DEVELOPMENTAL

BIOLOGY

137, lo!+lk!d

(1990)

Target Influences on Transmitter Choice by Sympathetic Neurons Developing in the Anterior Chamber of the Eye LESLIE

M. STEVENS’ AND STORY C. LANDIS

Center for Neurosciences, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106; and Departmat of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115 Accepted August 25, 1989 In contrast to the majority of sympathetic neurons which are noradrenergic, the sympathetic neurons which innervate sweat glands are cholinergic. Previous studies have demonstrated that during development the sweat gland innervation initially contains catecholamines which are lost as cholinergic function appears. The neurotransmitter phenotype of sweat gland neurons further differs from the majority in that they contain vasoactive intestinal peptide (VIP) rather than neuropeptide Y (NPY). In the experiments described here, we addressed the question of whether sympathetic targets influence the neurotransmitter-related properties of the neurons which innervate them; in particular, do sweat glands play a role in reducing the expression of noradrenergic properties and inducing the expression of cholinergic properties and VIP in sympathetic neurons? This was accomplished by cotransplanting to the anterior chamber of the eye of host rats the superior cervical ganglia (SCG) which contains neurons that normally innervate targets other than the sweat glands and differentiate noradrenergically and footpad tissue from neonatal rats. Sweat glands developed in the transplanted footpad tissue and became innervated by the cotransplanted SCG neurons. The transplanted neurons and sweat gland innervation initially exhibited catecholamine histofluorescence which declined with further development in the anterior chamber. After 4 weeks, choline acetyltransferase (ChAT) and VIP immunoreactivities were evident, These observations suggest that as in the neurons which innervate the glands in situ, noradrenergic properties were suppressed and cholinergic function was induced in the neurons which innervated the glands in oculo. To distinguish a specific influence of the sweat glands on transmitter choice, SCG were also cotransplanted with the pineal gland, a normal target of the ganglion. Neurons cotransplanted with the pineal gland continued to exhibit catecholamine histofluorescence and contained NPY immunoreactivity. At least some neurons in SCG/pineal cotransplants, however, developed ChAT immunoreactivity. The target-appropriate expression of catecholamines and peptides in these experiments is consistent with the hypothesis that some transmitter properties are influenced by target tissues. The indiscriminant expression of ChAT, however, suggests that at least in oculo, additional factors can influence transmitter choice. 0 1990 Academic Press, Inc.

traditional neurotransmitters, they also affect the expression of neuropeptides in cultured sympathetic Studies of sympathetic. neurons developing iiz vitro neurons. For example, levels of Substance P (SP) are have demonstrated that they are’plastic with respect to decreased by growth under depolarizing conditions and transmitter choice and that their ultimate transmitter increased by coculture with nonneuronal cells, nonphenotype can be influenced by environmental factors. neuronal cell-conditioned medium, and increased cell When sympathetic neurons from neonatal rats are density (Kessler et aZ., 1984; 1986; Kessler, 1984a,b; 1985; grown in the absence of other cell types (Mains and Adler and Black, 1985,1986). In addition, the proportion Patterson, 1973), in defined medium (Iacovitti et al., of neuronal cell bodies immunoreactive for SP, neuro1982; Wolinsky et al., 1985), or under conditions in which peptide Y (NPY), vasoactive intestinal peptide (VIP), they are chronically depolarized (Walicke et al, 1977; and calcitonin gene-related peptide (CGRP) increases Hefti et al., 1982; Raynaud et aH, 1987), they continue to medium differentiate noradrenergically. However, when they following growth in heart cell-conditioned (Sah, 1987). Fractionation of conditioned medium reare grown in the presence of certain nonneuronal cells veals that distinct neuropeptide-inducing activities can or medium conditioned by nonneuronal cells (Patterson and Chun, 1974, 1977a,b; Kessler, 1984a; 1985; Swerts et be identified (Nawa and Patterson, 1988). Although the majority of sympathetic neurons in the al., 1983), the neurons decrease their expression of norrat are noradrenergic, a subpopulation is cholinergic, adrenergic properties and differentiate cholinergically. including those which innervate the sweat glands. In Environmental factors not only influence the choice of adult rats, homogenates of footpad tissue contain choline acetyltransferase (ChAT) activity, the synthetic 1 Present address: Max Planck Institut fur Entwicklungsbiologie, Tubingen, Federal Republic of Germany. enzyme for acetylcholine (Leblanc and Landis, 1986); INTRODUCTION

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0012-1606190 $3.00 Copyright All rights

0 1990 by Academic Press. Inc. of reproduction in any form reserved.

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the fiber plexus in the glands exhibits ChAT immunoreactivity (Leblanc and Landis, 1986); and stimulation of the sciatic nerve evokes a cholinergically mediated secretory response (Stevens and Landis, 1987). The innervation also possesses acetylcholinesterase (AChE) activity (Landis and Keefe, 1983) and immunoreactivity for VIP (Yodlowski et al., 1984; Landis et al., 19SS), a neuropeptide which has been associated with cholinergic function in the sympathetic (Lundberg et al, 1979, 1982a) and parasympathetic nervous systems (Lundberg et al., 1980). In addition, the gland innervation exhibits immunoreactivity for calcitonin gene-related peptide (Landis and Fredieu, 1986). Developmental studies of the sweat glands and their innervation have shown that the cholinergic and peptidergic properties which characterize the mature innervation do not appear until the second postnatal week (Leblanc and Landis, 1986; Stevens and Landis, 1987; Landis et al., 1988). Instead, the developing innervation initially displays noradrenergic properties, the expression of which is either lost or significantly decreased during the third and fourth weeks of postnatal development (Landis and Keefe, 1983; Landis et ah, 1988). The existence in vivo of cholinergic sympathetic neurons which initially express noradrenergic properties raises the possibility that as in the case of sympathetic neurons developing in vitro, the change in neurotransmitter phenotype may result from exposure to environmental influences which promote cholinergic and peptidergic differentiation and suppress noradrenergic differentiation. In addition to their choice of transmitter, one of the properties which distinguishes these neurons is their target, the sweat glands, raising the possibility that the sweat glands influence the transmitter choice of the neurons which innervate them. Experimental evidence consistent with this hypothesis has been obtained in previous studies. In one series of experiments, when the normal developmental interaction between the glands and the neurons which innervate them is disrupted and the arrival of the innervation in the target tissue is delayed, the normal decline in endogenous catecholamines and the appearance of cholinergic function are also delayed (Stevens and Landis, 1988). In a second series of experiments, sweat glandcontaining skin is transplanted to hairy skin regions of early postnatal rats so that neurons which would normally innervate piloerectors and remain noradrenergic instead innervate the sweat glands (Schotzinger and Landis, 1988). The sympathetic innervation displays transmitter properties characteristic of the new target; that is, catecholamines are lost and choline acetyltransferase and acetylcholinesterase appear. To examine further the question of whether the sweat glands are capable of influencing the transmitter prop-

VOLUME 137,199O

erties of the neurons which innervate them, sympathetic neurons which normally innervate other targets and differentiate noradrenergically were induced to innervate sweat glands. This was accomplished by cotransplanting superior cervical ganglia (SCG) and footpad tissue from neonatal rats to the anterior eye chamber of adult host rats. Previous studies have indicated that the anterior chamber supports the growth of a number of developing and differentiated tissues, including transplanted iris (Olson and Malmfors, 1970), heart (Olson and Seiger, 1976; Taylor et al, 1978), pineal gland (Moore, 1975; Backstrom et al., 1976, Lingappa and Zigmond, 1987), and adrenal medulla (Unsicker et al., 1978). To distinguish between effects on the SCG neurons that were the result of contact with the novel target and effects due to transplantation to and growth in the anterior chamber, the SCG was also cotransplanted with the pineal gland, a normal target of the SCG that receives sympathetic innervation which is noradrenergic (Bondareff and Gordon, 1966; Owman, 1964; Schrier and Klein, 1974) and that expresses immunoreactivity (IR) for NPY (Schon et al., 1985) but not VIP. Neurons in the SCG/footpad cotransplants innervated the glands and went through a phenotypic transition in which catecholamine expression declined and ChAT and VIP immunoreactivities developed. In contrast, neurons cotransplanted with the pineal gland maintained catecholamine expression and exhibited NPY immunoreactivity. Surprisingly, some neurons in the SCG/pineal cotransplants developed ChAT immunoreactivity. Thus, in the anterior chamber the expression of some, but not all, properties is regulated in a target-appropriate fashion. MATERIALS

AND

METHODS

Animals and Surgery Rats were obtained from Charles River (CD strain, Wilmington, MA). Most host animals were 6-week-old males obtained directly from Charles River but occasionally male or female rats between the ages of 5 and 8 weeks, received from Charles River at various ages and raised in our animal facility, were used. Tissue for transplantation was taken from l-day-old rats, obtained as litters which were born and shipped the same day (Day 0). Host rats were anesthetized and the iris of the right eye was dilated by applying a drop of 1% atropine solution to the eye. Footpads, SCG, and pineal glands were dissected from l-day-old rats and placed immediately in phosphate-buffered saline (PBS) at room temperature. The transplants were performed essentially as described by Olson and Malmfors (1970). Briefly, a slit was made in the cornea of the right eye of the host rat using the corner of a razor blade and one

edge of the cornea was lifted up with forceps while the transplant was inserted into the anterior chamber with another pair of forceps. The cornea was then released, and the tissue was moved onto the dilated iris at the outer edge of the eye chamber by applying pressure to the outside of the cornea; the ganglion and target organ were positioned so that they were in contact. Footpad transplants were examined at 10 to 12 days and 4 weeks and pineal transplants were examined at 4 weeks after transplantation. In most cases, 3 to ‘7 days prior to examining the transplants, the host rats were anesthetized and the superior cervical and ciliary (Malmfors and Nilsson, 1964) ganglia were removed from the transplant side. Removal of the ciliary ganglion was verified by identifying the ganglion with the light microscope at the time of extirpation and confirming prior to sacrifice that the iris was dilated. Removal of the SCG was verified by the absence of catecholamine-containing fibers in stretch preparations of the iris after treatment with glyoxylic acid.

treated glass slides, and fixed for 10 min in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, rinsed briefly in PBS, incubated for 30 min in 10% horse serum in PBS, rinsed briefly in PBS, and incubated overnight at room temperature in monoclonal supernatant diluted 1:l with PBS. The sections were rinsed with PBS and then incubated at room temperature for 30 min with fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin (Antibodies Inc., Davis, CA) in PBScontaining 25% rat serum and 1% goat serum. Following the final incubation, the sections were rinsed in PBS and mounted in glycerol:ethanol (1:l). For the localization of neuropeptide immunoreactivities, rats were killed with an overdose of chloral hydrate and perfused through the heart with 4% paraformaldehyde in 0.1 Mphosphate buffer, pH 7.4, for 10 min at room temperature. After perfusion, the transplants were removed from the eye and fixed for an additional hour by immersion in 4% paraformaldehyde at room temperature. The tissues were rinsed in several changes of 0.1 M phosphate buffer and then equilibrated overnight with 30% sucrose in phosphate buffer at 4°C. The Histochemistry and Immunohistochemistry tissue was frozen at -20°C and lo-pm cryostat sections For some transplants, alternating lo-pm sections of were cut and mounted on gelatin-coated slides. VIP anfresh frozen tissue were examined for the presence of tiserum was prepared by injecting rabbits with synglyoxylic acid-induced fluorescence, AChE activity, and thetic porcine VIP (Boehringer-Mannheim, Indianapo~65, a synaptic vesicle antigen (Matthew et al., 1981; lis, IN) conjugated to bovine serum albumin through a Bixby and Reichardt, 1985). Host rats were killed by carbodiimide linkage. NPY antiserum, also of rabbit carbon dioxide inhalation, and the transplanted tissue origin, was obtained from Amersham Corp. (Arlington was removed from the eye and frozen on a cryostat Heights, IL). Sections were preincubated for at least 1 chuck at -20°C. Individual glands could be identified in hr in incubation buffer, which contained 0.5 M NaCI, adjacent sections on the basis of their position within 0.01 M phosphate buffer, pH 7.3, 0.2% Triton X-100, the transplants and the characteristic organization of 0.1% sodium azide, and 5% bovine serum albumin the secretory tubules within the gland. (Sigma). Sections were then incubated overnight in Glyoxylic acid fluorescence was carried out according humid chambers at room temperature in incubation to de la Torre (1980). Cryostat sections were picked up buffer containing antisera directed against VIP or NPY. Following incubation, the sections were rinsed on warm glass slides, dipped in a solution containing 1% glyoxylic acid (Sigma Chemical Co.) and 0.2 M su- with PBS, incubated for 2 hr at room temperature with isothiocyanate-conjugated goat crose in 0.24 M potassium phosphate buffer, pH 7.4, and tetramethylrhodamine anti-rabbit immunoglobulin (Tago, Burlingame, CA) in then dried under a cool blower for at least 15 min. After the sections were dried, they were covered with a drop incubation buffer. The sections were rinsed again with of mineral oil, heated in a 95°C oven for 2.5 min and PBS and then stained for p65 as described above. Preincubation of the VIP and NPY antisera with 10 pM coverslipped. or 5 pM synFor AChE localization, cryostat sections were cut, synthetic VIP (Boehringer-Mannheim) picked up on warm slides, and fixed for 5 min in 1% thetic NPY (Amersham), respectively, abolished speparaformaldehyde and 1.25% glutaraldehyde in 0.1 M cific staining. Tissue was stained for ChAT immunoreactivity using phosphate buffer, pH 7.4. The sections were reacted for AChE and staining was intensified with a dilute solu- an antiserum generated in mouse against ChAT purition of methylene blue in saline as previously described fied from pig brain (Eckenstein et al., 1981) (the kind (Landis and Keefe, 1983). The sections were dehydrated, gift of Drs. F. Eckenstein and R. Baughman). Animals were perfused through the heart with PBS as described cleared with xylene, and mounted in Permount. p65 immunoreactivity was detected using a monoclo- above and then perfused for 10 min with 4% parafornal antibody which was the generous gift of Dr. W. maldehyde and 15% picric acid in 0.1 M phosphate Matthew. Cryostat sections were cut, picked up on un- buffer, pH 7.4. Transplants were removed from the eye

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and postfixed for 30 min in the same fixative. The tissue was rinsed in 0.1 M phosphate buffer at 4°C and then frozen. Twenty-micrometer sections were cut, picked up with cold forceps, and floated into wells containing 100 mM Tris, 150 mM NaCl, pH 7.2 (TBS). All subsequent steps were carried out as the sections floated in wells. All antisera except the primary were pre incubated for 1 hr at room temperature in the following solution (TBS, 0.1% Triton X-100, 1% BSA, 10% rabbit serum, 10% rat serum). The sections were rinsed with TBS, incubated for 1 hr in rabbit anti-mouse (Cappel, West Chester, PA), rinsed with TBS, incubated for 90 min in mouse peroxidase anti-peroxidase (mouse PAP; Sternberger-Meyer Immunochemicals, Inc., Jarrettsville, MD), rinsed with TBS, and then incubated again in rabbit anti-mouse IgG and mouse PAP. The sections were rinsed with TBS and then with 100 mMTris, pH 7.2, and preincubated for 5 min in 0.5 ml 50 mlMTris, pH 7.2, plus 1 mg/ml diaminobenzidine. The reaction was activated by adding 1 X of hydrogen peroxidase to the well. Sections were stored at 4°C in 100 mM Tris, then dehydrated, cleared with xylene, and mounted in Permount. In all experiments, sections of stellate ganglia from normal adult rats were processed as a positive control. Results were analyzed in transplants only if immunoreactive neuronal cell bodies were present in the stellate sections. Results are not reported for terminals in transplanted sweat glands because we could not reliably obtain staining in control or experimental footpad tissues. Fifty-seven transplants were examined during the course of these studies. Five transplants of footpad tissue alone were assayed after 21 days for the presence of glyoxylic acid-induced fluorescence, AChE, and ~65 staining. Eight SCG/footpad transplants were examined after lo-12 days: 5 were assayed for glyoxylic acid-induced fluorescence, AChE, and ~65 staining and three for VIP IR. Twenty-five SCG/footpad transplants were examined after 21 days: 11 for glyoxylic acid-induced fluorescence, AChE, and ~65 staining; 4 for VIP IR; 5 for NPY IR; and 5 for ChAT IR. Nineteen pineal/ SCG transplants were examined; 4 were assayed for glyoxylic acid-induced fluorescence, AChE, and ~65 staining; 5 for VIP IR; 6 for NPY IR; and 4 for ChAT IR. RESULTS Development

of Transplanted

Sweat Glands

In most cases, morphologically normal glands developed in the footpads when they were transplanted both with or without an accompanying ganglion. The footpad tissue was taken from l-day-old postnatal rats and included the epidermis and underlying dermis. At this age, sweat gland primordia are present but glands have

not begun to form (Landis and Keefe, 1983). When the transplants were positioned in the anterior chamber so that the dermis was adjacent to the iris, sweat glands formed in the majority of the pads. The transplants were vascularized by blood vessels growing out from the host iris and appeared healthy up to 4 weeks posttransplantation. The development of the transplanted glands paralleled that of the glands in situ, so that after 4 weeks in oculo, the glands appeared morphologically mature and contained secretory, myoepithelial, fat, and mast cells. Although most tissues transplanted to the anterior chamber become innervated by sympathetic or parasympathetic fibers growing out from the host iris (Olson and Malmfors, 1970; Moore, 1975; Backstrom et al., 1976; Olson and Seiger, 1976; Taylor et al., 1978; Unsicker et al., 1978; Lingappa and Zigmond, 1987), this proved not to be the case for transplanted footpads. Although catecholamine fluorescent and acetylcholinesterase-positive fibers followed the irideal blood vessels which vascularized the transplants and formed a dense plexus around the perimeter of the transplant, these fibers, however, never grew into the glands themselves. When footpads were transplanted to the anterior chamber without an accompanying ganglion, neither glyoxylic acid-induced catecholamine fluorescence nor acetylcholinesterase staining was evident in the transplanted glands, nor did the glands become innervated by fibers which exhibited immunoreactivity for ~65, an antigen associated with synaptic vesicles of both sympathetic and parasympathetic neurons (Matthew et al., 1981; Bixby and Reichardt, 1985). Transmitter Phenotype of Sympathetic Neurons Transplanted to the Anterior Chamber with Sweat Glands

When a sympathetic ganglion from a newborn rat, the SCG, was transplanted to the anterior chamber with the footpad tissue, sympathetic neurons in the cotransplanted ganglion survived and innervated the footpads. After transplantation, the ganglion and footpad coalesced to form one piece of tissue and a small group of neurons was evident at the periphery of the transplant. The number of neurons which survived after 4 weeks in the SCG/footpad cotransplants was small; in most transplants, the number appeared to be between 10 and 20 neurons. To determine whether the glands became innervated by the cotransplanted ganglion, the glands were examined for ~65 immunoreactivity. At 11 days and at 4 weeks after transplantation, a plexus of p65-immunoreactive fibers was evident in the glands (Figs. lb, Id), although the density of the gland innervation appeared somewhat decreased at 4 weeks.

STEVENS AND LANDIS

Target Injbwnces

As the transplanted glands and their innervation developed in the anterior chamber, the transmitter-related properties of the innervation changed. When SCG/footpad transplants were examined after lo-12 days in oculo, the gland innervation possessed bright catecholamine fluorescence (Fig. la). After 4 weeks in oculo, however, catecholamine fluorescence was no longer detectable (Fig. lc). However, the presence of p65-positive fibers in adjacent sections through the same glands indicated that the glands remained innervated (Fig. Id). The innervation of the transplanted glands was also examined for the presence of AChE activity, a characteristic of the mature sweat gland innervation in vivo. AChE was detectable lo-12 days post-transplantation (Fig. 2a), and had intensified after 4 weeks in oculo (Fig. 2b). The characteristics of the gland innervation were unchanged when the superior cervical and ciliary ganglia of the host were removed 3 to 7 days prior to examining the transplant. This finding indicates, as expected from studies of footpads transplanted alone, that the cotransplanted SCG and not the host’s own ganglia was the source of innervation to the glands. The changes detected in the innervation of the transplanted glands were evident in the cell bodies of the cotransplanted neurons as well. When the transplants were examined lo-12 days after transplantation, catecholamine fluorescent cell bodies were present in aggregates at the edge of the tissue (not shown). Four weeks after transplantation, however, catecholamine fluorescent cell bodies were no longer detectable. Correspondingly, at lo-12 days post-transplantation, clusters of moderately AChE-positive cell somas were observed which resembled the catecholamine-containing neurons seen at this stage in size and distribution. By 4 intense AChE activweeks in o&o, neurons exhibiting ity were located in small groups on the periphery of the transplants (Fig. 3a). The results described above suggested that like the neurons which innervate the glands in situ, the neurons which innervated the transplanted glands underwent a transition in which they lost their noradrenergic properties and acquired cholinergic function. However, although AChE activity is correlated with the development of cholinergic function in the sweat gland innervation in situ, its presence in the gland innervation in oculo is not necessarily indicative of cholinergic differentiation. Therefore, we also examined the cotransplanted neurons for the presence of ChAT. Four weeks after transplantation, small groups of ChAT-immunoreactive neurons were detectable at the periphery of the transplants (Fig. 3b). All neurons identified in the transplants contained ChAT immunoreactivity but we were not able to exclude the possibility that some

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transplanted neurons lacked immunoreactivity because unlabeled neurons were difficult to visualize and not every section taken from each transplant was examined. The presence of ChAT immunoreactivity in neurons which were cotransplanted with footpad tissue indicates that the decline in catecholamine fluorescence in the neurons and gland innervation was accompanied by a concomitant development of cholinergic function. To determine whether peptide expression in the innervation of the transplanted glands reflected the normal peptide expression of the neurons which innervate sweat glands or of the neurons in situ, the SCG/footpad cotransplants were examined for the presence of VIP and NPY immunoreactivity. In the adult rat, VIP is uniformly expressed by the sweat gland innervation in situ (Yodlowski et ah, 1984; Landis et al, 1988), but by only a rare neuron in the SCG (Hokfelt et ah, 1977; Sasek and Zigmond, 1989). In contrast, NPY is expressed by the majority of SCG neurons in situ (Jarvi et al., 1986), but is not evident in either the developing or the mature innervation of the sweat glands (Landis et al., 1988). After 4 weeks in oculo, the innervation of the transplanted glands invariably expressed VIP immunoreactivity (Fig. 4b). When the same glands were double-stained for the presence of p65 immunoreactivity, the correspondence between the fiber plexuses revealed by the two labels suggested that most, if not all, fibers were immunoreactive for VIP. Consistent with this observation, neurons exhibiting VIP immunoreactivity were detected at the periphery of the transplant (Fig. 4a). It is not clear whether all of the neurons present 4 weeks after transplantation expressed VIP immunoreactivity; as it was not always possible to examine the entire transplant, not every neuron present could be identified. However, the invariable expression of VIP immunoreactivity by the gland innervation suggests that neurons in every transplant contained VIP. When three transplants were examined lo-12 days posttransplantation, VIP immunoreactivity was present already. Thus, the onset of VIP expression occurred in at least some of the fibers during the first 10 days in oculo. In contrast to VIP no NPY immunoreactivity was detectable in neuronal cell bodies in ganglion/footpad cotransplants and only an occasional faintly NPY-immunoreactive fiber was observed in the transplanted glands. Since the intrinsic innervation of the iris did not invade the footpad transplants, the rare, faintly immunofluorescent NPY-containing fibers appear to have arisen from transplanted neurons with very low levels of perikaryonal NPY. The findings described above suggested that the transplanted SCG neurons which innervated the sweat glands in oculo went through a transition in transmitter phenotype which was very similar to that seen in the

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Target Influences on Transmitter Choice

FIG. 2. The development of AChE activity in the innervation of the transplanted glands. Tubules of the developing glands appear larger than do those of mature glands and contain more densely stained cells. (a) AChE activity is detectable in the gland innervation by 12 days post-transplantation. (b) At 4 weeks post-transplantation, the fiber plexus which innervates the transplanted glands exhibits intense AChE activity. X512.

gland innervation. Since the neurons in the SCG normally innervate other targets, such as the iris and pineal gland, and differentiate noradrenergieally, this is consistent with the idea that the decline in catecholamine expression and the appearance of ChAT and VIP immunoreactivities in the transplanted SCG neurons were promoted by contact with sweat glands. It was also possible, however, that the shift in transmitter phenotype exhibited by the transplanted neurons did not reflect a specific influence of the sweat glands but was rather a consequence of explantation to the anterior chamber, such as denervation or exposure to a factor(s) present in the anterior chamber, and did not reflect a specific influence of the sweat glands. To address

the specificity of the sweat gland effect in altering transmitter phenotype, the SCG was cotransplanted with the pineal gland, a sympathetic target organ which in situ receives only noradrenergic sympathetic innervation from the SCG (Bondareff and Gordon, 1966; Owman, 1964; Bowers et al., 1984) and does not receive a cholinergic parasympathetic innervation (Schrier and Klein, 1974).

Transmitter Phenotype of Sympathetic Neurons Transplanted to the Anterior Chamber with Pineal Gland When neonatal SCG and pineal glands were transplanted to the anterior chamber, the pineal transplants

FIG. 1. Catecholamine fluorescence and p65 immunoreactivity in the innervation of transplanted sweat glands at 11 days (a, b) and 4 weeks (c, d) post-transplantation. Alternating lo-pm sections were reacted with glyoxylic acid to induce catecholamine histofluorescence (a, c) or processed to visualize ~65, a synaptic vesicle antigen (b, d). (a) At 11 days post-transplantation catecholamine fluorescent fibers are present in the gland innervation. The transplanted glands contain many fat cells (asterisk). X320. (b) An adjacent section from the same gland as in a reveals the presence of a p65-immunoreactive fiber plexus. X320. (c) Four weeks after transplantation, the gland innervation no longer exhibits detectable catecholamine fluorescence. X296. (d) The presence of p65-immunoreactive fibers in an adjacent section from the same gland as in c indicates that the gland is innervated and that the absence of catecholamine fluorescence is not due to a lack of innervation. X296.

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in sympathetic neurons 4 weeks after cotransplantation with footpad tissue to the FIG. 3. AChE activity and ChAT immunoreactivity neurons (arrow) are detectable in small clusters on the edge of the footpad transplants. Ten-microm eter anterio br chamber. (a) AChE-positive section. X400. neurons (arrow) are present in small I groups on the transplant periphery. Twenty-micrometer section (b) ChAT-immunoreactive

were vascularized and became innervated by both the host and the transplanted SCGs. Because pineal glands transplanted to the anterior chamber are known to receive ingrowth of sympathetic fibers from the host iris (Moore, 1975; Backstrom et al., 1976; Lingappa and Zigmond, 198’7), the host SCG was removed from the transplant side to eliminate sympathetic innervation to the transplanted pineal gland from the host. When the host SCG was removed at the time of transplantation, the transplanted tissue appeared less well vascularized and unhealthy compared to transplants in which the host SCG was intact. Therefore, the host SCG was not removed until 3 to 7 days prior to examining the transplant. In most cases, the ciliary ganglion was removed at the same time. The number of neurons which survived transplantation with the pineal gland was 10 to 20, similar to the number observed with footpad transplants.

The expression of catecholamines, NPY, and VIP in sympathetic neurons cotransplanted with pineal gland differed markedly from their expression in neurons which were transplanted with footpad tissue. After 4 weeks in the anterior chamber, both neuronal cell bodies and their fibers which innervated the pineal gland exhibited catecholamine fluorescence (Fig. 5). NPY-immunoreactive neurons were invariably present, and NPY-immunoreactive fibers could be detected coursing through the pineal gland (Fig. 6). In contrast, no VIP-immunoreactive cell bodies or fibers were present. Thus, both catecholamines and the two neuropeptides examined were expressed in a different and target-appropriate manner in the pineal and footpad transplants. The expression of AChE activity and ChAT immunoreactivity by the SCG neurons cotransplanted with the pineal gland, however, was similar to that observed in

STEVENS AND LANDIS

FIG. 4. VIP immunoreactivity in SCG/footpad transplants neurons are detectable on the periphery of the transplant. immunoreactivity. X576.

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to the anterior chamber 4 weeks post-transplantation. (a) VIP-immunoreactive X550. (b) The innervation of the transplanted glands invariably exhibits VIP

FIG. 5. Catecholamine fluorescence in SCG/pineal cotransplants to the anterior chamber 4 weeks after transplantation. (a) Catecholaminecontaining neurons (arrowheads) are present. (b) Fibers exhibiting catecholamine fluorescence are detectable in the transplanted pineal gland. The diffuse appearance of the fluorescent fibers is typical of the sympathetic innervation of the pineal gland in situ. X400.

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V0~~~~137,1990

FIG. 6. NPY immunoreactivity in the SCG/pineal cotransplants 4 weeks after transplantation. (a) Numerous NPY-immunoreactive are present. (b) Fibers exhibiting NPY immunoreactivity are detectable in the transplanted pineal gland. X320.

the footpad/SCG transplants. At 4 weeks post-transplantation, groups of intensely AChE-positive cell bodies were detected (Fig. ‘7a). Further, several ChATimmunoreactive neurons were also present in some, but not all, cotransplants after 4 weeks in oculo (Fig. 7b). In the transplants in which ChAT-immunoreactive neurons were detectable, the number of neurons exhibiting detectable ChAT immunoreactivity was approximately five, considerably less than the number usually seen exhibiting either AChE activity or NPY immunoreactivity. Although the presence of ChAT-immunoreactive neurons indicates that at least some of the neurons which were transplanted with the pineal gland developed cholinergic function, the findings that not all transplants contained ChAT-positive neurons and that not all neurons appeared to be ChAT-positive suggest that the cotransplantation with the pineal gland was not as effective in inducing cholinergic properties as cotransplantation with sweat glands.

neurons

DISCUSSION

Sympathetic ganglia were transplanted to the anterior chamber with either a cholinergic target, sweat glands, or a noradrenergic target, the pineal gland, to examine the role of the target in influencing neurotransmitter properties during development. The innervation of sweat and pineal glands differs not only in the expression of a classical transmitter but also in the neuropeptides which they contain. When footpads and SCG from neonatal rats were cotransplanted, sweat glands developed in the footpad tissue and became innervated by sympathetic neurons in the accompanying SCG. The innervation of the glands initially exhibited catecholamine fluorescence and faint AChE activity. After 4 weeks in oculo, however, the innervation of the transplanted glands lacked detectable catecholamine fluorescence and contained intense AChE activity. In addition, the gland innervation invariably expressed

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Target Influences cm Transmitter Choice

119

FIG. 7. AChE and ChAT immunoreactivity in sympathetic neurons 4 weeks after transplantation with pineal gland to the anterior chamber. (a) Neurons exhibiting intense AChE activity are present (arrow) in the SCG/pineal transplants. X440. (b) ChAT-immunoreactive neurons (arrows) are detectable. The pineal transplants also contain many mast cells (arrowhead) which demonstrate nonspecific labeling with the immunoperoxidase reagents. X400.

VIP immunoreactivity. The neurotransmitter properties evident in the gland innervation were paralleled by those exhibited by the transplanted neurons, which expressed immunoreactivity for ChAT as well. When SCG were cotransplanted to the anterior chamber with pineal gland, like the neurons which were cotransplanted with the footpad, neurons in the SCG/pineal cotransplants developed AChE activity and some developed ChAT immunoreactivity. In contrast to the SCG/footpad transplants, however, catecholamine-containing neurons and fibers were still detectable in the SCG/pineal cotransplants after 4 weeks in oculo. In addition, NPY-immunoreactive neurons were invariably present after cotransplantation with the pineal gland, but neurons exhibiting VIP immunoreactivity were never observed. In the experiments reported in this study, striking differences were observed in the neurotransmitter properties of sympathetic neurons transplanted to the anterior chamber with different target tissues. One interpretation, discussed in detail below, is that the targets influenced the transmitter properties of the neurons. An alternative interpretation is that different subpopulations of sympathetic neurons survived in the

presence of the two targets. According to this interpretation, transplantation with sweat glands would result in the survival of VIP and ChAT-IR neurons, while transplantation with pineal gland would allow the survival of catecholamine-containing and NPY-IR neurons. The small number of neurons, 10 to 20, which survive transplantation to the anterior chamber would be consistent with this hypothesis. An estimate of the number of neurons present in the neonatal rat SCG is 39,000, and although this number is reported to decline to 25,000 during the first 2 postnatal weeks (Wright et al., 1983), it is still several orders of magnitude larger than the number of neurons present 4 weeks after transplantation to the anterior chamber. In addition, although only a very small proportion of neurons in the adult SCG have detectable levels of either VIP IR or ChAT IR (Sasek and Zigmond, 1989; Landis, unpublished observations), their number would be consistent with the number surviving transplantation. However, the interpretation that differences in transmitter expression reflect selective survival presumes that there are distinct trophic factors which are specific for subpopulations of neurons expressing different transmitter phenotypes. In fact, studies of sympathetic neurons

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both in viva (Levi-Montalcini and Booker, 1960; LeviMontalcini and Angeletti, 1966; Gorin and Johnson, 1979, 1980; Landis et ab, 1985; Hill et aL, 1988) and in vitro (Chun and Patterson, 1977a,b; Raynaud et al, 1988) indicate that nerve growth factor is both necessary and sufficient for the survival of both noradrenergic and cholinergic sympathetic neurons in the rat. Rather, a large body of evidence summarized in the Introduction and discussed below supports the notion that environmental factors can regulate the expression of transmitter properties in postmitotic postnatal sympathetic neurons like those transplanted in the present study. Thus, although we cannot exclude this possibility, it seems highly unlikely that selective survival accounts for our observations. The expression of catecholamines by the neurons cotransplanted with footpad or with pineal gland differed markedly and in both cases resembled that seen in the innervation of these targets in situ. In the SCG/footpad cotransplants, at 10 to 12 days post-transplantation the gland innervation and many neurons were brightly catecholamine histofluorescent; after 4 weeks in oculo, however, catecholamines were undetectable in both the gland innervation and surviving neurons. This change resembles the loss of catecholamines’ fluorescence seen during the normal development of sweat gland innervation in situ (Landis and Keefe, 1983) and that following transplantation of sweat glands containing skin to hairy skin regions (Schotzinger and Landis, 1988). In contrast, in the SCG/pineal cotransplants, catecholamine-containing neurons and fibers could still be detected 4 weeks after transplantation. These results suggest that the transplanted targets influenced the expression of noradrenergic properties in the neurons which innervated them; catecholamine expression was apparently suppressed in the presence of the sweat glands, whereas it was maintained, and perhaps promoted, in the presence of the pineal gland. Like the classical transmitter, norepinephrine, neuropeptide expression by neurons developing in the anterior chamber varied according to whether they were transplanted with footpad or pineal gland and similarly reflected the pattern of expression exhibited by the fibers which innervate the target in situ. VIP is normally present in the cholinergic sympathetic innervation of sweat glands (Lundberg et al., 1979; Yodlowski et al., 1984; Landis et al., 1988), whereas NPY is evident in many noradrenergic SCG neurons (Lundberg et al., 1982b; Jarvi et ab, 1986) including those that innervate the pineal gland (Schon et al., 1985). The coexpression of neuropeptide and traditional transmitter, in particular of VIP with acetylcholine and of NPY with norepinephrine, has been described as an organizational principal in the sympathetic nervous system (Lundberg et al.,

1982a). The simultaneous expression of even these particular neurotransmitters and neuropeptides does not appear to be obligatory, however, since in other parts of the autonomic nervous system, NPY can be expressed in cholinergic neurons and VIP in noncholinergic neurons (Gu et ak, 1984; Furness et al, 1984; Hassall and Burnstock, 1984; Leblanc et al, 1987, Leblanc and Landis, 1988). Further, when cultured sympathetic neurons are induced to become cholinergic by treatment with heart cell-conditioned medium, they continue to express high levels of NPY (Marek and Mains, 1989). In the present experiments, most, if not all, of the neurons in the SCG/pineal cotransplants expressed NPY and some expressed ChAT immunoreactivity, suggesting that, as in the culture studies, cholinergic sympathetic neurons may express NPY. The results of the present study raise the possibility that the developmentally relevant relationship is, in fact, between a neuropeptide and target organ rather than between a classical neurotransmitter and a neuropeptide. Numerous examples exist of the restriction of neuropeptide expression to a subpopulation of neurons which innervate a particular target. For example, NPY is present in sympathetic neurons which innervate the vasculature but not the parenchyma of glands (Lundberg et aZ.,1982; Horn et ak, 1987; see also Morris and Gibbins, 1987; O’Connor and van der Kooy, 1988; Leblanc and Landis, 1988 for other examples). Thus, target-appropriate expression of neuropeptides may be influenced directly by the target tissue and be independent of the classical transmitter. The differential expression of NPY and VIP in SCG neurons cotransplanted with pineal and sweat glands, respectively, in the present study is consistent with the hypothesis that target tissues produce factors which determine the neuropeptides synthesized by neurons which innervate them. The observation that Substance P immunoreactivity appears in muscle sensory afferent fibers induced to innervate skin is also consistent with this hypothesis (McMahon and Gibson, 1987). Target regulation of peptide expression in sympathetic neurons has previously been suggested by studies in which SCG neurons cocultured with pineal or salivary glands exhibited increased expression of Substance P, whereas coculture with heart and intestine did not exert this effect (Kessler et aZ., 1984). However, since Substance P is not normally expressed at detectable levels by sympathetic neurons in who, the significance of these findings for peptide choice in situ is unclear. Candidates for factors that influence neuropeptide expression are being revealed in cell culture studies. A membrane-associated protein, MANS or membrane-associated neurotransmitter stimulating factor, has been shown to elevate SP levels (Wong and Kessler, 1987).

STEVENS AND LANDIS

Target Ir&ences

Ciliary neuronotrophic factor (CNTF) has been found to elevate levels of VIP and the number of VIP-immunoreactive neurons in cultures of chick sympathetic ganglia (Ernsberger et al, 1989). Finally, preliminary studies reveal the presence of several components in heart cellconditioned medium that differentially regulate peptide expression in cultures of dissociated sympathetic neurons (Nawa and Patterson, 1988). At present, data linking peptide expression and target projections in viva are largely correlative and this relationship could be achieved by a number of developmental mechanisms; additional experimental manipulation is required to establish a role for the target in this process in vivo and to define the relevant factors. Unlike the differential expression of catecholamines and neuropeptides, AChE was expressed in sympathetic neurons cotransplanted with both footpad and pineal gland. In situ, AChE activity is present in the sympathetic innervation of both sweat and pineal glands (Landis and Keefe, 1983; Eranko et al., 1970). Thus, although the expression of AChE was similar in the two types of transplants, like the expression of catecholamines and neuropeptides, it was target appropriate. In contrast to the target-appropriate expression of catecholamines, peptides and AChE, ChAT-immunoreactive neurons were present in cotransplants with the pineal gland, an adrenergic target, as well as with a cholinergic target, the sweat glands. The sympathetic neurons which innervate sweat glands normally acquire cholinergic function during development (Leblanc and Landis, 1986; Stevens and Landis, 1987). The role of the target in this induction was suggested in studies in which the innervation of sweat glands was experimentally disrupted causing a delay in both the arrival of nerve fibers in the sweat glands and the appearance of cholinergic function in the innervation (Stevens and Landis, 1988) and confirmed in studies in which sweat gland containing skin was transplanted to the hairy skin region of the thorax (Schotzinger and Landis, 1988). In these experiments, noradrenergic neurons which would normally innervate piloerectors and blood vessels in the hairy skin instead innervated sweat glands and acquired ChAT activity. The present experiments provide additional experimental evidence for the ability of sweat glands to induce cholinergic function in sympathetic neurons. Conversely, there is no evidence to suggest that the sympathetic innervation of the pineal gland contains ChAT in situ. Several observations suggest that the level of cholinergic induction was lower in the pineal than in the footpad cotransplants. In SCG transplanted with footpads, ChAT IR was observed in all transplants examined and most, if not all, neurons were detectably ChAT IR. In contrast, when SCG neurons were transplanted with pineal gland, not all

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transplants contained detectable ChAT IR, and in transplants where ChAT IR was evident, not all neurons appeared to express ChAT IR. Further, the neurons cotransplanted with sweat glands apparently lost their noradrenergic properties as they developed cholinergic function, whereas catecholamine expression is likely to have been maintained in ChAT-IR neurons cotransplanted with pineal gland. Studies of sympathetic neurons developing in vitro have indicated that the stronger the cholinergic induction, the more complete the suppression of noradrenergic properties (Wolinsky and Patterson, 1983; Raynaud et ah, 1987); under conditions in which the induction of cholinergic function is relatively weak, neurons continue to express noradrenergic properties (Higgins et al., 1981; Iacovitti et al, 1981; Wolinsky and Patterson, 1983; Raynaud et aZ., 1987). Taken together, these observations suggest that sweat glands are more effective at inducing cholinergic function than the pineal gland. Given that the expression of catecholamines, neuropeptides, and AChE appeared to be regulated strictly according to the phenotype of the innervation of the development of target in situ, the target-inappropriate even some cholinergic function in neurons cotransplanted with the pineal gland was surprising. An explanation for this result may be found in considering that although the target organ was the most prominent developmental variable in these experiments, several other aspects of the environment of both the neurons and their targets were altered relative to what they experienced in situ. These factors may also have played a role in the transmitter choice of the neurons. For example, it is possible that a cholinergic inducing activity, similar to that described in rat serum (Wolinsky and Patterson, 1985), is present in the anterior chamber and that the development of cholinergic function in the transplanted neurons resulted from exposure to this factor and not solely from influences exerted by the target organ. A possible candidate for this activity would be CNTF. Originally purified from chick eye tissues (Adler et al., 1979), it is also present in rat sciatic nerve (Manthorpe et ak, 1986) and is able to induce cholinergic function in cultured rat sympathetic neurons (Saadat et al., 1989). In addition, the sympathetic neurons transplanted to the anterior chamber were deprived of their normal preganglionic innervation which could have made them less susceptible to cholinergic factors. Studies of sympathetic neurons developing in vitro have demonstrated that chronic depolarization of the neurons decreases their responsiveness to cholinergic induction (Walicke et ak, 1977; Raynaud et al, 1987). Given the apparent differences in the degree of cholinergic induction apparent in neurons cotransplanted with sweat glands and pineal gland, it appears unlikely

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that either the lack of preganglionic innervation or the presence of a general hormonal factor in the anterior chamber is entirely responsible for the development of ChAT IR in neurons cotransplanted with sweat glands as well as in the pineal cotransplants. A more likely explanation for the appearance of ChAT IR in neurons cotransplanted with pineal gland is that although the pineal gland does not promote cholinergic differentiation in situ, when growing in oculo the pineal gland may have been exposed to factors present in the anterior chamber which stimulated it to promote cholinergic differentiation in the transplanted neurons. Consistent with this possibility, it has been shown in cell culture that the ability of nonneuronal cells to induce cholinergic development can be altered by hormones (Fukada, 1980; McLennan et ak, 1980). Thus, although the anterior chamber has been regarded as providing a normal environment for organotypic development of neurons and their targets, the induction of ChAT in SCG neurons cotransplanted with pineal gland in the present experiments is reminiscent of previous cell culture studies in which coculture of SCG neurons and pineal cells (Rowe and Parr, 1980) and growth in medium conditioned by pineal cells (Kessler, 1984a, 1985) were found to result in the development of ChAT activity. In these studies, as in the SCG/pineal transplants to the anterior chamber, the increase in ChAT activity was relatively small and either no or only a small reduction in tyrosine hydroxylase, a catecholamine synthetic enzyme, was observed. Thus, the pineal gland developing in oculo may have promoted a small induction of cholinergic function in the cotransplanted SCG which did not result in a concomitant suppression of their noradrenergic properties. In contrast, the sweat glands, independent of growth in the anterior chamber, may have exerted a strong cholinergic influence which led to a more complete noradrenergic to cholinergic transition. In previous studies of target tissues transplanted to the anterior chamber, transplanted organs were found to be innervated by fibers growing out from the host iris in a manner generally appropriate for that target based on innervation in situ. For example, hearts transplanted from fetal rats are innervated by both noradrenergic sympathetic and cholinergic parasympathetic fibers (Olson and Seiger, 1976; Taylor et ak, 1978), pineal glands are innervated by noradrenergic sympathetic fibers which are capable of inducing the activity of Nacetyltransferase in the pineal cells (Moore, 1975; Backstrom et ab, 1976) although not capable of establishing a diurnal rhythm of normal amplitude (Lingappa and Zigmond, 1987), and adrenal chromaffin cells receive only cholinergic innervation (Unsicker et cd., 1978). These results suggest that target tissue transplanted to the anterior chamber may select and/or pro-

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mote innervation by mature fibers from the host iris which express the appropriate transmitter. The present study provides evidence that when target tissues and developmentally plastic sympathetic neurons are transplanted to the anterior chamber together, the target plays a role in determining the appropriate expression of both neuropeptides and classical neurotransmitters by the neurons which innervate it. We thank Dr. Jaisri Lingappa for demonstrating the anterior chamber transplantation technique and Drs. Bill Matthew and Felix Eckenstein for the kind gift of antibodies. We also 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 NS023678, NS07112, and MH18012. Leslie Stevens was the recipient of a predoctoral fellowship from the National Science Foundation. REFERENCES ADLER, J. E., and BLACK, I. B. (1985). Sympathetic neuron density differentially regulates transmitter phenotype expression in culture. Proc. NatL Acad. Sci. USA 82,4296-4300. ADLER, J. E., and BLACK, I. B. (1986). Membrane contact regulates transmitter phenotypic expression. Dev. Brain Res. 30,23’7-241. ADLER, R., LANDA, K., MANTHORPE, M., and VARON, S. (1979). Cholinergic neuronotrophic factors. II. Intraocular distribution of trophic activity for ciliary neurons. Science 204,1434-1436. BACKSTROM, M., OLSON, L., and SEIGER, A. (1976). N-Acetyltransferase and hydroxyindole-0-methyltransferase activity in intraocular pineal transplants: Diurnal rhythm as evidence for functional sympathetic adrenergic innervation. Acta PhysioL Stand 96.64-71. BIXBY, J. L., and REICHARDT, L. F. (1985). The expression and localization of synaptic vesicle antigens at neuromuscular junctions in vitro. J. Neurosci. 5,3070-3080. BONDAREFF, W., and GORDON, B. (1966). Submicroscopic localization of norepinephrine in sympathetic nerves of rat pineal. J. PhamacoL Exp. Ther. 153,42-47. BOWERS, C. W., BALDWIN, C., and ZIGMOND, R. E. (1984). Sympathetic reinnervation of the pineal gland after postganglionic nerve lesion does not restore normal pineal function. J. Neurosci. 4,2010-2015. CHUN, L. L. Y., and PATTERSON, P. H. (1977a). Role of nerve growth factor in the development of sympathetic neurons in vitro. I. Survival, growth and differentiation of catecholamine production. J. Cell BioL 75,694-704. CHUN, L. L. Y., and PATTERSON, P. H. (197713). Role of nerve growth factor in the development of sympathetic neurons in vitro. III. Effects on acetylcholine production. J. Cell Biol. 75, 712-718. DE LA TORRE, (1980). An improved approach to histofluorescence using the SPG method for tissue monoamines. J. Neurosci. Methods 3,1-5. ECKENSTEIN, F., BARDE, Y. A., and THOENEN, H. (1981). Production of specific antibodies to choline acetyltransferase purified from pig brain. Neuroscience 6,993-1000. ERANKO, O., REICHARDT, L., ERANKO, L., and CUNINGHAM, A. (1970). Light and electron microscopic histochemical observations on cholinesterase-containing sympathetic nerve fibers in the pineal body of the rat. Histochem. J 2,479-489. ERNSBERGER, U., SENDTNER, M., and ROHRER, H. (1989). Proliferation and differentiation of embryonic chick sympathetic neurons: Effects of ciliary neurotropbic factor. Neuron 2.1275-1284. FUKADA, K. (1980). Hormonal control of neurotransmitter choice in sympathetic neurone cultures. Nature (Londm) 287,553-555.

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