Norepinephrine Transporter Expression in Cholinergic Sympathetic Neurons: Differential Regulation of Membrane and Vesicular Transporters

Developmental Biology 220, 85–96 (2000) doi:10.1006/dbio.2000.9631, available online at http://www.idealibrary.com on

Norepinephrine Transporter Expression in Cholinergic Sympathetic Neurons: Differential Regulation of Membrane and Vesicular Transporters Beth A. Habecker,* ,1 Michael G. Klein,* Brian C. Cox,† and Benjamin A. Packard‡ *Department of Physiology and Pharmacology, Oregon Health Sciences University, Portland, Oregon 97201; †George Fox University, Newberg, Oregon 97132; and ‡Mt. Vernon Nazarene College, Mt. Vernon, Ohio 43050

Sympathetic neurons that undergo a noradrenergic to cholinergic change in phenotype provide a useful model system to examine the developmental regulation of proteins required to synthesize, store, or remove a particular neurotransmitter. This type of change occurs in the sympathetic sweat gland innervation during development and can be induced in cultured sympathetic neurons by extracts of sweat gland-containing footpads or by leukemia inhibitory factor. Sympathetic neurons initially produce norepinephrine (NE) and contain the vesicular monoamine transporter 2 (VMAT2), which packages NE into vesicles, and the norepinephrine transporter (NET), which removes NE from the synaptic cleft to terminate signaling. We have used a variety of biochemical and molecular techniques to test whether VMAT2 and NET levels decrease in sympathetic neurons which stop producing NE and make acetylcholine. In cultured sympathetic neurons, NET protein and mRNA decreased during the switch to a cholinergic phenotype but VMAT2 mRNA and protein did not decline. NET immunoreactivity disappeared from the developing sweat gland innervation in vivo as it acquired cholinergic properties. Surprisingly, NET simultaneously appeared in sweat gland myoepithelial cells. The presence of NET in myoepithelial cells did not require sympathetic innervation. VMAT2 levels did not decrease as the sweat gland innervation became cholinergic, indicating that NE synthesis and vesicular packaging are not coupled in this system. Thus, production of NE and the transporters required for noradrenergic transmission are not coordinately regulated during cholinergic development. © 2000 Academic Press Key Words: norepinephrine transporter (NET); vesicular monoamine transporter (VMAT2); sympathetic development; cholinergic differentiation.

INTRODUCTION The specification of chemical phenotype is a critical aspect of neural development, since the proper matching of presynaptic neurotransmitter with postsynaptic receptor is required for functional signaling. Synthesis of the correct transmitter is not sufficient to ensure synaptic transmission, however, since transmitters must be packaged into vesicles for release and removed from the synapse to terminate signaling. Each neurotransmitter utilizes a particular complement of transport proteins, including a vesicular 1

To whom correspondence should be addressed at the Department of Physiology and Pharmacology, L334, Oregon Health Sciences University, Portland, OR 97201-3098. Fax: (503) 494-4352. E-mail: [email protected]. 0012-1606/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

transporter for storage into vesicles and a plasma membrane transporter for reuptake from the synapse. For example, noradrenergic neurons contain a vesicular monoamine transporter (VMAT2) (Liu et al., 1996) that transports newly synthesized dopamine into vesicles for conversion to norepinephrine (NE) and repackages NE that has been brought back into the cell by the plasma membrane NE transporter (NET) (Pacholczyk et al., 1991). To ensure that neurotransmitter synthesis is accompanied by storage into vesicles and reuptake from the synapse, expression of the synthetic enzymes and transporters could be coregulated during development. The mechanisms that control the developmental expression of proteins required to synthesize, store, or remove a particular neurotransmitter are not well understood, but coordinate regulation of synthetic enzymes and vesicular transporters has been

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observed in at least two neurotransmitter systems. In cholinergic neurons of many species the gene encoding the vesicular acetylcholine transporter (VAChT) is embedded in the gene encoding choline acetyltransferase (ChAT), the synthetic enzyme for acetylcholine (ACh), and the two are regulated by shared promoter elements (Bejanin et al., 1994; Erickson et al., 1994; Eiden, 1998). Most neurotransmitter systems do not share this type of genetic clustering, however, and it is not essential for coregulation of synthetic enzymes and vesicular transporters. In Caenorhabditis elegans the unc-25 gene, which encodes the ␥-amino butyric acid (GABA) synthetic enzyme glutamic acid decarboxylase, is coregulated with unc-47, which encodes the vesicular GABA transporter (Eastman et al., 1999). In contrast to the well-defined examples of coregulation between synthetic enzymes and vesicular transporters, little is known about developmental regulation of transmitter synthetic enzymes and expression of plasma membrane transporters. During the development of the sympathetic nervous system, the appearance of tyrosine hydroxylase (TH), the rate-limiting enzyme in NE synthesis, corresponds with the onset of vesicular storage as well as catecholamine reuptake (Rohrer and Ernsberger, 1998; Ernsberger et al., 1995), providing indirect evidence for coregulation of TH and NET. Studies in adult animals, however, indicate that plasma membrane transporters are often regulated in the direction opposite to that of synthetic enzymes and vesicular transporters. For example, in some noradrenergic neurons acute NE depletion increases catecholamine production and storage into vesicles by VMAT2, but decreases reuptake via NET, while increased synaptic NE levels decrease TH but increase NET expression (Cubells et al., 1995b; Xiao et al., 1995). This suggests that the mechanisms required to control the expression of enzymes and transporters during development are quite different from those that maintain homeostasis in adult animals. The subset of sympathetic neurons which undergo a noradrenergic to cholinergic change in transmitter phenotype provides a unique model system to investigate the developmental regulation of proteins involved in neurotransmission. Like other sympathetic neurons they initially acquire noradrenergic properties including NE synthesis, vesicular storage, and catecholamine reuptake (Iacovitti et al., 1982). These neurons undergo a change in neurotransmitter properties, however, as NE disappears and is replaced by ACh and vasoactive intestinal peptide (VIP) (Landis, 1990). The proteins associated with ACh production and packaging, ChAT and VAChT, are coordinately upregulated in cultured sympathetic neurons induced to acquire cholinergic properties (Berrard et al., 1993; Misawa et al., 1995), due to their regulation by the same promoter. The simultaneous onset of several noradrenergic properties during development suggests that expression of the enzymes and transporters associated with noradrenergic function are also coordinately regulated, and that these properties will likewise be suppressed as a group by cholinergic

differentiation factors (CDFs) which induce ACh synthesis in sympathetic neurons. To investigate whether cholinergic differentiation factors coordinately suppress all aspects of noradrenergic function, we have examined the cholinergic sympathetic innervation of rodent sweat glands and cultured cholinergic sympathetic neurons. When sympathetic axons first reach the developing sweat glands in the rear footpad they contain vesicular stores of NE (Guidry and Landis, 1998; Landis and Keefe, 1983), but interactions with the sweat glands inhibit noradrenergic and induce cholinergic function so that by P21 the gland sympathetic innervation contains ACh and VIP (Schotzinger et al., 1994; Schotzinger and Landis, 1988, 1990). The loss of NE in the developing rat sweat gland innervation is accompanied by decreases in the NE synthetic enzymes TH and dopamine ␤-hydroxylase (Landis et al., 1988), but it remains unclear whether the NE transporters VMAT2 and NET decline as well. Soluble extracts of sweat gland-containing footpads (Habecker and Landis, 1994), and the cytokines leukemia inhibitory factor (LIF) (Fukada, 1985; Nawa et al., 1991; Rao et al., 1992a; Yamamori et al., 1989) and ciliary neurotrophic factor (CNTF) (Ernsberger et al., 1989; Rao et al., 1992b; Saadat et al., 1989), induce the same set of phenotypic changes in cultured sympathetic neurons (Rao et al.,1992a; Rohrer, 1992). Although neither LIF nor CNTF is the sweat glandderived cholinergic differentiation factor (Francis et al., 1997), they provide an additional model to test whether this family of cytokines alters VMAT2 and NET content in cultured sympathetic neurons that have been induced to acquire cholinergic properties. We hypothesized that both VMAT2 and NET decrease as NE disappears in sympathetic neurons undergoing a noradrenergic to cholinergic switch. We used a variety of biochemical and molecular techniques to investigate the expression and activity of VMAT2 and NET in the developing sweat gland sympathetic innervation and in cultured sympathetic neurons. Consistent with our hypothesis, NET levels decreased significantly as sympathetic neurons became cholinergic, indicating that CDFs regulate the developmental expression of NE transporters via mechanisms distinct from those used for the maintenance of homeostasis. Surprisingly, VMAT2 levels did not decrease as TH expression and NE synthesis declined. This suggests that, in contrast to other neurotransmitter systems, production of NE is controlled independent of its storage during the cholinergic development of sympathetic neurons.

MATERIALS AND METHODS Primary Cell Culture Cultures of sympathetic neurons were prepared from the superior cervical ganglia of newborn rats as described by Hawrot and Patterson (1979) and modified by Rao and Landis (1990). To reduce the number of nonneuronal cells, neurons were preplated for at least 2 h and then were grown in the antimitotic agent cytosine

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arabinoside (10 ␮M) for 2 days after plating. Cytokines and tissue extracts were diluted in medium and filter sterilized before addition to cultures. Cells were grown in L15CO 2- or F12-complete supplemented with NGF (100 ng/ml), penicillin G (100 U/ml), streptomycin sulfate (100 ␮g/ml), and 5% rat serum with similar results.

Sympathectomy To prevent development of the sympathetic nervous system, newborn rats were injected for 7 days with 100 mg/kg of the sympathetic neurotoxin 6-hydroxydopamine (6OHDA) or an equal volume of vehicle (0.9% NaCl, 1 mM ascorbate). This treatment eliminates sympathetic innervation of sweat glands (Yodlowski et al., 1984).

Biochemical Assays Cholinergic function was determined by measuring the activity of ChAT, the synthetic enzyme for acetylcholine, using the method of Fonnum (1966) as modified by Rao and Landis (1990). NE uptake was assayed essentially as described (Pacholczyk et al., 1991). Cultured sympathetic neurons were rinsed with Krebs– Ringer–Hepes buffer (KRH; 120 mM NaCl, 4.7 mM KCl, 2.2 mM CaCl, 1.2 mM KH 2PO 4, 1.2 mM MgSO 4, 10 mM Hepes, 5 mM Tris base, pH 7.4) and incubated for 15 min in KRH containing 10 nM [ 3H]NE, 1 mM ascorbic acid, 50 ␮M pargyline at 37°C with or without 10 ␮M imipramine. Excess 3H was removed by washing with ice-cold buffer lacking [ 3H]NE, and cells were solubilized in a solution of 0.1% SDS/0.1 M NaOH to quantify radioactivity. Nonspecific uptake was defined as that remaining in the presence of 10 ␮M imipramine and was negligible in the neuron cultures. The data shown represent total uptake.

RNA Isolation and Analysis

Immunohistochemistry Rats of the appropriate age were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer for 10 min. Footpads were postfixed in 4% paraformaldehyde for 1 h and cryoprotected overnight in a 30% sucrose/0.1 M phosphate solution. Tenmicrometer cryostat sections were thaw mounted onto gelatincoated or charged slides, rinsed in phosphate-buffered saline (PBS), preincubated in dilution buffer (2% BSA, 0.1% sodium azide, and 0.3% Triton X-100 in PBS) for 1 h, and incubated overnight with primary antisera raised against TH (rabbit anti-TH diluted 1:200; Pel-Freez Biologicals), VIP (guinea pig anti-VIP diluted 1:300; gift from Dr. Story Landis, NINDS, Bethesda, MD), NET (rabbit antiNET diluted 1:3000; gift from Dr. Randy Blakely, Vanderbilt University Medical School, Nashville, TN), or VMAT2 (affinitypurified rabbit anti-VMAT2 diluted 1:300; generated against 19 amino acids of the COOH-terminus of VMAT2 by Quality Controlled Biochemicals, Inc., Hopkinton, MA). Sections were then rinsed in PBS, incubated for 1–2 h with species-specific fluorescent secondary antibodies diluted 1:300 in dilution buffer containing 5% rat serum or goat serum, and rinsed again with PBS before visualization by fluorescence microscopy.

Extract Preparation Soluble footpad extracts were prepared from the rear footpads of rats and mice as described previously (Habecker et al., 1995; Rao et al.,1992a), with a single modification. Prior to homogenization with a Polytron, frozen footpad tissue was pulverized with a stainless steel mortar and pestle on dry ice. Extracts were concentrated by centrifugation through Centricon 10-kDa filters, and protein concentrations were determined using the Pierce protein assay kit. Extracts for Western blot analysis of transporter expression were prepared from tissue that was frozen on dry ice and stored at ⫺80°C until use. Frozen tissue was crushed with a stainless steel mortar and pestle on dry ice and homogenized 40 times with a groundglass homogenizer in disruption buffer (7.5 mM NaHPO 4, 10 mM EDTA, 1% SDS) supplemented with the protease inhibitors leupeptin (1 ␮g/ml), pepstatin (1 ␮g/ml), and phenylmethylsulfonyl fluoride (100 ␮g/ml). Samples were incubated at 37°C for 10 min and then centrifuged at 10,000g for 10 min. Supernatants were collected and stored at ⫺20°C or diluted in loading buffer.

Cytoplasmic RNA was isolated from tissue or from cultured sympathetic neurons using Trizol (Gibco BRL). Antisense 32Plabeled RNA probes were generated to mRNAs encoding TH, NET, VMAT2, and cyclophilin (used to normalize for differences in total RNA loading). RNA probes were synthesized from linearized plasmids with RNA polymerases and gel purified using a Fullengther apparatus (Dwarf Science, Aloha, OR). Plasmids containing TH (280 bp), NET (660 bp), and cyclophilin (132 bp) cDNAs were obtained from Dr. Richard Simerly, Oregon Regional Primate Center (Portland, OR). These probes have been used together successfully by other investigators (Brooks et al., 1997), and we adopted the same hybridization conditions for our experiments. A rat VMAT2 cDNA (bases 71– 493; 422 bp) was generated by reverse transcription-polymerase chain reaction, subcloned into the pCR-II TOPO vector (Invitrogen, Carlsbad, CA), and sequenced prior to use. For reactions, 2– 4 ␮g of total RNA was hybridized in a single tube either with TH, NET, and cyclophilin probes or with NET, VMAT2, and cyclophilin probes for 16 h at 45°C. Ribonuclease T1 (4 U/␮l) was added to digest single-stranded RNAs, and samples were extracted and protected RNA bands resolved on a polyacrylamide gel. Each gel was exposed to film for visualization and to a phosphorimager screen for quantitation. Bands were quantitated from subsaturating exposures of the phosphorimager screen on a Bio-Rad phosphorimager with Multi-Analyst software. Briefly, a rectangle of uniform size was placed over each band on the screen and the optical density per area of the rectangle was calculated. The background was subtracted from each measurement, and the ratio of the TH, NET, or VMAT OD/mm 2 to the cyclophilin OD/mm 2 was calculated. Multiple exposures of a single gel were used when necessary, in order to ensure that all samples analyzed were within the linear range of detection. TH, NET, and VMAT mRNAs were normalized to the cyclophilin control. NET:cyclophilin ratios were similar in reactions containing the TH probe and those containing the VMAT2 probe.

Immunoblotting Membrane extracts were diluted in loading buffer and denatured at 37°C for 30 min, and equivalent amounts of protein from each extract were separated on 8.5% SDS–polyacrylamide gels and transferred to nitrocellulose for identification of NET, VMAT2, and neuron-specific enolase (NSE). Blots were blocked 1 h with 5% nonfat dry milk in Tris-buffered saline–Tween 20 and incubated

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FIG. 1. Developmental expression of NET in whole footpad. (A) Representative Western blot. 5-␮g samples of total protein from postnatal day 6 (P6), 10, 15, and 21 and adult rat footpad were blotted, incubated with anti-NET diluted 1:1000, and visualized with chemiluminescence. Data shown are representative of results obtained with tissue from three or more animals at each age. (B) Western blot quantitation. Tissues from three different sets of animals were assayed on duplicate Western blots, and the blots were quantified as described under Materials and Methods. Data shown are the means ⫾ SEM, n ⫽ 3. #Two P10 samples were assayed only once.

overnight in blocking solution with guinea pig anti-VMAT2 diluted 1:2000 (Calbiochem, San Diego, CA), affinity-purified rabbit anti-NET diluted 1:1000 (gift from Dr. Susan Amara, The Vollum Institute, Portland, OR; Nguyen and Amara, 1996), or rabbit anti-NSE diluted 1:1000 (Polysciences, Inc., Warrington, PA). Bound antibodies were detected using IgG conjugated to horseradish peroxidase (1:10,000) and visualized by chemiluminescence (NEN-DuPont, Wilmington, DE). Blots were exposed onto film for visualization and onto a chemiluminescence phosphorimager screen for quantitation using MultiAnalyst software (Bio-Rad) as described for the RPA experiments. In some cases, films were scanned and the pixel density of the image was analyzed using the Multi-Analyst software. Dark film exposures have been shown for ease of visualization, but for purposes of quantitation only exposures that were within the linear range of the film were used. In each case, equal amounts of protein were loaded onto gels. Similar results were obtained with an affinity-purified anti-peptide VMAT2 antiserum from Quality Controlled Biochemicals, Inc. (data not shown) and the VMAT2 antiserum from Calbiochem.

[ 3H]Nisoxetine Binding NET was identified by [ 3H]Nisoxetine binding in membrane homogenates essentially as described by Zhu and Ordway (1997). Sweat glands were dissected from adult footpads and collected in ice-cold PBS. Sweat gland and brain tissue were homogenized in ice-cold buffer (10% wt/vol; 50 mM Tris, pH 7.4, 120 mM NaCl, 5 mM KCl) using a Polytron. Homogenates were washed twice in ice-cold homogenization buffer after centrifugation at 4°C, 40,000g for 30 min. Final pellets were resuspended in 500 ␮l ice-cold incubation buffer (50 mM Tris, pH 7.4, 300 mM NaCl, and 5 mM KCl) and duplicate 100-␮l aliquots assayed for total or background binding in the presence of 5 nM [ 3H]Nisoxetine (250 ␮l final reaction volume). Background binding was defined as that remaining in the presence of 10 ␮M imipramine. Reactions were incubated at 0°C for 3– 4 h and stopped by the addition 5 ml ice-cold incubation buffer. Samples were then filtered through glass fiber filters, presoaked in 0.3% polyethylenimine, using a Brandel cell harvester. Reaction tubes and filters were rinsed three times with cold incubation buffer, and radioactivity was determined by liquid scintillation counting. Protein concentrations were determined using the Pierce protein assay kit.

RESULTS NET Disappears from the Sweat Gland Sympathetic Innervation Neurotransmitter transporters are usually expressed in neurons which produce the corresponding transmitter substrate. Thus, we hypothesized that NET levels would decrease as NE content declined in cholinergic sympathetic neurons. To test whether NET levels decreased in the sweat gland innervation as those neurons ceased production of NE, footpad extracts from postnatal day 6 (P6), 10, 15, and 21 and adult rats were assayed for NET by Western blot. Surprisingly, no significant decrease in NET protein was detected in footpad extracts during the developmental switch from a noradrenergic to a cholinergic phenotype in the sweat gland innervation (Fig. 1; P6 NET, 0.07 ⫾ 0.03 OD/mm 2; adult NET, 0.09 ⫾ 0.02 OD/mm 2; n ⫽ 3; ⫾ SEM). Footpad extracts contain several different tissues, however, including two populations of sympathetic neurons: noradrenergic neurons which innervate the vasculature and cholinergic neurons which innervate the sweat glands. We used immunohistochemistry to localize NET within the footpad and determine if NET levels changed specifically within the sweat gland innervation. At the same time, anti-smooth muscle actin was used to identify myoepithelial cells within the developing glands. Although the pattern of actin immunoreactivity (actin-IR) did not change during development, NET immunoreactivity (NET-IR) was present in the gland innervation early when those neurons produced NE (Figs. 2A–2C), but was not detectable in neurons innervating sweat glands by P21 (Fig. 2E). To confirm that this reflected decreased NET expression rather than deficient axonal transport, NET-IR was identified in neuron cell bodies in the stellate ganglion, which innervate the sweat glands in the front footpad as well as other targets that induce cholinergic function (Asmus et al., 1994). To identify the subset of neurons within the ganglion which acquired cholinergic and peptidergic properties, sections were labeled with guinea pig anti-VIP in addition to

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NET and VMAT2 in Cholinergic Sympathetic Neurons

FIG. 2. Developmental expression of NET in the sweat gland innervation. Developmental time course of NET (A–E) and smooth muscle actin (F–J) immunoreactivity. Footpad sections from P7 (A, F), P9 (B, G), P12 (C, H), P15 (D, I), and P21 (E, J) rats were double-labeled with anti-NET antiserum diluted 1:3000 and anti-smooth muscle actin antiserum diluted 1:300 and visualized by fluorescence microscopy. Arrowheads denote NET-IR nerve fibers surrounding sweat glands (A–C) or NET-IR myoepithelial cells (D, E). Block arrows identify smooth muscle actin in gland myoepithelial cells. Selected sweat glands are identified by asterisks, and selected blood vessels are labeled with a “v” (A, B, F, G). Vascular smooth muscle is actin-IR while the vascular innervation is NET-IR. Data shown are representative of results obtained with at least four animals of each age.

rabbit anti-NET. Sympathetic neurons in P21 rat stellate which were VIP-IR no longer contained detectable NET-IR, while noradrenergic neurons within the ganglion maintained NET expression but did not contain VIP (Fig. 3), suggesting that NET was undetectable in cholinergic/ peptidergic sympathetic neurons. The apparent discrepancy between data suggesting that NET levels in the footpad did not change and data indicating that neuronal NET decreased significantly during development was clarified by the observation that NET-IR appeared in the maturing sweat glands (Figs. 2D, 2E, and 4A). NET was first detected in glands at P15 (see arrowheads in Figs. 2D and 2E), and expression was maintained in adult animals (Fig. 4). The pattern of staining suggested that NET was expressed in myoepithelial rather than secretory cells (Habecker et al.,1995), and this was confirmed by doublelabeling sections with anti-NET and anti-smooth muscle actin (Figs. 2F–2J). Because the presence of NET-IR in myoepithelial cells was unexpected, we wanted to confirm that the staining was not simply cross-reactivity with an unknown epithelial transporter. To determine independently whether NET was present in myoepithelial cells, radioligand binding studies were carried out using the NET-specific ligand [ 3H]Nisoxetine. Membrane homogenates from adult footpad and brain were assayed for [ 3H]Nisoxetine binding with or without 10 ␮M imipramine to identify nonspecific binding (Fig. 4B). The detection of specific Nisoxetine binding sites in footpad tissue indicated that the NET-IR observed in myoepithelial cells was not an artifact, but reflected the presence of functional norepinephrine transporter.

The onset of NET-IR in sweat glands coincided with innervation-dependent changes that are required for the glands to become competent to sweat (Grant et al., 1995). This raised the possibility that NET expression in myoepithelial cells was dependent on the innervation and might be involved in the development of functional responsiveness. To test whether the expression of NET in gland cells was induced by the innervation, newborn pups were injected for the first postnatal week with the sympathetic neurotoxin 6OHDA, to destroy the developing sympathetic nervous system, or were injected with saline vehicle as a control. Tissue was collected from P21 and adult animals for immunohistochemistry and catecholamine histofluorescence. NET-IR was unchanged in 6OHDA-treated animals at P21 and adulthood, indicating that NET expression in myoepithelial cells was innervation-independent (Fig. 5). Histochemical analysis of catecholamines (data not shown) and the absence of NET-IR innervation to the smooth muscle actin-IR footpad vasculature (Fig. 5) confirmed that the sympathetic innervation was absent in 6OHDA-treated animals.

The Sweat Gland-Derived Cholinergic Differentiation Factor Suppresses NET Activity and mRNA in Cultured Sympathetic Neurons Sympathetic neurons from the superior cervical ganglia are all noradrenergic, but culturing these neurons in the presence of LIF, CNTF, or footpad extracts decreases catecholamine production while increasing synthesis of ACh

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FIG. 3. NET- and VIP-IR in P21 stellate ganglion. Sections were double-labeled for VIP-IR (red; A, C) and NET-IR (green; B, C). Arrowheads identify a VIP-IR cholinergic cell within the sympathetic ganglion which no longer contains NET, and block arrows identify a NET-IR noradrenergic cell. Data shown are representative of results obtained with at least four animals of each age.

and VIP (Raynaud et al., 1987; Swerts et al., 1983; Wolinsky et al., 1985). LIF and CNTF also decrease NET in cultured sympathetic neurons (Matsuoka et al., 1997). To test whether the sweat gland-derived CDF decreased NET activity as part of the noradrenergic to cholinergic switch, sympathetic neurons were grown in control medium, LIF (10 ng/ml), or extracts from sweat gland-containing footpads (300 ␮g/ml) for 5–7 days. Cells from each treatment group were then assayed for the ability to take up [ 3H]NE as a measure of NET activity, while other cells were assayed for ChAT activity as a measure of their acquisition of cholinergic properties (Fig. 6A). NE uptake was significantly lower in the cells which had been induced to acquire cholinergic function (percentage of control: LIF, 33 ⫾ 6; footpad extract, 34 ⫾ 2; n ⫽ 3; ⫾ SEM; P ⬍ 0.01). A similar decrease in NE uptake occurred in sympathetic neurons cocultured with sweat gland cells (19 ⫾ 6% of control; n ⫽ 3; ⫾ SEM). To determine if the loss of NE uptake in cholinergic sympathetic neurons resulted from alterations in NET gene

expression, we asked whether NET mRNA expression decreased as sympathetic neurons became cholinergic. Cells grown in control medium, LIF, or footpad extracts were assayed for NET, TH, and cyclophilin mRNA. The ratio of NET to cyclophilin mRNA decreased significantly in cells undergoing a change in phenotype (Fig. 6B) (percentage of control: LIF, 39 ⫾ 8; footpad extracts, 47 ⫾ 6; n ⫽ 7; ⫾ SEM; P ⬍ 0.01). Tyrosine hydroxylase mRNA, which also decreased in cells treated with LIF and footpad extracts, was quantified in some experiments as a control for the suppression of noradrenergic properties (Fig. 6C; percentage of control: LIF, 43 ⫾ 14; footpad extracts, 29 ⫾ 14; n ⫽ 3; ⫾ SEM; P ⬍ 0.05). These data are consistent with the decreases in NET (Matsuoka et al., 1997) and TH (Rao and Landis, 1990; Rao et al., 1992a) previously observed in sympathetic neurons treated with CDFs. Although these data represent steady-state mRNA levels, they suggest that cholinergic differentiation factors suppress NET mRNA expression as part of the noradrenergic to cholinergic switch in sympathetic neurons.

FIG. 4. NET expression in adult sweat glands. (A) NET immunohistochemistry. Sections of adult footpad were incubated anti-NET diluted 1:3000. NET-IR was absent from the innervation and present in gland myoepithelial cells. (B) Nisoxetine binding. Membrane homogenates of adult brain and footpad were incubated with 5 nM [ 3H]Nisoxetine with or without 10 ␮M imipramine to identify nonspecific binding. Data shown are the means ⫾ SEM of three independent experiments, each assayed in duplicate.

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NET and VMAT2 in Cholinergic Sympathetic Neurons

sympathetic neurons within the footpad. Therefore, we used immunohistochemistry to determine whether any change could be detected in VMAT2-IR within the gland innervation specifically. VMAT2 was detected in sympathetic neurons throughout the footpad (Fig. 8), and the level of VMAT2-IR did not change significantly in the sweat gland innervation as those neurons acquired cholinergic and lost noradrenergic properties. The apparent increase in VMAT2 identified by Western blot contrasted with histological data suggesting no apparent alteration in transporter expression. Because the tissue distribution of VMAT2 did not change during development, we examined expression of the panneuronal marker NSE. Western blot analysis indicated that NSE levels also increased during development (Fig. 7) and suggested that a larger fraction of the footpad homogenate from an older animal is derived from neuronal protein. Thus, VMAT2 protein levels increased twofold, from 0.07 ⫾ 0.01 OD/mm 2 (n ⫽ 3; ⫾ SEM) at P7 to 0.152 ⫾ 0.03 OD/mm 2 (n ⫽ 3; ⫾

FIG. 5. NET expression in sympathectomized footpad. NET and smooth muscle actin immunohistochemistry. Sections of sympathectomized P21 (A, B) and adult (C, D) footpad were doublelabeled with anti-smooth muscle actin diluted 1:400 (A, C) or anti-NET diluted 1:3000 (B, D). Arrowheads highlight actin- and NET-IR myoepithelial cells. Block arrows denote blood vessels which are smooth muscle actin-IR but lack NET-IR sympathetic innervation. Data shown are representative of results obtained with two animals of each age.

VMAT2 Is Present in the Cholinergic Sweat Gland Innervation While NET provides the primary mechanism for terminating the actions of NE, the vesicular monoamine transporter VMAT2 packages NE for regulated release. This protein transports newly synthesized dopamine into vesicles where it is converted to NE by dopamine ␤-hydroxylase and transports NE into vesicles after it has been removed from the synapse by NET. We hypothesized that CDFs, which decrease NE in the sweat gland innervation, would simultaneously lower VMAT2 levels. To test whether the sweat gland-derived differentiation factor decreased VMAT2 expression in the gland sympathetic innervation during development, footpad tissue was collected before, during, and after the change in neuronal phenotype and was assayed for VMAT2 by Western blot and immunohistochemistry. Western blot analysis indicated that VMAT2 levels in footpad did not decrease during development as we had hypothesized (Fig. 7), but appeared to increase as the sympathetic innervation of sweat glands became predominantly cholinergic. This result was reminiscent of the initial NET data and was similarly complicated by the presence of both noradrenergic and cholinergic

FIG. 6. NET expression in cultured cholinergic sympathetic neurons. (A) NE uptake in cultured neurons. Cultured noradrenergic sympathetic neurons were treated with 10 ng/ml LIF, or 300 ␮g/ml soluble extracts of sweat gland-containing footpads (FP Ext.), and assayed for [ 3H]NE uptake to assess NET activity and ChAT to confirm the acquisition of cholinergic function. Data shown are averages of three independent experiments, each assayed in duplicate or triplicate. (*P ⬍ 0.01; significantly less than control; ChAT activity was significantly greater than control in each individual experiment, P ⬍ 0.05). (B and C) NET and TH mRNA in cultured neurons. Sympathetic neurons were grown under control conditions or treated with 10 ng/ml LIF or 300 ␮g/ml footpad extracts and RNA was isolated for quantitation by RNase protection assay. NET and TH mRNAs were normalized to cyclophilin mRNA in the same hybridization reaction and calculated as the percentage of control. (B) shows the means ⫾ SEM of NET:cyclophilin mRNA in seven independent experiments (LIF, 39 ⫾ 8; footpad extract, 47 ⫾ 6, *P ⬍ 0.01; significantly less than control). In three of the experiments summarized in (B) TH and NET mRNA levels were quantified simultaneously, and (C) shows the means ⫾ SEM of TH:cyclophilin mRNA in these three independent experiments (LIF, 43 ⫾ 14; footpad extract, 29 ⫾ 14; *P ⬍ 0.05; significantly less than control).

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SEM) in adults, but the level of VMAT2 normalized to NSE changed less than 20% in that time, from 1.07 to 0.89. This suggests that a general increase in neuronal protein can account for the elevated VMAT2 observed in adult footpad.

Cholinergic Differentiation Factors Do Not Decrease VMAT2 Expression in Cultured Sympathetic Neurons

FIG. 7. VMAT2 expression in whole footpad. Western blot analysis of VMAT2 and NSE. (A) (Top) 5-␮g samples of total protein from P4, 7, 14, and 21 and adult rat footpad were blotted, incubated with guinea pig anti-VMAT2 diluted 1:2000, and visualized with chemiluminescence. (Bottom) 5-␮g samples of total protein from P6, 15, and 21 and adult rat footpad were blotted, incubated with rabbit anti-NSE diluted 1:1000, and visualized with chemiluminescence. (B) Footpad samples from three independent sets of animals were assayed for VMAT2 content on duplicate Western blots, and the blots were quantified as described under Materials and Methods. Data shown are the means ⫾ SEM, n ⫽ 3 (P7, 0.07 ⫾ 0.02 OD VMAT2/mm 2; adult, 0.152 ⫾ 0.03 OD VMAT2/mm 2). (C) VMAT2 levels from (B) normalized to neuron-specific enolase expression at the same age (P7 ratio 1.07; adult ratio 0.89).

The retention of VMAT2 in the cholinergic gland innervation was surprising because those neurons no longer produced its substrate NE. To quantify the effects of CDFs on sympathetic neurons, neurons were cultured with control medium, LIF (10 ng/ml), or footpad extracts (300 ␮g/ml). The acquisition of cholinergic function was assayed by measuring ChAT activity (data not shown), and VMAT2 protein levels were assayed by Western blot (Fig. 9). VMAT2 content did not change significantly (P ⬎ 0.05), although cholinergic properties were induced in the cells treated with LIF and footpad extracts. Similarly, VMAT2 mRNA did not change relative to cyclophilin mRNA in cells treated with LIF or footpad extracts (Fig. 9), even as NET mRNA decreased in the same cells (see Fig. 6). These results are consistent with the observation that VMAT2 is present in the cholinergic sweat gland innervation and with tetrabenazene binding studies indicating that VMAT2 levels do not decrease upon the acquisition of cholinergic properties in cultured sympathetic neurons (Scherman and Weber, 1987).

DISCUSSION Sympathetic neurons can undergo a change in neurotransmitter and neuropeptide phenotype when grown under the appropriate conditions. For example, exposure to neuropoietic cytokines such as LIF and CNTF reduces catechol-

FIG. 8. Developmental expression of VMAT2 in the sweat gland innervation. (A–C) Developmental time course of VMAT2-IR. Footpad sections from P10 (A), P14 (B), and adult (C) rats were incubated with affinity-purified rabbit anti-VMAT2 antiserum diluted 1:200 and visualized by fluorescence microscopy. Arrowheads denote VMAT2-IR fibers innervating the sweat glands. Data shown are representative of results obtained with at least four animals of each age.

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FIG. 9. VMAT2 expression in cultured sympathetic neurons. (A and B) Western blot analysis of VMAT2 expression in cultured sympathetic neurons. Neurons were treated with 10 ng/ml LIF or 300 ␮g/ml soluble footpad extract (FP Ext.). After 5 days of treatment equal amounts of protein from cells grown under each condition were blotted, incubated with guinea pig anti-VMAT2 diluted 1:2000, and visualized with chemiluminescence. (A) Representative samples from two different cultures. (B) Quantitation of four independent experiments, means ⫾ SEM (no significant difference between treated and control). (C) RPA analysis of VMAT2 mRNA. RNA was isolated from sympathetic neurons grown under control conditions or treated with 10 ng/ml LIF or 300 ␮g/ml footpad extracts. VMAT2 mRNA was normalized to cyclophilin mRNA within each reaction and calculated as the percentage of control. Data shown are the means ⫾ SEM of four independent experiments (LIF, 91 ⫾ 3; footpad extract, 92 ⫾ 4).

amine production and induces expression of ACh and VIP in cultured sympathetic neurons. When neurotransmitter plasticity was first observed in these neurons in culture, several investigators tested whether noradrenergic and cholinergic properties were coordinately regulated. Those early experiments, which utilized conditioned medium from a variety of sources to induce cholinergic function, produced conflicting results. The data concerning regulation of the transporters involved in noradrenergic function were particularly confusing. Although the methods used to assess uptake were indirect and did not distinguish between transport into the cell via NET and transport into vesicles via VMAT2, all of the studies showed that [ 3H]NE or other catecholamines

such as 5OHDA could be taken up into cholinergic sympathetic neurons and packaged into vesicles. Some reports concluded, however, that there was no difference in the ability of cholinergic and noradrenergic sympathetic neurons to take up and store NE (Reichardt and Patterson, 1977; Wakshull et al., 1978), while others reported significantly less uptake and storage of catecholamines in neurons that had become cholinergic, both in culture (Johnson et al., 1976, 1980; Landis, 1976, 1980) and in vivo (Landis and Keefe, 1983). We used a variety of biochemical and molecular techniques to test directly whether VMAT2 and NET were suppressed along with other noradrenergic properties in cholinergic sympathetic neurons. Our results indicated clearly that NET mRNA, protein, and activity decreased in cultured sympathetic neurons that acquired cholinergic function and that NET-IR disappeared from the developing sweat gland innervation as it acquired cholinergic properties. These results provide an explanation for the decreased uptake of exogenous catecholamine observed in the cholinergic sweat gland innervation of adult rats (Landis and Keefe, 1983) as well as the relative resistance of the cholinergic gland innervation to 6OHDA toxicity (Yodlowski et al., 1984). The loss of NET and TH from the gland innervation is also consistent with our hypothesis that developmental regulation of NET expression would differ from the pattern of expression observed following acute changes in transmitter levels, when TH and NET are regulated in opposing manners (Cubells et al., 1995a; Xiao et al., 1995). Perhaps the most puzzling result of our studies was the observation that NET-IR appeared in sweat gland myoepithelial cells at P15, as it disappeared from the gland innervation. Binding studies with [ 3H]Nisoxetine confirmed the presence of NET in adult sweat gland tissue, indicating that the immunoreactivity was not simply an artifact. Although the onset of NET-IR in gland cells coincided with innervation-dependent changes within the glands (Grant et al., 1995), NET expression was innervation-independent. The function of NET in myoepithelial cells remains unknown. Since the mature gland innervation no longer produces NE, it seems unlikely that NET functions as a NE transporter in these cells. Circulating epinephrine is a potential substrate, although immunohistochemistry suggests that a significant fraction of the NET present in myoepithelial cells is cytosolic rather than in the plasma membrane. Whether its distribution is altered in response to conditions that stimulate epinephrine release remains to be determined. In contrast to the loss of neuronal NET, VMAT2 levels did not decrease as TH-IR and NET-IR declined in the sweat gland innervation and in cultured sympathetic neurons. This uncoupling of transmitter synthesis and packaging is strikingly different from the coordinate regulation of synthetic enzymes and vesicular transporters observed in cholinergic and GABAergic systems and from the simultaneous appearance of vesicular uptake and NE production during noradrenergic sympathetic differentiation (Eastman et al.,

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1999; Rohrer and Ernsberger, 1998; Eiden, 1998). The detection of VMAT2-IR in the sweat gland innervation is consistent, however, with the observation of reserpine-sensitive catecholamine stores (Guidry and Landis, 1998) and NEcontaining small granular vesicles in those neurons (Landis and Keefe, 1983) but conflicts with Shafer et al., who report that VMAT2-IR is not present in the sweat gland innervation (Schafer et al., 1997). The discrepancy in VMAT2 staining may be due to differing immunohistochemistry conditions, since in our hands VMAT2 immunohistochemistry varied with perfusion efficiency. The retention of VMAT2 in the sweat gland innervation and in cultured cholinergic sympathetic neurons is surprising in comparison with other transmitter systems, but consistent with the observation that cholinergic and noradrenergic sympathetic neurons contain similar levels of reserpine-sensitive binding sites for the VMAT2-specific ligand [ 3H]dihydrotetrabenazine (TBZ) (Scherman and Weber, 1987). The subcellular distribution of TBZ binding sites suggests that a similar fraction of the VMAT2 protein is inserted into vesicles whether the cells make ACh or NE and raises the possibility that a single vesicle could contain both VAChT and VMAT2. The function of VMAT2 in neurons lacking both NE and dopamine is unclear, but this may provide an interesting system to investigate vesicular targeting of transporters. On the surface, these results appear inconsistent with the reported decrease in vesicular accumulation of exogenous catecholamines in the cholinergic sweat gland innervation (Landis and Keefe, 1983). Those studies did not distinguish between plasma membrane uptake and vesicular transport, however, and in order for exogenous catecholamines to be detected in vesicles they must first be taken into the neuron via NET. Our data, and earlier experiments using the sympathetic neurotoxin 6OHDA (Yodlowski et al., 1984), indicate that NET levels and transport activity decrease significantly as the sweat gland innervation acquires cholinergic properties. This prevents the accumulation of exogenous catecholamines into a compartment where they are available for transport by VMAT2. Therefore, the lack of NET can account for the decreased accumulation of catecholamines into vesicles, even in the continued presence of vesicular VMAT2. We hypothesized that developmental regulation of neurotransmitter transporter expression would differ from the pattern of expression observed following acute changes in transmitter levels, when expression of VMAT2 and NET is regulated in an opposing manner (Xiao et al., 1995). We predicted that both NET and VMAT2 would be suppressed by cholinergic differentiation factors, but found that NET decreased along with TH while VMAT2 remained the same. This indicates that regulation of enzymes and transporters by CDFs during chronic suppression of NE differs significantly from their regulation by catecholamines during acute alterations in NE content. It also indicates that CDFs can suppress some proteins associated with a particular neurochemical phenotype while not changing the expres-

sion of others. LIF, CNTF, and related ligands play important roles in the development of the central nervous system in addition to their effects on peripheral development (Bonni et al., 1997; Marmur et al., 1998; Li et al., 1995; Ware et al., 1995), and their ability to selectively regulate transporter expression may be important there as well as in the periphery. The presence of vesicular transporters for both ACh and NE within the same neuron suggests that transporter expression is not as restricted to neurons expressing the substrate neurotransmitter as was previously thought. Future studies will examine the mechanisms by which cholinergic differentiation factors selectively downregulate proteins required for noradrenergic function.

ACKNOWLEDGMENTS VMAT2 immunohistochemical studies were carried out in the Neural Development Section, National Institute of Neurological Disorders and Stroke, NIH. This work was sponsored by MRF Oregon No. 9724 and American Heart Association Grant-in-Aid 9750083N (B.A.H.), an NIH summer undergraduate fellowship to B.A.P., and a Murdock Charitable Trust/OHSU Heart Research Center undergraduate fellowship to B.C.C. The authors thank Dr. Richard Simerly for TH, NET, and cyclophilin plasmids; Regeneron Pharmaceuticals, Inc., for Axokine (a modified form of recombinant CNTF); Drs. Susan Amara and Randy Blakely for anti-NET antisera; Dr. Story Landis for anti-VIP antiserum and for critically reading the manuscript; and Drs. Steve Asmus and Christine Brennan for critically reading the manuscript.

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