Regulation of Substance P Is Similar to that of Vasoactive Intestinal Peptide after Axotomy or Explantation of the Rat Superior Cervical Ganglion

M. S. Rao, Y. Sun, U. Vaidyanathan, S. C. Landis, and R. E. Zigmond*

Department of Neurosciences, Case Western Reserve University School of Medicine, Cleveland, Ohio 441 06-4975

SUMMARY

The regulation of the expression of substance P (SP) in the rat superior cervical ganglion was compared to that of vasoactive intestinal peptide (VIP) in vivo after axotomy and in vitro after explantation. Previous studies have demonstrated that both neuropeptides increase after ex- plantation, depolarization, and decentralization; how- ever, whereas VIP expression increases after postgangli- onic axotomy, S P expression reportedly does not. To compare the effect of axotomy on these two peptides di- rectly, the content of both was determined in individual ganglia at various times after surgery. The level of VIP- like immunoreactivity (IR) is increased at 2 days, reaches a peak at 6 days, and then declines by 14 days to approximately half its peak value. The level of SP-IR also increases 2 days after axotomy, but returns to con- trol values by day 6. The increase in SP-IR is accompa- nied by an increase in @-preprotachykinin mRNA, sug- gesting that the elevation in SP content is due, at least in part, to enhanced peptide synthesis. Immunocytochemi- cal localization of SP-IR revealed the presence of immu-

noreactive principal neurons in axotomized, but not in sham-operated ganglia. Similarities in the regulation of these two neuropeptides were also investigated in organ culture by examining the effects of dexamethasone and interleukin-l@ on VIP content, since the former has been shown to prevent the increase in SP in culture, while the latter has been found to enhance this increase (Kessler, Adler, Bell, et al., 1983, Neuroscience 9:309-321; Frei- din and Kessler, 1991, Proc. Natl. Acad. Sci. USA 88:3200-3203; Hart, Shadiack, and Jonakait, 1991, J. Neurosci. Res. 29:282-291). As with SP expression, dexamethasone reduces the increase in VIP expression, while interleukin-l@ increases it. Thus, both in vivo and in vifro, similar changes in VIP and SP expression are observed following a number of experimental manipula- tions, suggesting that expression of the two peptides is regulated by qualitatively similar mechanisms in sympa- thetic neurons. 0 1993 John Wiley & Sons, Inc. Keywords: substance P, vasoactive intestinal peptide, ax- otomy, superior cervical ganglion.

INTRODUCTION

Sympathetic neurons of neonatal and adult rats are plastic with respect to their expression of VIP and SP. The superior cervical ganglion, for example, normally contains little vasoactive intestinal pep- tide- (VIP) or substance P- (SP) like immunoreac- tivity (IR). While sparse plexuses of both VIP- and

Received November 10, 1992; accepted December 2 1, 1992 Journal of Neurobiology, Vol. 24, No. 5 , pp. 57 1-580 (1993) 0 1993 John Wiley & Sons, Inc. CCC 0022-3034/93/05057 1-10

* To whom correspondence should be addressed.

SP-IR fibers form in the ganglion, only occasional neuronal cell bodies are VIP-immunoreactive, and none contain detectable SP-IR (Hokfelt, Elfvin, Schultzberg, et al., 1977a; Hokfelt, Elfvin, Schultz- berg, et al., 1977b; Sasek and Zigmond, 1989). However, the expression of both neuropeptides can be induced by a number of manipulations in vitro and in vivo. Thus, levels of VIP- and SP-IR rise dramatically when either neonatal or adult gan- glia are placed in explant culture (Kessler, Adler, Bohn, et al., 1981; Adler and Black, 1984; Sun, Rao, Landis, et al., 1992a; Zigmond, Hyatt-Sachs, and Baldwin, et al., 1992). Within 48 h, there is a 30-fold increase in VIP-IR (Sun, Rao, Zigmond, et

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572 Rao et al.

al., 1992a; Zigmond et al., 1992) and a similar change in SP-IR (Kessler et al., 1983; Adler and Black, 1984). Immunohistochemically, there is a dramatic increase in the number of immunoreac- tive neuronal cell bodies and fibers (Kessler et al., 1981; Sun et al., 1992a; Zigmond et al., 1992). These increases in VIP- and SP-IR are accompa- nied by increases in their respective mRNAs (Roach, Adler, and Black, 1987; Hart et a]., 199 1 ; Zigmond et al., 1992 ). Neither decreases in nerve growth factor (NGF) levels nor the presence of serum appear to be responsible for elevating neuro- peptide levels (Jonakait and Schotland, 1990; Zig- mond et al., 1992). Depolarization of sympathetic ganglion explants with 30 m M potassium or a low concentration of veratridine (1.5 p M ) further in- creases both VIP and SP levels (Sun et al., 1992a). The effects of dexamethasone and interleukin- 10 (IL-10) have been examined on SP, but not VIP, expression in explant culture: dexamethasone de- creases SP and IL-1P increases it (Kessler et al., 1983; Freidin and Kessler, 199 1; Hart et al., 199 1 ). Thus, VIP and SP expression appear to change in concert in explant cultures under each condition where previous studies allow a comparison to be made.

Changes in neuropeptide expression in the supe- rior cervical ganglion are also seen in vivo, after surgical transection of afferent input and efferent outflow. After the preganglionic cervical sympa- thetic trunk is cut, both VIP- and SP-IR increase. The magnitude of these changes is small, for VIP- IR twofold and for SP-IR 1.5-fold in adults (Kessler and Black, 1982; Hyatt-Sachs, Schreiber, Bennett, et al., 1993) and twofold in neonates (Kessler et al., 198 1 ). The doubling of VIP content after decentralization is paralleled by a small in- crease in VIP mRNA (Hyatt-Sachs et al., 1993).

In contrast to the similarities in the regulation of expression of VIP and SP in explant culture and following denervation, the responses of the two neuropeptides to axotomy appear to differ. A dra- matic change is seen in VIP levels after postgangli- onic nerve section: 48 h after the two major post- ganglionic trunks are severed, peptide is increased 22-fold and mRNA fivefold (Hyatt-Sachs et al., 1993). In contrast, no change in SP content was observed when ganglia were assayed 12 days after section of the postganglionic trunks (Kessler and Black, 1982). The apparent difference in the re- sponses of the two peptides to axotomy could, how- ever, reflect the fact that they were assayed at dif- ferent times after axotomy.

Thus, VIP and SP expression in the superior cervical ganglion appear to be regulated similarly in several, but not all, instances. To clarify the ex- tent to which their expression is controlled by simi- lar environmental factors, we have examined the effects of axotomy on SP expression in more detail. In addition, we have determined whether dexa- methasone and IL- 10 also influence ganglionic content of VIP-IR, as they have been shown to modulate expression of SP-IR. We report that SP- IR does increase after axotomy, that the magnitude of the increase is much closer to that observed in explant culture than is the increase previously re- ported after decentralization ( Kessler and Black, 1982), and that dexamethasone and IL-lP modu- late VIP- and SP-IR in a qualitatively similar fash- ion. Our findings suggest that the expression of these two neuropeptides is regulated similarly in sympathetic neurons.

MATERIALS AND METHODS

Rats were obtained from Zivic Miller (Zelienople, PA). Cell culture reagents were obtained from Gibco (Grand Island, NY) and culture plates from Corning (Corning, NY). Nerve growth factor was provided by Dr. K. Neet (Department of Biochemistry, Chicago Medical School, Chicago, IL) or by Dr. Gary Landreth (Department of Neurology, Case Western Reserve University, Cleve- land, OH) . Protein assay kits were obtained from Pierce (Rockford, IL). SP radioimmunoassay kits were ob- tained from Peninsula (Belmont, CA). SP antisera used for immunocytochemistry were the kind gifts of Dr. Su- san Leeman (Boston University School of Medicine, Boston, MA) and Dr. John Maggio (Harvard Medical School, Boston, MA) or obtained from Incstar (Still- water, MI) . For the VIP radioimmunoassay, the '25VIP was obtained from New England Nuclear (Boston, MA) and the rabbit anti-VIP. goat anti-rabbit IgG, and nor- mal rabbit serum from Peninsula Laboratory (Belmont, CA). Fluorescein-conjugated goat anti-rabbit antiserum was from Tago (Burlingame, CA). IL- 1 @ was obtained from Cistron Biotechnology (Pine Brook, NJ). Dexa- methasone and other chemicals were purchased from Sigma (St. Louis, MO).

Axotomy

Animals (200 g) were anesthetized with chloral hydrate, and the superior cervical ganglion was exposed. The ma- jor outflow tracts, the external and the internal carotid trunks, were identified and cut. The sham-operated ani- mals were similarly anesthetized, the ganglia were ex- posed, and the trunks were identified but not cut. At

Substance P und VIP E.upres.cion in SCG 573

different times after surgery, superior cervical ganglia from operated animals were collected and processed for immunocytochemistry, Northern blot analysis, or pep- tide radioimmunoassay, as described below. In certain experiments, ganglia from unoperated animals were also examined.

Explant cultures

Superior cervical ganglia were dissected from neonatal rats and put in organ culture on Millipore filters placed on stainless-steel rafts as described previously (Zigmond et al., 1992). Ganglia were cultured for 24 h in F12-de- fined medium containing 100 ng/ml NGF. In some ex- periments, dexamethasone (0.1 p M ) or IL-IP ( 10 units/ ml) was added to the medium. Ganglia were then frozen at -80°C prior to radioimmunoassay, as described below.

lmmunocytochemistry

Animals were perfused with 4% paraformaldehyde in 0. I M phosphate buffer, pH 7.4, for 10 min, and the ganglia were dissected and post-fixed for an additional 50 min. After rinsing with phosphate-buffered saline (PBS), the tissue was equilibrated with 30% sucrose in PBS. Cryo- stat sections (7 pm) were thaw-mounted onto gelatin- coated slides and processed for indirect immunofluores- cence. Sections were incubated with the SP primary anti- serum ( 1: 1000 or 2000) overnight, rinsed, incubated for 2 h with goat anti-rabbit antiserum conjugated to fluores- cein, and mounted in PBS and glycerol ( 1 : 1 ). Neuron cell body and fiber staining was absent when the primary antibody was omitted. The relative frequency of immu- noreactive cell bodies and fibers was determined by vi- sual inspection.

Radioimmunoassay

Neuropeptide levels were determined by radioimmuno- assay as previously described (Sun et al., 1992a; Zig- mond et al., 1992). In brief, ganglia were boiled in 2N acetic acid for 20 min. Samples were then centrifuged for 1 min in an Eppendorf microfuge. The supernatants were lyophilized under vacuum and stored at -80°C for subsequent assays. The primary antisera have been previ- ously shown to display minimal cross-reactivity with other peptides.

Northern Blot Analysis

Total RNA was prepared from control and axotomized ganglia using the single-step guanidinium-isothiocya- nate method (Chomczynski and Sacchi, 1987). The RNA was denatured on a 1.5% agarose gel containing 12.2 M formaldehyde and blotted onto a positively

charged nylon membrane (Boehringer Mannheim). RNA from six ganglia ( 18 p g ) was loaded per lane. The blot was probed for 6-preprotachykinin mRNA (Krause, Chirgwin, Carter, et al., 1987) and glucose 6-phosphate dehydrogenase (GAPDH) mRNA. The P-preprotachy- kinin probe was labeled by in vitro transcription using a riboprobe kit from Promega. Hybridization was per- formed at 65°C overnight. The blot was washed at room temperature in 2X SSC/O.l% SDS followed by 0.1X SSC/O.I% SDS at 65°C and exposed to Kodak XAR-5 film for 3-5 days. The 32P-labeled DNA probe for GAPDH was made from a cDNA for GAPDH (a gift from Dr. J. M. Blanchard; Fort, Marty, and Piechaczyk, et al., 1985) by random hexanucleotide priming using the Random Primed DNA labeling kit (Boehringer Mannheim), and hybridization was performed for 1 h at 65°C in QuikHyb (Stratagene). The amounts of p-pre- protachykinin mRNA were determined by scanning den- sitometry and normalized against GAPDH mRNA.

RESULTS

Axotomy Increases SP Levels

SP- and VIP-IR were measured by radioimmunoas- say in unoperated, sham-operated, and axoto- mized ganglia. The levels of both peptides did not differ significantly between unoperated and sham- operated ganglia (data not shown). Within 2 days after transection of the major postganglionic trunks of the superior cervical ganglion, however, there was a 12-fold increase in SP-IR in the gan- glion [Fig. 1 (A)]. By 6 days after axotomy, levels of SP-IR were no longer distinguishable from those found in ganglia from unoperated animals. As in the case of SP-IR, the content of VIP-IR also in- creased rapidly [ Fig. 1 (B)] . In contrast to SP-IR, levels of VIP-IR continued to rise after 2 days, and, although reduced from their peak value at 6 days, remained elevated at 14 days compared to levels in control ganglia. Thus, the ganglionic contents of both SP- and VIP-IR increase rapidly following ax- otomy and subsequently decline-SP to basal lev- els and VIP to a level intermediate between basal values and the maximum values seen after axotom y.

The Increase in SP Is Due in Part to an Increase in Tachykinin mRNA

We examined 0-preprotachykinin mRNA levels with Northern blot analysis to determine whether the changes in SP-IR were correlated with an in- crease in this mRNA. While P-preprotachykinin

574 Raorral

1.0 k l d a y 1-Zday k6day td4day

'0°] B 600

.t

I

1-0 k l d a y t=6day

Figure 1 SP and VIP levels are increased after axotomy. Ganglia were removed from unoper- ated ( t = 0) and from operated animals at the times indicated after surgery. SP ( A ) and VIP levels (B) were determined by radioimmunoassay. Data represent the mean k S.E.M. of four ganglia at each time point. The values indicated by * are significantly different from t = 0 withp < 0.0001 by one-way Anova plus posthoc t test. The values indicated by ** are significantly different from t = 0 with p < 0.05 by one-way Anova plus posthoc t test.

mRNA was barely detectable in the superior cervi- cal ganglia of unoperated or sham-operated ani- mals, a distinct hybridizing band at 1.2 kb was found in axotomized ganglia (Fig. 2). When scan- ning densitometry was performed and the amount of P-preprotachykinin mRNA was normalized to the amount of GAPDH mRNA, a twofold increase was seen as a result of axotomy.

Localization of SP lmmunoreactivity

The finding that P-preprotachykinin mRNA was increased after axotomy suggested that the SP-IR induced by axotomy was synthesized locally and present in neuronal cell bodies in the superior cer- vical ganglion. To examine the localization of SP- IR, sections of superior cervical ganglia from con-

trol and axotomized rats were reacted with antisera directed against SP. In ganglia of unoperated or sham-operated rats, no neurons were present that contained detectable immunoreactivity for SP [ e.g., Fig. 3( A)]. As described previously (Hokfelt et al., 1977b), the ganglia contained a sparse plexus of SP-IR fibers thought to be sensory in origin (Gamse, Wax, Zigmond, et al., 198 1 ) . After axot- omy, however, a subset of principal neurons scat- tered throughout the ganglion contained SP-IR, and there was an increase in the number of SP-IR fibers [Fig. 3 (B) ] . In neuronal cell bodies that con- tained SP-IR, the intensity of the immunoreactiv- ity varied significantly from neuron to neuron, ranging from barely above background to moder- ately intense. This variability in immunofluores- cence intensity is similar to that previously ob-

Subslance P and VIP Expression in SCG 575

Figure 2 Levels of detectable preprotachykinin mRNA are increased after axotomy. RNA ( 18 pg) was obtained from six unoperated and six axotomized sympathetic ganglia and was transferred to a nylon membrane. The blot was probed for 6-preprotachykinin mRNA (arrow) and for glucose 6-phosphate dehydrogenase (GAPDH) mRNA (not shown). When the hybridization to 0-pre- protachykinin mRNA was quantitated by scanning den- sitometry and normalized to the amount of GAPDH mRNA, a twofold increase was evident in the axoto- mized ganglia. The arrowheads indicate the localization of the 28s and 18s ribosomal RNA.

served for SP-IR in explanted ganglia (Sun et al., 1992a).

VIP and SP Are Regulated by Dexamethasone and Il-lp SP-IR (Kessler et al., 198 1 ; Adler and Black, 1984) and VIP-IR (Zigmond et al., 1992) both have been shown to increase in superior cervical ganglia placed in organ culture. The present finding that SP-IR, like VIP-IR, also increases in vivo after axot- omy raises the possibility that the increases in both peptides in organ culture are primarily responses to axotomy. If the increases in SP and VIP in vitro share a common stimulus, axotomy, then factors that regulate SP levels in explant cultures might also regulate VIP levels. We, therefore, examined the effects on VIP- and SP-IR of dexamethasone and IL- I / ? in individual ganglia. Dexamethasone

completely blocked the normal increase in SP-IR [Fig. 4( A)] and partially prevented the increase in VIP-IR [Fig. 4(B)]. IL-1P enhanced the increase in both peptides, although the magnitude of the effect of IL- 1 P on VIP-IR [Fig. 5 (B)] was smaller than on SP-IF4 [Fig. 5 (A)].

DISCUSSION

We have shown that a large increase in SP-IR oc- curs in the superior cervical ganglia of adult rats 2 days after axotomy. The increased SP-IR is present in the cell bodies and processes of a subset of prin- cipal neurons and is accompanied by a rise in the mRNA encoding SP. Treatment of ganglia in ex- plant culture with dexamethasone and IL- I P, which have been shown to decrease and increase SP content respectively, also causes qualitatively similar changes in VIP. Thus, both in vivo after axotomy and in vitro in explant cultures, VIP and SP levels are regulated in a similar fashion. Not all neuropeptides expressed by sympathetic neurons exhibit such alterations. For example, neuropep- tide Y, present in many principle neurons in the superior cervical ganglion (Lundberg, Terenius, Hokfelt, et al., 1982; Schon, Allen, Yeats, et al., 1985), shows only small changes in response to explantation or axotomy (Zigmond et al., 1992; Hyatt-Sachs et a]., 1993).

Our finding that the levels of SP-IR are elevated after axotomy is in apparent contrast to the finding in a previous report that axotomy does not alter SP-IR (Kessler and Black, 1982). The experimen- tal observations, however, are not contradictory. We observed a rapid but transient increase in SP. Kessler and Black examined the level of SP-IR at 12 days after axotomy, a time at which our data suggest that peptide levels would be indistinguish- able in axotomized and sham-operated ganglia. The increase in the number of neurons showing SP-IR and in the level of P-preprotachykinin mRNA observed in the present study provide fur- ther evidence for an increase in SP expression after axotomy.

Our data raise the possibility that the increase in SP, previously reported when sympathetic ganglia from neonatal or adult rats are placed in explant culture (Kessler et al., 1981; Kessler and Black, 1982), is primarily the consequence of this axot- omy, as has been proposed for the increase in VIP expression that occurs under the same conditions (Sun et al., 1992a; Zigmond et al., 1992; Hyatt-

576 Rao et al.

Figure 3 Principal neurons contain SP-immunoreactivity. In sham-operated superior cervi- cal ganglia, fibers containing SP-IR course through the ganglion (arrowhead). This field was selected specifically to show SP-IR fibers. The overall density of these fibers in control ganglia is, however, lower than seen here. At 2 days after axotomy, a small proportion of principal neurons contain SP-IR (arrows). In addition, there is an increase in the number of SP-IR fibers in thc ganglion. 300X.

Sachs et al., 1993). This induction of SP was ini- tially attributed to preganglionic denervation and the consequent loss of neuronal activity (Kessler et al., 1981; Kessler and Black, 1982; Adler and Black, 1984); however, a decrease in neural activ- ity is unlikely to explain these changes for two rea- sons. First of all, the magnitude of the increase in SP-IR after explantation (Kessler et al., 1981; Kessler et al., 1983; Adler and Black, 1984), which is approximately 40-fold, is much larger than the approximately 1.5-fold increase seen after decen- tralization (Kessler and Black, 1982), even though both manipulations are thought to render the neu- rons totally inactive electrically. The increase in SP-IR seen after axotomy, on the other hand, is approximately 12-fold, which is of the same order of magnitude as that seen after explantation. Sec- ond, treatment of explanted ganglia with low con- centrations of either potassium chloride or veratri- dine, two depolarizing agents, causes a substantial, additional increase in SP-IR (Sun et al., 1992a), rather than a blockade of the increase as predicted by the decentralization hypothesis (Kessler et al.,

198 1 ). These observations make it likely that the induction of SP is not due to a lack of neuronal activity but rather to axotomy.

Comparison of the changes in SP and VIP levels after axotomy reveal several similarities. The time course of induction is rapid; the ganglionic content of both neuropeptides is significantly elevated 48 h after axotomy. The peptide levels are reduced dur- ing the subsequent 2 weeks. The elevation of both SP and VIP content is accompanied by increases in the mRNA encoding each neuropeptide. Finally, following axotomy, there is an appearance of neu- rons containing detectable SP-IR (Fig. 3), an in- crease in the number of neurons with VIP-IR ( Hyatt-Sachs et al., 1993 ) and increases in the num- ber of both types of peptide-IR fibers (Fig. 3; Hyatt-Sachs et al., 1993). In both cases, only a sub- set of the neurons that are axotomized when the two postganglionic trunks are cut contain detect- able immunoreactivity. The increases in SP- and VIP-IR following explantation are also modulated in a qualitatively similar fashion. Dexamethasone completely blocks the increase in SP-IR in explant

Substance P and VIP Expression in SCG 577

tro Fl2,24h Dex, 24h

t:o F12,24h Dex, 24h

Figure 4 Effect of dexamethasone on SP and VIP levels in explant cultures. Superior cervical ganglia from newborn rats were dissected. The ganglia were either extracted immediately ( t = 0) or placed in culture for 24 h in normal F12 medium (F12, 24 h ) or in F12 medium supplemented with 0.1 pM dexamethasone (Dex. 24 h) . The ganglia were extracted and their content of SP ( A ) and VIP ( B ) was determined by radioimmunoassay. The results are ex- pressed as mean levels f S.E.M. and represent at least 14 ganglia. The values indicated by * are significantly different from both t = 0 and Dex, 24 h with p < 0.001 by one-way Anova plus a posthoc t test. The values indicated by ** are significantly different from t = 0 and those indicated by *** are significantly different from Dex, 24 h by one-way Anova plus a posthoc t test with p < 0.00 1.

cultures, as reported earlier by Kessler et al. ( 1983), and partially prevents the increase in VIP- IR. IL- 1 @ enhances the increases in SP-IR, as previ- ously observed (Jonakait and Schotland, 1990; Freidin and Kessler, 199 1 ; Hart et al., 199 1 ) and has a similar effect on VIP-IR. In addition, as we have reported, depolarization elevates both SP- and VIP-IR in organ culture (Sun et al., 1992a).

The similarities in the regulation of the two pep- tides after axotomy suggest that a common fac- tor( s ) plays a role in both inductions. In principle, trauma associated with the operation could induce the rise; however, this seems unlikely since no changes are evident in sham-operated animals and

only small changes are seen after preganglionic de- nervation (Kessler and Black, 1982; Hyatt-Sachs et al., 1993). Instead, the stimulus leading to the alter- ation of expression of SP and VIP is likely to be separation of sympathetic neurons from their tar- gets and/ or the loss of their axonal arbors or both. The signal ( s ) involved could be the loss ofan inhib- itory factor derived from target tissues o r the pro- duction of a stimulatory factor in the ganglion or both. Studies of neuropeptide expression in cul- tured sympathetic neurons have identified two mol- ecules, cholinergic differentiation factor or leuke- mia inhibitory factor (CDF/LIF) and ciliary neu- rotrophic factor (CNTF), that increase the

578 Rao et a1

loo 1 B

F12,24h IL-I, 24h

F12.24h IL-l124h

Figure 5 Effect of dexamethasone on SP and VIP levels in explant cultures. Superior cervical ganglia from new- born rats were dissected. The ganglia were placed in cul- ture for 24 h in normal F12 medium (F12, 24 h ) or in F12 medium supplemented with 10 units of human re- combinant IL-lp (IL-1, 24 h) . The ganglia were ex- tracted and their content of SP ( A ) and VIP (B) was determined by radioimmunoassay. The results are ex- pressed as mean levels +- S.E.M. and represent at least 10 ganglia. The value indicated by * is significantly different from F12, 24 h withp < 0.0001 by a student t test. The value indicated by ** is significantly different from F12, 24 h with p < 0.01 with a student t test.

expression of both SP and VIP (Ernsberger, Sendtner, and Rohrer, 1989; Nawa and Patterson, 1990; Nawa and Sah, 1990; Freidin and Kessler, 199 1 ; Nawa, Yamamori, Le, and Patterson, 199 1 ; Rao, Tyrrell, Landis, et al., 1992). Consistent with the possibility that one or both of these factors me- diate the changes observed after axotomy, CDF/ LIF and CNTF increase preprotachykinin and VIP mRNA levels and cause VIP and SP peptide levels to rise in as little as 24 h in culture (Nawa et al., 199 1; Lewis, Rao, Dauer, et al., 1992). Further- more, when CDF/LIF is removed from disso-

ciated sympathetic neuron cultures, SP content falls rapidly (Nawa et al., 199 I ) . Both proteins are likely to be localized in or secreted from cell types present in sympathetic ganglia, namely fibroblasts and Schwann cells (Patterson and Chun, 1977; Lin, Mismer, Lile, et al., 1989; Stockli, Lottspeich, Sendtner, et al., 1989; Rende, Muir, Ruoslahti, et al., 199 I ; Dobrea, Unnerstall, and Rao, 1992; Sha- diack, Hart, and Jonakait, 1992; Sun, Rao, Zig- mond, et al., 1992b). Finally, it is of interest that in other systems, CDF/LIF is up-regulated by IL-lp, consistent with the possibility that CDF/ LIF is also involved in the increase in these peptides pro- duced by IL-lp (Wetzler, Talpaz, Lowe, 1991; Lubbert, Mantovani, Lindemann, et al., 199 1 ). Ev- idence that CDF/LIF plays an important role in inducing both SP and VIP when ganglia are placed in explant culture is provided by two preliminary reports (Shadiack et al., 1992; Sun et al., 1992b).

Although many similarities exist in the regula- tion of SP and VIP, we have also found differences. First, levels of VIP-IR continue to rise when levels of SP-IR have begun to fall, and, while SP-IR has returned to normal at 1 week, VIP-IR continues to be elevated. Second, while dexamethasone com- pletely blocks the increase in SP-IR in explants, it only partially blocks that of VIP-IR. Third, IL-10 causes a larger increase in SP-IR than in VIP-IR. These differences raise the possibility that in addi- tion to a common inductive signal for the two neu- ropeptides that appears in response to injury, a sec- ond inductive signal exists that is specific for VIP. Elucidation of this issue must await identification of the molecule( s ) involved.

This work was supported by NIH grants NS 175 12 to R.E.Z. and HD2568 1 to S.C.L., a postdoctoral fellow- ship from the Northeast Ohio Affiliate of the American Heart Association to M.S.R., and a Research Scientist Award to R.E.Z. (MH00162). Theauthors thank Hilary Hyatt-Sachs for performing the Northern blot analysis.

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