons growing in culture is plastic and can be influenced by environmental factors raises the possibility that the transmitter phenotype of sympathetic neurons in viva may be similarly regulated. For the cholinergic sympathetic neurons that innervate sweat glands, one obvious candidate for the source of such a factor is their target, the sweat glands. To determine whether the developing sweat gland innervation is plastic and to test the hypothesis that the sweat glands promote a change from noradrenergic to cholinergic function in the neurons which innervate them, we have interfered with the normal developmental interaction between the sweat glands and their innervation by delaying the growth of sympathetic fibers into the glands. A delay of 7 to 10 days in the onset of sympathetic innervation was achieved by treating rats with a single dose of the adrenergic neurotoxin 6-OHDA on Postnatal Day 2. We found that the sympathetic fibers which innervated the glands after a delay underwent a change in neurotransmitter properties which was both delayed relative to its occurrence in untreated animals, and incomplete in terms of the suppression of catecholamine expression and the extent of cholinergic induction. In addition to effects on the developing innervation, delay in the innervation of the sweat glands and in the appearance of cholinergic function delayed the acquisition of secretory competence by the sweat glands. Our findings indicate that the transmitter phenotype of the developing sweat gland innervation is plastic, are consistent with the hypothesis that the target can influence the neurotransmitter properties of sympathetic neurons developing in vivo, and suggest that complex developmental interactions can exist between neurons and their targets for establishment of functional connections.

STEVENS AND LANDIS MATERIALS

AND

Developmental

Interactims

METHODS

Animals

Litters were born and shipped the same day from Charles River (CD strain, Wilmington, MA). The day of arrival was counted as 0. On Day 2, rat pups were injected subcutaneously with 100 mg/kg 6-OHDA (Sigma Chemical Co., St. Louis, MO) in 0.9% sodium chloride with 0.5 mg/ml sodium ascorbate to retard oxidation of the drug. Control animals were either untreated or injected with vehicle alone. Since no differences were observed in initial studies of the neurotransmitter properties of animals injected with vehicle and untreated rats, most comparisons reported here were made with untreated animals. Morphological

Assays

Glyoxylic acid fluorescence was carried out according to de la Torre (1980). Briefly, animals were killed by carbon dioxide inhalation and rear footpads were frozen in Tissue-Tek on cryostat chucks. Ten-micrometer cryostat sections were picked up on warm glass slides, dipped in a solution containing 1% glyoxylic acid (Sigma Chemical Co.) and 0.2 Msucrose in 0.24 Mpotassium phosphate buffer, pH 7.4, and then dried under a cool blower for at least 15 min. After the sections were dried, they were covered with drops of mineral oil, heated in a 95°C oven for 2.5 min, and coverslipped. For acetylcholinesterase (AChE) localization, lo-pm cryostat sections were cut, picked up on warm glass slides, and fixed for 5 min in 1% paraformaldehyde and 1.25% glutaraldehyde in 0.1 Mphosphate buffer, pH 7.4. The sections were reacted for AChE and staining was intensified with a dilute solution of methylene blue in saline as previously described (Landis and Keefe, 1983). The sections were dehydrated, cleared with xylene, and mounted in Permount. VIP and ~65, a synaptic vesicle antigen (Matthew et ah, 1981; Bixby and Reichardt, 1985), were localized with immunocytochemical techniques. Rats were deeply anesthetized with chloral hydrate and ether and perfused through the heart first with phosphate-buffered saline (PBS) to clear the blood and then with 4% paraformaldehyde in 0.1 Mphosphate buffer, pH 7.4, for 10 min at room temperature. After perfusion, hind footpads were removed and fixed for an additional hour. The tissues were rinsed with 0.1 1M phosphate buffer and then equilibrated overnight with 30% sucrose in 0.1 M phosphate buffer at 4°C. The tissue was then frozen in Tissue-Tek on a cryostat chuck and IO-pm cryostat sections were mounted on gelatin-coated slides. The sections were preincubated for at least 1 hr in an incubation buffer which contained 0.5 M NaCl, 0.1 M phosphate buffer, pH 7.3, 0.2% Triton X-100, 0.1% sodium

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azide, and 5% bovine serum albumin (Sigma) and then incubated overnight in humid chambers at room temperature in incubation buffer containing rabbit antiserum directed against VIP. The antiserum directed against VIP was prepared by injecting rabbits with synthetic porcine VIP (Boehringer-Mannheim) conjugated to bovine serum albumin through a carbodimide linkage. The sections were rinsed with PBS, and then incubated with tetramethylrhodamine isothiocyanate-conjugated goat anti-rabbit immunoglobulin (Tago) in incubation buffer. The sections were rinsed with PBS and then mounted in glycerol:PBS (1:l) and examined with epifluorescence and a rhodamine/fluorescein filter set appropriate for double-label studies. Incubation of the primary antiserum with 10 rig/ml of the appropriate peptide abolished specific staining. In some cases, sections were double-stained for the presence of a synaptic vesicle antigen, P65, as well as the neuropeptide using a mouse monoclonal antibody (Matthew et al, 1981; the kind gift of Dr. W. Matthew). After staining sections with the rabbit antiserum as described above, sections were incubated at room temperature for 30 min in 10% horse serum in PBS, rinsed in PBS, and then incubated at room temperature for at least 1 hr in monoclonal supernatant diluted 1:l with PBS. The sections were rinsed and then incubated with fluorescein-conjugated goat anti-mouse immunoglobulin (Antibodies, Inc.) preabsorbed with 25% rat serum prior to use. In some cases, sections of fresh frozen tissue were stained for ~65. Ten-micrometer cryostat sections were cut, picked up on untreated glass slides, and fixed for 10 min with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The sections were then rinsed in PBS and staining was carried out as described above. Tissue was prepared for electron microscopy following fixation with potassium permanganate to localize vesicular stores of norepinephrine (Richardson, 1966). Rats were killed by carbon dioxide inhalation and rear footpads were removed and placed in a drop of ice-cold 3.5% potassium permanganate in 0.1 M phosphate buffer, pH 7.3. The epidermis and dermis were removed and the remaining tissue was cut into small pieces and fixed for an hour at 4°C. The tissue was rinsed extensively in ice-cold acetate buffer, stained overnight with 1% uranyl acetate in 0.05 M sodium acetate at 4”C, dehydrated, and embedded in Epon 812. Thin sections were cut and examined in the electron microscope without further staining. Sweating Assay

Sweating was assayed by making a mold of the plantar surface with a silicone elastic material (“Syringe Elasticon,” Kerr Co., Romulus, MI) as described by Kennedy et al. (1984). Base material (0.1 to 0.2 ml) was

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mixed with two or three drops of hardener and then applied to the plantar surface. The impression material is immiscible with water, and as it hardens the sweat droplets form pores in the mold. Each pore represents the activity of an individual sweat gland and the number of active glands can be determined by counting the pores using a dissecting microscope. Sweating was evoked either by stimulating the sciatic nerve or by local injection of agonists. Animals were anesthetized with chloral hydrate. The sciatic nerve was exposed and fitted with a Silastic cuff in which two platinum electrodes had been embedded, as described by Sauter et al., (1933). After fitting the cuff, the nerve and cuff were coated with mineral oil. The nerve was stimulated at 15 V at 5.5 Hz with alternating current pulses. At the beginning of an experiment, the nerve was stimulated for a 3-min period to allow the ducts to fill. The paw was then wiped dry, the impression material was applied, and the nerve stimulated for another 2.5 min. After the material hardened, approximately 2-3 min after the cessation of stimulation, the mold was removed by grasping an end with forceps and peeling it off the plantar surface. The footpads to be tested were then injected with the drug of interest, and another coat of impression material was applied immediately in the case of agonists, 5 min postinjection in the case of antagonists, and at the beginning of a second 2.5min period of nerve stimulation in the case of antagonists.

VOLUME 130,1988

All pharmacological agents tested were dissolved in 0.9% NaCl or 0.9% NaCl plus 0.5 mg/ml sodium ascorbate (isoproterenol). Drugs were administered locally; volumes of 2-10 ~1 were injected into individual footpads using a IO ~1 Hamilton syringe. Atropine, isoproterenol, propranolol, methacholine, and muscarine were obtained from Sigma. 6-Fluoronorepinephrine was obtained from Research Biochemicals. Clonidine (Sigma) and phentolamine (Ciba-Geigy) were the kind gifts of Dr. Randall Pittman. Concentrations of drugs to be used were determined in a previous study (Stevens and Landis, 1987). RESULTS

Development and Properties of the Sweat Gland Innervation in &OHDA-Treated Rats We first determined the extent to which the growth of sympathetic fibers into the sweat glands was delayed by a single injection of 6-OHDA on Day 2. Because the sympathetic innervation of the sweat glands is initially noradrenergic, glyoxylic acid-induced catecholamine fluorescence was used to detect sympathetic fibers in the developing footpad. When the footpads of normal 7-day-old animals were examined, fluorescent fibers were present in nerves and were associated with the forming glands (Fig. la) and blood vessels. In contrast, no catecholamine fluorescence was present in nerves or in association with the glands (Fig. lb) or blood vessels

FIG. 1. Catecholamine fluorescence in the sweat glands of ‘I-day-old normal and 6-OHDA-treated rats. (a) In the untreated animal, the developing glands are innervated by catecholamine-fluorescent sympathetic fibers, which are closely associated with the glands (asterisk). (b) No catecholamine-containing fibers are evident in the footpads of treated animals. Catecholamine-containing sympathetic axons were visualized with glyoxylic acid-induced fluorescence in lo-Nrn frozen sections. X400.

STEVENS AND LANDIS

Developmental

Interactions between Neurms and Their Targets

of 6-OHDA-treated animals. Catecholamine-containing fibers were first observed in footpads of 6-OHDAtreated animals at Day 10, and by 14 days, these fibers had reached the glands and begun to innervate them (Fig. 2b). Blood vessels, however, remained uninnervated. The extent of gland innervation was reduced in the treated rats relative to normal animals, which exhibited an extensive plexus of catecholamine fluorescent sweat gland fibers at Day 14 (Fig. 2a). Since previous studies have shown that in normal animals sympathetic axons first become associated with the developing sweat glands on Postnatal Day 4 (Landis and Keefe, 1983), these observations indicate that treatment with 6-OHDA on Day 2 delayed the growth of sympathetic fibers into the glands for 6 to 10 days. The density of the gland innervation increased greatly between Days 14 and 21, but then stabilized so that subsequently there was no significant ingrowth of new fibers into the glands. Many, but not all, of the glands in 6-OHDA-treated animals became innervated by sympathetic fibers. Examination of sections stained for ~65, a synaptic vesicle antigen (Matthew et al., 1981; Bixby and Reichardt, 1985) which is present in sympathetic axons but absent from peripheral sensory terminals in adult rats, revealed that one-quarter to one-half of the glands in mature 6-OHDA-treated animals were

707

sympathetically innervated. The amount of innervation varied between footpads; in some pads, none of the glands became innervated, while in others the majority of the glands received innervation. In contrast to the glands, the blood vessels in the footpads of 6-OHDAtreated animals were devoid of p65 staining and therefore did not appear to be innervated by sympathetic fibers even in mature rats. The changes in the expression of AChE and catecholamines which normally occur in the developing sweat gland innervation were delayed in 6-OHDA-treated rats. In normal rats, AChE activity first appears in the gland innervation at 7 days, and staining intensity increases with age, while endogenous catecholamine stores begin to decline after 14 days, and are not detectable in the adult innervation (Landis and Keefe, 1983). When the footpads of 21-day-old normal and 6-OHDAtreated rats were examined for the presence of endogenous cateeholamines, fluorescence was barely detectable in the innervation of the control glands, while the fibers in the glands of 6-OHDA-treated animals were still brightly fluorescent (Figs. 3a and 3b). In 6-OHDAtreated rats, AChE activity was present in the gland innervation as early as Day 14; as in normal rats, it increased in intensity during development. At 21 days, the AChE reaction product in 6-OHDA-treated rats

FIG. 2. Catecholamine fluorescence in the sweat gland innervation of 14-day-old normal and GOHDA-treated sympathetic fibers have formed a catecholamine-fluorescent plexus around the secretory tubules (asterisk). (b) fluorescent fibers have grown into the connective tissue surrounding the glands and begun to form a plexus asterisk. Catecholamine-containing sympathetic axons were visualized with glyoxylic acid-induced fluorescence

rats. (a) In the normal animal, In the 6-OHDA-treated animal, in the gland. Secretory tubule, in lo-pm frozen sections. ~500.

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STEVENS AND LANDIS

Developmental Interactions between Neurons and Their Targets

(Fig. 3d) was not as intense as that in control (Fig. 3c), but since the extent of gland innervation in 6-OHDAtreated animals was generally less than that in untreated rats, it was not possible to determine if the amount of AChE per axon differed in 6-OHDA-treated and normal rats. Thus, at 21 days, in normal animals, catecholamines had disappeared almost entirely from the innervation and staining for AChE activity was very intense (Figs. 3a and 3c), while in 6-OHDA-treated animals the innervation of sweat glands was brightly catecholamine fluorescent and relatively weakly AChE positive (Figs. 3b and 3d). Although catecholamines were still present at 21 days in the sweat gland innervation of 6-OHDA-treated rats, their expression subsequently declined. The sympathetic innervation was still brightly fluorescent at 25 days, but at 29 days the fluorescence appeared less intense than at 21 and 25 days. By 6 weeks of age, the expression of catecholamines in the gland innervation, although variable, was significantly less than that at 21 days. In some glands, the nerve fibers continued to express catecholamine fluorescence (Fig. 4a). In others, however, catecholamines were no longer detectable (Fig. 4b); the presence of p65-positive fibers in adjacent sections from the same glands indicated that the absence of catecholamine fluorescence was not due to lack of sympathetic innervation (Fig. 4~). These results indicate that in 6-OHDA-treated rats, the disappearance of endogenous catecholamines was delayed relative to its occurrence in untreated animals, and was in some instances incomplete, such that the innervation of some glands continued to express variable but detectable levels of catecholamines in the adult. It is of interest that previous studies of regenerating sympathetic fibers in a number of noradrenergic targets after 6OHDA treatment have not provided any evidence for a subsequent loss of catecholamines (Haeusler et al., 1969; de Champlain, 1971; Jonsson and Sachs, 1972; Lorez et cd, 1975). In addition to examining the effects of delayed innervation on catecholamine fluorescence and AChE activity, it was of interest to determine how peptide expression was affected. In normal adult rats, the sweat gland innervation possesses immunoreactivity for VIP (Yodlowski et ab, 1984; Landis et ah, 1988). In approxi-

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mately 25% of the 6-OHDA-treated rats in which the glands examined were sympathetically innervated, the innervation exhibited VIP immunoreactivity (Figs. 5a and 5b). In the majority of 6-OHDA-treated rats, however, no VIP immunoreactivity was detectable in the glands examined (Fig. 5c), although double staining for p65 confirmed that the glands were innervated by sympathetic fibers (Fig. 5d). Properties of the Secretory Response in Sweat Glands of 6-OHDA-Treated Rats and Its Development In normal developing and adult rats, nerve-evoked sweating is mediated through a cholinergic mechanism (Stevens and Landis, 1987) and can therefore be used to assay cholinergic function and its development. We examined the effects of both cholinergic and adrenergic antagonists on nerve-evoked secretion in the 6-OHDAtreated rats. In general, the secretory response in 6OHDA-treated rats was less robust than that in untreated rats, presumably because of the lower density of gland innervation. Active glands were identified by their response to nerve stimulation and those footpads which contained responsive glands were injected with a saline solution containing the antagonist(s) or saline alone as a control. Five minutes after the injection, the nerve was stimulated again and sweating was assayed. As shown in Table 1, the cholinergic muscarinic antagonist, atropine, was highly effective in blocking the secretory response to nerve stimulation in the 6-OHDAtreated animals. In contrast, a combination of the a- and ,&adrenergic antagonists, phentolamine and propranolol, had no effect. Thus, the nerve-evoked secretory response in 6-OHDA-treated animals was mediated cholinergically, indicating that acetylcholine was present in the gland innervation. In those glands in which catecholamines were present, they appeared to play no detectable role in the secretory response. To determine whether the onset of cholinergic function was delayed in 6-OHDA-treated rats, the nerveevoked secretory responses of 21-day-old and adult (6 weeks and older) rats were assayed and compared with those obtained in a previous study of normal development (Stevens and Landis, 1987). In untreated rats, we found that nerve-evoked sweating is first detectable in

FIG. 3. Catecholamine fluorescence and acetylcholinesterase activity in the sweat gland innervation of 21-day-old normal (a,c) and 6OHDA-treated rats (b,d). (a) Catecholamines are barely detectable in the innervation of the glands, but the innervation of a nearby blood vessel is intensely catecholamine fluorescent (arrow). (b) The glands of the treated animal are surrounded by a plexus of brightly fluorescent fibers. (c) The fiber plexus in the glands of control animals is uniformly labeled with acetylcholinesterase reaction product (arrowheads). (d) Acetylcholinesterase reaction product (arrowheads) of varying intensity can be detected in the fibers innervating glands of treated animals. (a,b) Glyoxylic acid-induced catecholamine fluorescence in lo-pm frozen sections. X500. (c,d) Ten-micrometer frozen sections reacted 90 min for the presence of acetylcholinesterase and intensified with methylene blue. X388.

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FIG. 4. Catecholamine fluorescence and p65 immunoreaetivity in the gland innervation of adult 6-OHDA-treated rats. The expression of catecholamines in the gland innervation of 6-OHDA-treated animals was variable. (a) In some glands, the fibers exhibit eatecholamine fluorescence. The fluorescence intensity shown here represents the brightest observed. (b) In other glands, catecholamine-containing fibers are not detectable. In the footpad which contained this gland, none of the glands contained fibers which exhibited detectable levels of catecholamines, even though almost all of the glands were innervated by p65-positive fibers. (c) Two of the glands from the same footpad as that pictured in (b) contain a sparse plexus of p65-immunoreactive sympathetic fibers. (a,b) Ten-micrometer frozen sections reacted with glyoxylic acid to induce catecholamine fluorescence. X400. (c) Ten-micrometer freshly frozen section stained for the presence of ~65. X400.

one-quarter of lkday-old animals, increases rapidly, and is present in all 21-day-old animals. In contrast, in 21-day-old 6-OHDA-treated animals, only one-quarter of the rats tested exhibited a response to nerve stimulation (Table 2), and the number of active sweat glands

was only 20% of the number active following stimulation of untreated rats at 21 days. In the representative assay of 21-day-old 6-OHDA-treated animals shown in Fig. 6a, only two glands responded, a number characteristic of 14-day-old untreated rats. Thus, although a

STEVENS AND LANDIS

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in the sweat gland innervation of adult 6-OHDA-treated rats. (a) In this gland, VIP-immunoreactiv FIG 5. VIP immunoreactivity e fibers (arrov vhead) are present. (b) The same section as in (a) stained for the presence of p65 immunoreactivity. The correlation between thee itaining indicates that most, if not all, of the fibers innervating this gland are VIP immunoreactive. (c) In this for VI :P and p65 immunoreactivities are not detectable. (d) The same section as in (c) stained for the presence of p65 immunl oreactigland, fibers exhibiting VIP immunoreactivity vity. 1Ihe presence of an p65-positive fiber plexus indicates that the gland is innervated by sympathetic fibers. X450.

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TABLE 1 EFFECTSOFADRENERGICAND CHOLINERGICANTAGONISTSON SWEATINGIN ADULT RATS TREATEDWITH 6-OHDA AS NEONATES

Antagonist Atropine 1.0 pM 0.1 pM Phentolamine + propranolol 10.0 PM Saline

Total no. glands responding to nerve stimulation

Percentage glands blocked by antagonist(s)

No. rats

64 37

100 84

4 4

19 82

0 4

3 6

Note. The sciatic nerve was stimulated for a period of 3 min, and then sweating was assayed while the nerve was stimulated for an additional 2.5 min. Footpads in which glands responded to nerve stimulation were injected with a saline solution containing the antagonist(s) or saline alone as a control. Five minutes after the injection, sweating was assayed again during a second 2.5-min period of nerve stimulation. The total number of glands responding before and after injection of the antagonist was determined by adding the numbers from each rat. The percentage of inhibition was calculated by taking the difference between the total number of glands respnding during the first and second assays, and dividing that by the total number activated during the first assay. For 10 pMphentolamine + propran0101,the total number of glands responding during the second assay was higher than that during the first, so the percentage of inhibition was considered to be 0.

secretory response was present in some 6-OHDAtreated rats at 21 days, the percentage of rats sweating and the proportion of glands activated at this age were similar to those seen at 14 days in untreated rats, suggesting that the development of cholinergic function was delayed about a week in the 6-OHDA-treated rats. During subsequent development, nerve-evoked secretion increased both with respect to the number of rats sweating and the number of glands activated per rat. By 6 weeks of age, almost all 6-OHDA-treated rats exhibited a secretory response to nerve stimulation (Table 2). There was a wide range in the number of glands which responded to nerve stimulation: although in some rats over 50% of the approximately 300 glands present were activated, in the majority of rats tested the percentage was less than 30. An assay from a 6-OHDA-treated animal with a relatively high number of active glands is shown in Fig. 6b. Although the number of active glands in adult 6-OHDA-treated rats was variable, it was always considerably higher than that at 21 days. Thus, although the onset of cholinergic transmission was delayed in the sweat glands of 6-OHDA-treated rats, as in untreated rats, it became more robust during subsequent development. The delayed development of cholinergic transmission in the sweat glands of 6-OHDA-treated rats provided

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an opportunity to examine the relationship between the onset of cholinergic function in the gland innervation and the development of sensitivity to cholinergic agonists in the gland cells. In immature rats, glands do not develop sensitivity to cholinergic agonists until the gland innervation begins to differentiate cholinergitally (Stevens and Landis, 1987). This finding raised the possibility that during normal development glands require cholinergic innervation in order to develop responsiveness to cholinergic agonists. It was not possible, however, to rule out the possibility that the gland cells are simply immature and therefore not competent to secrete at earlier ages. In both 21-day-old and adult 6-OHDA-treated rats, many footpads were devoid of active glands when sweating was assayed during nerve stimulation. To determine whether glands in 6-OHDAtreated animals which were not activated by nerve stimulation would respond to an exogenous cholinergic agonist, we injected nonresponsive footpads with muscarine. In no case were glands in nonresponsive footpads activated by muscarine. In untreated rats at these ages, all footpads contain glands which are capable of secreting. These results indicate that the failure of some glands in 6-OHDA-treated rats to secrete in response to nerve stimulation or muscarine did not represent an age-related inability to produce sweat but rather the lack of responsiveness in the glands appeared to result from an insensitivity to cholinergic agonists. To determine whether a relationship existed between insensitivity and the state of the sympathetic innervation of the glands, the glands in nonresponsive footpads were examined for immunoreactivity to p65 and catecholamine fluorescence. Although many of the nonresponsive glands were not sympathetically innervated, others contained fibers which exhibited catechol-

TABLE 2 NERVE-EVOKEDSWEATINGIN RATS TREATED WITH 6-OHDA AS NEONATES

Age of rat

Percentage sweating in response to nerve stimulation

No. tested

21 days 6 weeks and older

27 95

22 62

Note. The sciatic nerve was stimulated for a period of 3 min to fill the ducts of the glands, and then sweating was assayed while the nerve was stimulated for an additional 2.5 min. Rats were considered to have a secretory response if any glands secreted, even though their number may have been only a fraction of the number of glands activated hy nerve stimulation in control animals. If no glands were activated during the initial assay, in most cases a second assay was done during a second 2.5-min period of stimulation to confirm the absence of sweating.

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FIG. 6. Nerve-evoked secretory responses in a Zl-day-old rat (a) and an adult rat treated with 1 injection of 6-OHDA on Day 2 (b). (a) This mold shows the sweating response in three interdigital footpads of a 21 day-old animal. Only two glands (arrow) responded to nerve stimulation. The number of glands activated in this rat is typical of 6-OHDA-treated rats which exhibited a response at this age. (b) This mold shows the sweating response in three interdigital pads of an adult animal that exhibited a relatively strong secretory response to nerve stimulation. Many glands in each footpad were activated by nerve stimulation. The sciatic nerve was stimulated for a 3-min period to fill the ducts of the glands, then the elastic assay material was applied, and the nerve was stimulated for an additional 2.5 min. The activity of each gland is represented by a pore in the hardened mold. X10.

amine histofluorescence. The presence of detectable catecholamines does not exclude the possibility of cholinergic development as well but it may reflect a lesser extent of cholinergic differentiation. Such a reciprocity between noradrenergic and cholinergic functions has been well documented in studies of sympathetic neurons grown in the presence of a strong cholinergic inducing influence (Patterson and Chun, 1977a; Wolinsky and Patterson, 1983; Potter et al, 1986; Raynaud et al., 1987). Our observations suggest that in 6-OHDAtreated rats, the lack of innervation, and of cholinergic innervation in particular, resulted in the failure of the glands to develop sensitivity to cholinergic agonists. Although the nerve-evoked secretory response in 6OHDA-treated rats did not have a detectable adrenergic component, since the innervation of some glands in adult 6-OHDA-treated rats expressed catecholamines, it was of interest to examine the relative sensitivity of the glands to cholinergic and adrenergic agonists and to compare it with that previously obtained in a study of normal rats (Stevens and Landis, 1987). In normal adult rats, some glands respond to adrenergic agonists, particularly when they are administered simultaneously with VIP. However, the adrenergic response is much smaller than the response to cholinergic agonists both with respect to volume of sweat and number of glands sweating. To test responsiveness to cholinergic and

adrenergic agonists in 6-OHDA-treated rats, glands which were capable of sweating were identified by their response to sciatic nerve stimulation. Footpads which contained active glands were injected with a saline solution containing the agonist(s) or with saline alone as a control, and sweating was assayed 1 to 3 min postinjection. As shown in Table 3, all active glands tested were sensitive to 50 PM muscarine. In contrast, only 3% of glands tested responded to a combination of the CY-and /3-adrenergic agonists, 6-fluoronorepinephrine and isoproterenol. However, when VIP was simultaneously injected with the adrenergic agonists, the percentage of glands responding rose to 33. VIP alone produced a secretory response in 9% of the glands tested. In normal rats, although VIP alone elicits a cholinergically mediated response from 7% of the glands, the primary effect of VIP administered in combination with adrenergic agonists is to enhance the adrenergic secretory response, such that the percentage of glands responding increases from 13% with adrenergic agonists alone to 25% when they are coinjected with VIP (Stevens and Landis, 1987). By analogy, it seems likely that the effect of VIP when administered with adrenergic agonists in 6-OHDA-treated animals was to promote the adrenergic response. Although catecholamines persisted in the innervation of some glands in mature 6-OHDA-treated rats, the sensitivity of the glands to adrenergic agonists

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TABLE 3 EFFECTSOFMUSCARINE,ADRENERGICAGONISTS, ANDVIP ON SWEATINGINADULT RATSTREATEDWITHB-OHDAAS NEONATES

that the presence of VIP and a cholinergic secretory response were not necessarily coincident, it was not possible to establish this from such experiments. To test this directly, VIP immunoreactivity was examined in No. glands Percentage responding glands the innervation of glands in which a nerve-evoked seto nerve responding No. cretory response had been established. Rats were asDrugs stimulation to agonist(s) rats sayed for nerve-evoked sweating and footpads containing active glands were identified. The animals were alMuscarine 40 103 5 lowed to recover for at least 24 hr and then the sweat 6-Fluoronorepinephrine + isoproterenol 192 3 11 gland innervation in the identified footpads was examVIP 56 9 4 ined immunocytochemically for the presence of VIP. In 6-Fluoronorepinephrine untreated animals in which sweating was assayed 24 hr + isoproterenol + VIP 36 33 7 prior to sacrifice, VIP immunoreactivity was as intense Saline 60 0 4 as in unstimulated rats, indicating that the sweating Note. The concentrations used were as follows: 50 +%f muscarine, 50 assay did not interfere with VIP localization. In 6@6-fluoronorepinephrine, 50 N isoproterenol, and 3 fl VIP. The OHDA-treated rats, in the majority of footpads which sciatic nerve was stimulated for 3 min, and then sweating was asdid exhibit a secretory response, the gland innervation sayed during a subsequent 2.5-min period of stimulation. Footpads in lacked detectable VIP immunoreactivity. These results which glands responded to nerve stimulation were injected with a indicate that in the sweat gland innervation of 6saline solution containing the agonist(s) or saline alone as a control. Sweating was assayed 1 to 3 min after injection. The total number of OHDA-treated rats, VIP was not necessarily coexglands responding to either nerve stimulation or agonist(s) was de- pressed with acetylcholine and further that VIP was termined by adding the numbers from each rat. To calculate the not required for effective cholinergic transmission in percentage of glands responding to the agonist, the total number of the glands. glands responding to an agonist or agonist combination was divided by the total number of glands activated by nerve stimulation in the footpads injected with that agonist or agonist combination.

was not increased relative to that of the glands in untreated animals. Thus, in contrast to acetylcholine, whose presence in the gland innervation appears to induce cholinergic responsiveness in the gland cells, catecholamines, even when expressed in the adult innervation, did not evoke increased responsiveness to adrenergic agonists in our assay. Correlation between the Secretory Response and VIP Immunoreactivity VIP is present in the sweat gland innervation of cats and rats (Lundberg et al., 1979; Yodlowski et al, 1984; Landis et aL, 1988) and it has been suggested that in the sympathetic nervous system, VIP expression may be linked to cholinergic function (Lundberg et aL, 1982). Although 95% of adult 6-OHDA-treated rats sweated in an atropine-sensitive fashion in response to nerve stimulation, indicating the presence of cholinergic function, VIP immunoreactivity was detected in the gland innervation of only about 25% of the rats examined. This suggested that in these rats VIP was not expressed by acetylcholine-secreting sweat gland fibers. In many rats, however, active glands were restricted to only a few of the 11 hind footpads and only the two medial interdigital pads were examined for VIP immunoreactivity. Thus although the results suggested

Ultrastructural

Studies

The results presented above provide evidence for the existence of cholinergic and adrenergic properties in the sweat gland innervation of adult 6-OHDA-treated rats. To determine whether this duality represented two separate populations of fibers, one cholinergic and one adrenergic, or a single population of dual-function axons containing both acetylcholine and catecholamine, we examined the gland innervation of 6-OHDA-treated rats with the electron microscope after fixation with potassium permanganate, which precipitates vesicular stores of norepinephrine and therefore reveals norepinephrine as a small granular vesicle (SGV) (Richardson, 1966; Hokfelt, 196% Hokfelt and Jonsson, 1968). In normal adult animals, the nerve terminals of the cholinergic innervation of the sweat glands contain only clear synaptic vesicles after permanganate fixation (Fig. 7a) (Landis and Keefe, 1983), whereas the terminals of the noradrenergic innervation of adjacent blood vessels contain almost exclusively SGV. We examined the sympathetic gland innervation in nine footpads from seven 6-OHDA-treated animals and found that the terminals exhibited a wide range of vesicle profiles which varied from those containing no SGV to others in which almost 50% of the vesicles exhibited dense cores (Figs. 7b and 7~). The terminal phenotype most commonly observed contained a small percentage of SGV similar to the terminals of adrenergic/cholinergic dual-function neurons identified in microculture

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F‘IG. 7. Ultrastructural characteristics of the gland innervation in adult normal and 6-OHDA-treated rats following permanganate fixatic m. (4 The nerve terminals associated with the sweat glands in untreated rats contain only clear vesicles. X42,000. (h) This terminal from a 6-OIHDA-treated rat contains numerous synaptic vesicles, of which only a few possess dense cores (arrows). X37,000. (c) Small granular vesicl #es con uprise a substantial fraction of the vesicles present in the varicosities (arrowheads) contained within this nerve bundle. ~37,000.

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(Landis, 1976; Potter et al, 1986). These results are consistent with the histochemical and physiological results which suggested that catecholamines and acetylcholine coexisted in the sweat gland innervation of mature 6OHDA-treated rats, and suggest that the gland innervation consisted of a single population of nerve fibers which was heterogeneous in its relative expression of noradrenergic and cholinergic properties.

1897; Purves and Thompson, 1979; Bowers et ak, 1984; Hendry et al., 1986). For example, neurons which originally innervate the iris may regenerate to innervate the submandibular gland (Hendry et al. 1986). “Cross-innervation” of sympathetic targets almost certainly occurred in our experiments as well. Two sympathetic targets exist in footpads. The principal target is the sweat glands which receive innervation which is cholinergic and which contains the neuropeptides VIP and CGRP. In addition, blood vessels which receive innerDISCUSSION vation which is noradrenergic and which contains In the study reported here, the normal developmental neuropeptide Y are also present (Leblanc and Landis, interaction between sweat glands and their innervation 1986; Landis et ak, 1988). The observation that blood was disrupted to investigate neurotransmitter plastic- vessels in footpads of mature 6-OHDA-treated rats ity of sympathetic neurons developing in viva and possi- were not innervated by sympathetic fibers makes it ble target influences on transmitter choice. This was likely that in the treated animals sympathetic neurons accomplished by a single injection of 6-OHDA on Post- which would normally have innervated blood vessels natal Day 2. In the treated animals, catecholamineinnervate sweat glands instead. If this is in fact the containing axons became associated with the glands be- case, our results suggest that these neurons exhibited tween Postnatal Days 10 and 14 instead of Day 4, a developmental changes in transmitter properties apdelay of 6 to 10 days. With subsequent development, as propriate to their new target. Such changes would be in the gland innervation of normal rats, the expression consistent with the postulated neurotransmitter plasof endogenous catecholamines declined, cholinergic ticity in viva and a target role in determining transmitfunction was induced, and the development of sensitivter phenotype. The present study, however, does not ity to cholinergic agonists by the sweat glands was cou- provide direct evidence bearing on the issue of cross-inpled to the onset of functional cholinergic transmission. nervation and the following discussion presumes that The change in neurotransmitter properties, however, fibers which innervated the sweat glands following 6OHDA treatment are predominantly those which would occurred later in development than that in untreated animals and was not always complete. In adult animals, normally have done so. In a separate series of experisome fibers continued to express endogenous catechol- ments, the effect of cross-innervation between noradamines and many nerve terminals contained a small renergic and cholinergic sympathetic targets has been proportion of small granular vesicles after permanga- explicitly examined (Schotzinger and Landis, 1988); by nate fixation. In addition, immunoreactivity for VIP transplanting sweat gland containing footpad skin to was absent from the innervation of the majority of the presumptive hairy skin region of early postnatal rats, neurons which would normally innervate piloerecglands. Treatment of rat pups with a single dose of 6-OHDA tors and blood vessels and remain noradrenergic were on Postnatal Day 2 resulted in a significant delay in induced to innervate sweat glands instead. Catecholinnervation of the developing sweat glands by sympa- amines were initially present in the sympathetic axons which innervated the transplant. With further developthetic fibers. This experimental paradigm contrasts with a previous study in which sweat glands were per- ment, catecholamines significantly decreased and choactivity appeared. Taken tomanently deprived of sympathetic innervation by line acetyltransferase treatment of rat pups with multiple doses of 6-OHDA gether with the present experiments in which cross-induring the first postnatal week (Yodlowski et aL, 1984). nervation was likely to have occurred but could not be In the present experiments, innervation was delayed proven, these observations indicate that the sweat but not prevented. The exact mechanism by which the glands influence the neurotransmitter properties of the 6-OHDA acted to delay innervation is unclear but it neurons which innervate them. In 6-OHDA-treated animals, both the decline in cateseems likely that the noradrenergic neurotoxin was accumulated in growing sympathetic axons and caused cholamine fluorescence and the onset of cholinergic their destruction. If 6-OHDA did result in axotomy, function in the gland innervation occurred later in dethen the sympathetic fibers in the treated animals were velopment than those in untreated rats. In normal rats, the expression of catecholamines is maximal at 14 days, regenerating. In adult animals, when postganglionic nerves arising from the superior cervical ganglion are and then decreases until catecholamine fluorescence is cut and allowed to regenerate, the sympathetic axons barely detectable by 21 days of age (Landis and Keefe, 1983). In 6-OHDA-treated rats, catecholamine fluoresreinnervate target organs nonspecifically (Langley,

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De-velopwwntalInteractions between Neurons and Their Targets

cence was maximal around Day 25 and although it subsequently declined, some fibers exhibiting varying degrees of fluorescence intensity were still detectable in some glands in adults. Cholinergic transmission in normal rats is first detected in 25% of 14-day-old rats; during the next week of development the percentage of rats exhibiting nerve-evoked sweating increases to 100, and the number of glands responding increases lo- to 20-fold (Stevens and Landis, 1987). In 6-OHDA-treated rats, the onset of cholinergic transmission was delayed such that at 21 days, only 27% of the rats exhibited a response to nerve stimulation and the number of glands activated in 21-day-old 6-OHDA-treated rats was similar to that seen in untreated animals at 14 days. During subsequent maturation, however, the percentage of animals exhibiting nerve-evoked secretion increased to 95%. Thus, the decrease in catecholamine expression and the onset of cholinergic function were delayed in 6-OHDA-treated rats by 7 to 10 days, approximately the same delay as that observed for the arrival of sympathetic axons at the glands. Our results are consistent with the hypothesis that sympathetic neurons are plastic with regard to neurotransmitter choice and that the sweat glands promote a change from noradrenergic to cholinergic function in the neurons which innervate them. This is true for any neurons which innervated the sweat glands as a consequence of the 6-OHDA treatment. An alternative interpretation of our results with respect to the neurons which would normally have innervated the sweat glands is that the effect of 6-OHDA treatment was to set back a developmental clock within sympathetic neurons, possibly by shifting the neurons’ metabolic priorities and/or by delaying the establishment of preganglionic innervation (Black et ab, 1971; Thoenen et al., 1972; Hendry, 1973; Black, 1973; Black and Geen 1973, 1974; Cheah and Geffen, 1973). A delay in the transition in transmitter phenotype in the gland innervation would be predicted if such a setback did occur; once axons reached the target sweat glands, the developmental program would recommence. According to this model, although the transition would be delayed, when it did occur it would be expected to occur normally. However, not only was the developmental schedule for changes in neurotransmitter properties altered, but differences in their expression were observed as well. First, although the sympathetic innervation of the glands in 6-OHDA-treated rats did undergo a transition in which noradrenergic properties declined and cholinergic function developed, the extent of this transition was not as great as that in untreated rats. In the gland innervation of normal adult rats, endogenous catecholamine fluorescence is virtually undetectable, none of the terminals associated with the glands contain

717

small granular vesicles after permanganate fixation (Landis and Keefe, 1983), and sciatic nerve stimulation produces a robust cholinergically mediated secretory response in approximately 300 glands (Stevens and Landis, 1987). In contrast, in adult 6-OHDA-treated rats, some glands contained fibers which continued to express reduced but detectable levels of catecholamines and very few of the sympathetic terminals examined were entirely devoid of small granular vesicles. Correspondingly, the nerve-stimulated secretory response was weaker than normal and some innervated glands did not acquire sensitivity to cholinergic agonists, presumably due to inadequate cholinergic differentiation in their innervation. Second, the expression of VIP was not tightly coupled with that of cholinergic function in 6-OHDA-treated rats. VIP is present in the cholinergic sympathetic innervation of sweat glands in both rats (Yodlowsi et al., 1984; Landis et ah, 1988) and cats (Lundberg et al, 1979), and it has been suggested that the expression of VIP by sympathetic neurons may be restricted to those that contain acetyleholine (Lundberg et al., 1982). During normal development of the sweat gland innervation, the appearance of VIP immunoreactivity is coincident with the development of cholinergic function (Leblanc and Landis, 1986; Landis et al., 1988). These observations raised the possibility that the developmental expression of the transmitter and neuropeptide might be coregulated. However, in the majority of glands in 6-OHDAtreated rats, including those in which cholinergic transmission was established, VIP immunoreactivity was not detectable. Thus, these results suggest that the expression of VIP in sympathetic neurons is not tightly linked to cholinergic function. The developmental differences between the VIP-immunoreactive and nonimmunoreactive neurons remain to be defined. It is possible, for example, that VIP appeared only in those neurons that normally innervate the sweat glands and not in those that normally innervate blood vessels. There are several possible explanations for the failure of the sweat gland innervation in the 6-OHDAtreated animals to undergo a complete transition in transmitter phenotype. These derive from our understanding of how environmental factors can regulate neurotransmitter choice by sympathetic neurons in vitro; it is important to note, however, that none of these cues has yet been shown to influence transmitter choice in vivo and thus the following discussion is necessarily speculative. In studies of sympathetic neurons developing in cell culture several factors including neuronal age, neuronal activity, and hormonal influences have been identified which may have modulated the postulated i7~ viva effect of sweat glands. In vitro, the ability of sympathetic neurons to respond to factors

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which induce cholinergic differentiation decreases with age (Ross et al, 1977; Patterson and Chun, 197713;Johnson et al, 1980b; Potter et cz&1986). Thus, by analogy, it is possible that during the delay in reaching the sweat glands, the continued maturation of the neurons resulted in decreased plasticity with respect to transmitter choice and a consequent reduction in their ability to respond to subsequent influences affecting that choice. A second factor which may have influenced the final phenotype of the neurons is their preganglionic innervation and the resulting pattern of activity to which they are exposed. Work in culture has demonstrated that chronic depolarization or increased electrical activity can inhibit the ability of neurons to respond to the cholinergic-inducing factor present in conditioned medium (Walicke et a& 1977; Walicke and Patterson, 1981; Raynaud et ab, 1987). Thus, after 6-OHDA treatment, if the neurons which innervated the sweat glands received inappropriate synaptic input which produced a significantly greater than normal level of electrical activity, their ability to respond to cholinergic induction could have been compromised, resulting in an incomplete transition. A third factor is that the strength of the stimulus-promoting cholinergic induction in vivo may decline during development. Studies in culture have demonstrated that the induction of cholinergic properties and the suppression of noradrenergic properties are inversely related and dependent upon the strength of the cholinergic stimulus (Patterson and Chun, 1977a,b; Wolinsky and Patterson, 1983; Raynaud et aZ., 1987). If a similar factor is present in the sweat glands in vivo, its levels may change over time such that they are highest early in development and subsequently decline. Further, the release of cholinergic factor by cultured heart cells has been shown to be regulated by hormones and growth factors; glucocorticoids inhibit it and epidermal growth factor is required (Fukada, 1980). If analogous regulation occurs in vivo, developmental changes in these substances could affect the levels of cholinergic factor. Finally, the neurons that innervated the glands may possess collateral fibers which innervated noradrenergic targets outside the footpads, such as blood vessels or piloerector muscles, which could inhibit their cholinergic development. The results of the experiments reported here provide additional insights into the development and regulation of the secretory response in the sweat glands and suggest that important reciprocal developmental interactions exist between the sweat glands and their innervation. In a previous study, evidence was obtained which suggested that the onset of responsiveness to cholinergic agents in the gland cells was linked to the development of cholinergic function in the gland innervation (Stevens and Landis, 1987). In adult 6-OHDA animals,

VOLUME130, 1988

glands that were either sympathetically uninnervated or in some cases were innervated by catecholaminecontaining fibers not only did not exhibit nerve-evoked secretion, but also did not respond to cholinergic agonists. These results support the notion that the presence of cholinergic innervation is essential to the development of agonist sensitivity in gland cells. Although the simplest explanation for the lack of cholinergic responsiveness in the glands of immature normal and mature 6-OHDA-treated rats is that the presence of acetylcholine is required to induce responsiveness in the glands, it is not possible to exclude the possibility that some other factor which is normally coexpressed with acetylcholine, and not acetylcholine itself, is responsible. VIP is one such candidate. In the 6-OHDA-treated animals, however, glands that which did not receive detectably VIP-immunoreactive innervation did exhibit nerve-evoked secretory responses, indicating that VIP was not required for the development of the secretory response. Thus, these experiments strongly suggest that it is the development of cholinergic function in the sweat gland innervation which is essential for a final maturational event in the gland cells which confers secretory responsiveness and that interaction with the sweat glands is responsible for induction of cholinergic function in the innervation. We wish to thank our colleagues from the Department of Neurobiology at Harvard Medical School, many of whom are now scattered throughout the country, for numerous interesting discussions concerning these studies. This research was supported by USPHS Grants NS23678, NS07112, and MH18012. Leslie Stevens was the recipient of a predoctoral fellowship from the National Science Foundation. S.C.L. was an Established Investigator of the American Heart Association, supported in part by funds contributed by the Massachusetts Affiliate. REFERENCES

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VOLUME 130.1988

SWERTS,J. P., LE VAN THAI, A., and WEBER,M. J. (1983). Regulation of enzymes responsible for transmitter synthesis and degradation in cultured rat sympathetic neurons. I. Effect of muscle conditioned medium. Dev. Biol. 100, l-11. WALICKE, P. A., CAMPENOT,R. B., and PATTERSON,P. H. (1977). Determination of transmitter function by neuronal activity. Proc. Natl. Acad. Sci. USA 74,3767-3771. WALICKE, P. A., and PATTERSON,P. H. (1981). On the role of CaC+in the transmitter choice made by cultured sympathetic neurons. J. Neurosci. 1,343-350. WEBER, M. J. (1981). A diffusible factor responsible for the determination of cholinergic functions in cultured sympathetic neurons: Partial purification and characterization. J. Biol. Chem. 256, 3447-3453. WEBER, M., RAYNPUD, B., and DELTEIL, C. (1985). Molecular properties of a cholinergic differentiation factor from muscle-conditioned medium. J. Neurochem 45,1541-X47. WOLINSKY,E. J., LANDIS, S. C., and PATTERSON,P. H. (1985). Expression of noradrenergic and cholinergic traits by sympathetic neurons cultured without serum. J. Neurosci. 5,1497-1508. WOLINSKY, E., and PATTERSON,P. H. (1983). Tyrosine hydroxylase activity decreases with induction of cholinergic properties in cultured sympathetic neurons. J. Neurosci. 3,1495-1500. WOLINSKY,E. J., and PATERSON, P. H. (1985). Rat serum contains a developmentally regulated cholinergic inducing activity. J. Neuro soi. 5,1509-1512. WONG, V., and KESSLER,J. A. (1987). Solubilization of a membrane factor that stimulates levels of substance P and choline acetyltransferase in sympathetic neurons. Proc Natl. Acad. Sci. USA 84, 8726-8729. YODLOWSKI,M., FREDIEU, J. R., and LANDIS, S. C. (1984). Neonatal 6-hydroxydopamine treatment eliminates cholinergic sympathetic innervation and induces sensory sprouting in rat sweat glands. J. New-o& 4.1535-1548.