Exp Brain Res (1996) 111:393-404 �9 Springer-Verlag 1996

Xu Zhang �9 Ru-Rong J i . Jan Arvidsson Jan M. Lundberg- Tamas Bartfai �9 Katarina Bedecs Tomas H6kfelt

Expression of peptides, nitric oxide synthase and NPY receptor in trigeminal and nodose ganglia after nerve lesions

Received: 6 March 1995 / Accepted: 17 April 1996

Abstract Using immunohistochemistry and in situ hy- bridization, the expression of galanin (GAL)/galanin message associated peptide (GMAP)-, neuropeptide Y (NPY)-, vasoactive intestinal polypeptide (VIP)/peptide histidine isoleucine (PHI)- and nitric oxide synthase (NOS)-like immunoreactivities and mRNAs, and NPY receptor mRNA was studied in normal trigeminal and nodose ganglia and 14 and 42 days after peripheral axo- tomy. In normal trigeminal ganglia about 11% of the counted neuron profiles contained GAL mRNA, 4% NOS mRNA, 5% NPY mRNA, 7% VIP mRNA, and 19% NPY receptor mRNA. Peptide mRNA- and NPY re- ceptor mRNA-positive neuron profiles were small in size. Fourteen days after axotomy a marked increase in the number of GAL mRNA- (34% of counted neuron profiles), NPY mRNA- (54%) and VIP mRNA- (31%) positive neuron profiles, and a moderate increase in the number of NOS mRNA- (22%) positive neuron profiles were observed in the ipsilateral trigeminal ganglia. The GAL/GMAR VIP- and NOS-positive profiles were mainly small, the NPY-positive ones mostly large. NPY receptor mRNA was expressed in some large neurons. In normal nodose ganglia, about 3% of the counted neuron profiles contained GAL mRNA, 3% NPY mRNA, 17% NOS mRNA and less than 1% VIP mRNA. Fourteen days after peripheral axotomy, a marked increase in the number of GAL mRNA- (78% of counted neuron pro- files), NOS mRNA- (37%) and VIP- (46%) mRNA-posi- five neuron profiles was seen in the ipsilateral nodose

X. Zhang �9 R.-R. Ji �9 J. Arvidsson I �9 T. H6kfelt (Bi~) Department of Neuroscience/Histology, Karolinska Institute, Box 60400, S- 17177 Stockholm, Sweden; Tel.: +46 8-728 7070, Fax: +46 8-33 16 92

J. M. Lundberg Department of Pharmacology, Karolinska Institute, Stockholm, Sweden

T. Bartfai �9 K. Bedecs Department of Neurochemistry and Neurotoxicology, Stockholm University, Stockholm, Sweden

1 During the final part of this study Dr. Jan Arvidsson tragically died from a cerebral insult.

ganglia. The number of NPY-positive (23%) neurons was moderately increased, mainly in small neuron profiles. There were no NPY receptor mRNA-positive neurons, either in normal nodose ganglia or in nodose ganglia ip- silateral to the axotomy. In contralateral nodose ganglia the number of GAL- and NPY-positive neuron profiles was slightly increased, and VIP cells showed a moderate increase. Immunohistochemical analysis revealed paral- lel changes in expression of peptides and NOS in both trigeminal and nodose ganglia, demonstrating that the changes in mRNA levels are translated into protein. Fi- nally, although not quantified, similar upregulations of peptide and NOS mRNA levels were observed in both ganglia 42 days after nerve injury provided that regener- ation was not allowed, suggesting that the changes are long lasting. The present results show that the effect of axotomy on peptide and NOS expression in the trigemi- nal and nodose ganglia is similar to that previously shown for lumbar dorsal root ganglia. However, no mRNA for the NPY Y1 receptor could be detected in the vagal system. In general the mechanism(s) for and the purpose(s) of the messenger regulation in response to axotomy may be similar in these different sensory sys- tems (dorsal root, trigeminal and nodose ganglia).

Key words Galanin - Neuropeptide Y �9 Vasoactive intestinal polypeptide �9 Plasticity �9 Axotomy

Introduction

Expression of peptides and their mRNAs in dorsal root ganglia (DRGs) can be markedly influenced by various experimental manipulations, particularly after peripheral axotomy. Thus, Jessell et al. (1979) reported a decrease in substance P levels in the dorsal horn after peripheral axotomy, subsequently shown to be due to downregula- tion of substance P synthesis in DRGs (Nielsch et al. 1987). A distinct increase in vasoactive intestinal poly- peptide (VIP) levels occurs in the dorsal horn after pe- ripheral axotomy (McGregor et al. 1984; Shehab and At-

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kinson 1984, 1986b; Xu et al. 1990; Kashiba et al. 1992b), and this is due to increased synthesis in DRGs (Shehab and Atkinson 1986a; Noguchi et al. 1989; Xu et al. 1990; Kashiba et al. 1992b). Similarly, galanin (GAL) (H6kfelt et al. 1987; Kashiba et al. 1992b; Villar et al. 1989, 1991; Xu et al. 1990; Zhang et al. 1995c), neuro- peptide Y (NPY) (Noguchi et al. 1993; Wakisaka et al. 1991, 1992; Zhang et al. 1993b), substance P (in a sub- population) (Noguchi et al. 1994, 1995), pituitary ade- nylate cyclase-activating polypeptide (PACAP) (Zhang et al. 1995b), and also the enzyme nitric oxide synthase (NOS) (Fiallos-Estrada et al. 1993; Verge et al. 1992; Zhang et al. 1993c) are markedly upregulated in DRGs after peripheral axotomy. In addition, the mRNA levels of two recently cloned peptide receptors, the Y1 subtype of the NPY receptor (Y1 receptor) (Eva et al. 1990; Her- zog et al. 1992; Larhammar et al. 1992) and the chole- cystokinin (CCK) B receptor (Wank et al. 1992), both change in DRG neurons in response to axotomy (Zhang et al. 1993a, 1994).

Most of the studies on the effect of axotomy on pep- tide expression have been carried out on spinal DRGs. However, an increase in NPY-like immunoreactivity (- LI) has been observed in medium-sized and large neu- rons in trigeminal ganglia (Wakisaka et al. 1993). Pep- tides are affected by axotomy in the visceral sensory system also. Thus, Helke and Rabchevsky (199l) have demonstrated an increase in VIP-LI in the nodose gangli- on and a parallel reduction in the number of tyrosine hy- droxylase immunoreactive cells after axotomy, whereas calcitonin gene-related peptide (CGRP) and substance P cells seemed relatively unaffected. Thus, there may be certain variations in the regulation of peptide expression in response to axotomy between different types of senso- ry ganglia. This question is of interest, since this type of sensory neuron monitors different stimuli as compared with, for example, DRG neurons, including increase in blood pressure, changes in blood gases and distension of internal organs such as heart, lung and stomach (Paintal 1973).

In the present study we have, as a comparison with our studies on L4 and L5 DRGs (see HOkfelt et al. 1994), carried out a more extensive analysis of the effect of axotomy on the expression of peptides, NOS and NPY receptor in a somatosensory ganglion, the trigeminal ganglion, as well as in a viscero-sensory ganglion, the nodose ganglion, using immunohistochemistry and in situ hybridization.

Materials and methods

Animals and operations

Twenty-four male Sprague-Dawley rats (body weight 200-250 g; Alab, Stockholm, Sweden) were anesthetized with an intraperito- heal injection of sodium pentobarbital (Mebumal; 60 mg/kg). Twelve rats received a unilateral transection of the submandibular branch of the trigeminal ganglion. Another 12 rats were subjected to unilateral, cervical vagotomy. They were allowed to survive for 14 or 42 days after surgery. We chose 14 days as the main post-

surgery interval for analysis because in previous studies on lumbar DRG neurons the effects of axotomy were maximal at that time point, but also because it is well within the period from 6 days to several months when dramatic effects can always be recorded.

The Principles of Laboratory Animal Care (NIH publication 86-23, revised 1985) were followed. The present experiments were approved by the local ethics committee (Stockholms norra djurf0rsiSksetiska n~mnd; application Dnr. 102193).

Immunohistochemistry

Three rats from each experimental group and each time point (14 and 42 days) as well as three normal control rats were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and perfused via the as- cending aorta with warm (37~ Ca2+-free Tyrode's solution (50 ml) followed by warm fixative containing 4% paraformalde- hyde and 0.2% picric acid (50 ml) (Pease 1962; Zamboni and De Martino 1967) and the same, but ice-cold fixative for 6 rain. The trigeminal and nodose ganglia were rapidly dissected out and im- mersed in the same fixative for 90 rain, then rinsed in 10% sucrose with added Bacitracin (0.01%; Bayer, Leverkusen, Germany) and sodium azide (0.02%; Sigma, St. Louis, Mo.) for at least 24 h.

The trigeminal ganglia were then mounted together on a chuck and the nodose ganglia on another chuck, frozen and cut at 14 gm thickness in a cryostat (Microm, Heidelberg, Germany) and pro- cessed for the indirect immunofluorescence technique (Coons 1958). Briefly, the sections were incubated with rabbit antiserum to GAL (1:400; Peninsula, Belmont, Calif.), GAL message-associ- ated peptide (GMAP) (1:400; H6kfelt et al. 1992), NPY (1:400; J. Walsh, unpublished), substance P (1:400; Christensson-Nylander et al. 1986), peptide histidine isoleucine (PHI) (1:800; Fahrenkrug and Pedersen 1984) or amino acids 845-864 of rat neuronal NOS (1:800; Riveros-Moreno et al. 1993). After rinsing in phosphate- buffered saline (PBS), the sections were incubated with fluores- cein isothiocyanate (FIIC)-conjugated goat anti-rabbit antibodies (1:80; Boehringer Mannheim Scandinavia, Stockholm, Sweden), rinsed in PBS, mounted in a mixture of PBS and glycerol (1:3) containing 0.1% p-phenylenediamine (Johnson and de C Nougue- ira Araujo 1981; Platt and Michael 1983) and examined in a Ni- kon Microphot-FX microscope equipped for epifluorescence with proper filter combinations. Kodak T-max 400 black-and-white film was used for photography.

Controls were carried out by preabsorption of antisera with an excess (10 -6 M) of the corresponding peptide, i.e., GAL, NPY, substance P (Peninsula, Belmont, Calif., USA or Bachem, Bis- sendorf, Switzerland) and GMAP (H6kfelt et al. 1992) and amino acids 845-864 of rat NOS (see Ceccatelli et al. 1993). None of the above immunoreactivities were observed after absorption with an excess of the respective peptide (10-6M).

Fig. 1 Immunofluorecence micrographs of control (white stars) trigeminal ganglia (a, e, g, j) and ipsilateral trigeminal ganglia (b-d, f, h, i, k) 14 days after unilateral axotomy, incubated with antiserum to GAL (a, e), GMAP (b, d), NOS (e, f), NPY (g-i) and PHI (j, k). a-d Some GAL-immunoreactive (IR) small neuron pro- files (arrowheads) are seen in a control ganglion. Many small neu- ron profiles and some medium-sized and large ones are GMAP- and GAL-IR in ipsilateral ganglia, e A few small (arrowheads) and medium-sized NOS-IR neuron profiles (arrows) in a control ganglion, f Some small (arrowheads) and medium-sized (arrows) neuron profiles are NOS-IR in an ipsilateral ganglion, g Only a single neuron is NPY-IR (arrowhead) in a control ganglion, h Many large neuron profiles in the ipsilateral ganglion contain NPY-LI. i High magnification of NPY-IR neurons in an ipsilateral ganglion, j There are no PHI-IR neurons in a control ganglion, k A few detectable PHI-IR neurons are observed in an ipsilateral ganglion (arrowheads point to small profiles, arrows to medium- sized ones). Scale bars represent 100 lain in a (a=b=e=f=g=h=j=k) and 50 gm in e (e=d=i)

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Table 1 Percentage of positive a DRG neuron profiles in the trigeminal and nodose ganglia of normal rats (Control) and in contralateral (Contra) and ipsilateral (Ipsi) ganglia 14 days after unilateral axotomy

Trigeminal ganglia Nodose ganglia

Control Ipsi Control Contra Ipsi

GAL 10.9 34.2 3.2 7.3 78.1 NOS 3.7 21.5 17.1 18.2 37.0 NPY 4.8 54.3 3.4 8.3 22.9 VIP 7.2 31.1 0.6 13.6 46.0 Y1-R 18.5 28.2 n.d. n.d. n.d.

a mRNAs for: GAL, galanin; NOS, nitric oxide synthase; NYP, neuropeptide tyrosine (NPY); VIP, vasoactive intestinal polypeptide; Y1- R, NPY1 receptors, n.d., not detectable

In situ hybridization

Three rats of each experimental group and each time point as well as three normal control rats were deeply anesthetized with an in- traperitoneal injection of sodium pentobarbital (60 mg/kg) and perfused via the aorta with 50 ml warm saline to clear the blood, followed by rapid dissection and freezing of the trigeminal and no- dose ganglia. Before sectioning, experimental and normal trigemi- nal ganglia were fused with saline on the same block and nodose ganglia on another block, so that all groups could be processed to- gether on the same slide. Sections (14 gin) were cut in a cryostat (Microm, Heidelberg, Germany), thawed onto "Probe-On" slides (Fisher Scientific, Pittsburgh, Pa.) and stored in sealed boxes at -20~ until hybridization.

Preparation of probes

Oligonucleotide probes were purchased from Scandinavian Gene Synthesis AB (KOping, Sweden). Sequences of the oligonucleo- tide probes were complementary to nucleotides 152-199 of rat GAL (Vrontakis et al. 1987), nucleotides 347-394 of rat VIP (Ni- shizawa et al. 1985), nucleotides 1671-1714 of rat NPY (Larham- mar et al. 1987), nucleotides 546-585 of the rat NPY (Y1) recep- tor (Eva et al. 1990), and nucleotides coding for amino acids 151-164 of rat brain NOS (Bredt et al. 1991). The oligonucleotide probes were labeled at the 3' end with [c~-35S]dATP (New England Nuclear, Boston, Mass.) using terminal deoxynucleotidyl transfer- ase (Amersham, Amersham, UK) in a buffer containing 10 mM cobalt chloride, 1 mM dithiothreitol (DTT), 300 mM TRIS base, and 1.4 M potassium cacodylate (pH 7.2). The labeled probes were purified through Nensorb-20 columns (New England). Activ- ities obtained were in the range 1Mx 10 6 cpm/ng oligonucleotide.

Hybridization procedure

Our procedure followed previously published protocols (Schalling 1990; Young 1990; Dagerlind et al. 1992). Sections were hybrid- ized after thawing without pretreatment for 16-18 h at 42~ in hu- midified boxes with 106 cpm of labeled probe per 100 gl of a mix- ture containing: 50% formamide (G.T. Baker Chemicals, Deven- ter, The Netherlands); 4xSSC (lxSSC=0.15 M sodium chloride and 0.0015 M sodium citrate); lxDenhardt~s solution (0.02% each of polyvinylpyrrolidone, bovine serum albumin and Ficoll); 1% sarkosyl (N-lauryl sarcosine; Sigma); 0.02 M phosphate buffer (pH 7.0); 10% dextran sulfate (Pharmacia, Uppsala, Sweden); 250 gg/ml yeast tRNA (Sigma); 500 ~tg/ml sheared and heat-denatured salmon testis DNA (Sigma); and 200 mM DTT (LKB, Bromma, Sweden).

After hybridization the sections were rinsed repeatedly (4x15 min) in lxSSC at 55~ then brought to room temperature over 30 min while in the final rinse, dipped twice in distilled wa- ter, dehydrated through 60% and 95% ethanol, and dried in air. The slides were dipped in NTB2 nuclear track emulsion (Kodak,

Rochester, NY, USA) diluted 1:1 with distilled water, exposed in the dark at -20~ for 4-6 weeks, developed in D-19 (Kodak) for 3 min, fixed in Kodak 3000A&B for 6 min, and rinsed for 30 rain in running water. Developed slides were mounted with glycerol and coverslipped for analysis in a Nikon Microphot-FX micro- scope equipped with a dark field condenser or stained with tolui- dine blue, mounted with Entellan (Merck, Darmstadt, Germany) and a coverslip for viewing under brightfield illumination.

Quantification

To determine the percentage of labeled neurons before and after injury, counts were done on the sections stained with toluidine blue. Four to eight sections of each ganglion from three control animals and three injured animals were randomly chosen and ana- lyzed 14 days after the lesions. The sections were examined under hrightfield illumination using a x20 objective lens. Neuron pro- files containing 3 times more grains than mean background grain densities were counted. Mean background grain densities were de- termined by averaging grain counts over defined areas of the neu- ropil devoid of positively labeled cell bodies (0-5 grains per 100 gin2). Two hundred and thirty to 430 neuron profiles from the sec- tions of each trigeminal ganglion and 145-200 neuron profiles from the sections of each nodose ganglion were analyzed. The to- tal number of labeled neuron profiles was divided by the total number of toluidine-blue-stained neuron profiles. Data were ana- lyzed with a one-tailed unpaired t-test, and results expressed as the mean+standard error of mean.

Fig. 2 Darkfield (a-k) and brightfield (l-n) micrographs of con- �9 trol (stars) trigeminal ganglia (a, c, e, g, i, 1) and ipsilateral tri- geminal ganglia 14 (b, d, f, h, j, m) or 42 (k, n) days after unilat- eral axotomy, hybridized with probes against GAL (a, b), NOS (c, d), NPY (e, f), HVIP (g, h) and Y1 subtype of NPY receptor (Y1) (i-n) mRNAs, a A few neurons in a control ganglion are GAL mRNA-positive (arrowheads). b Numerous GAL mRNA-positive neurons are seen in an ipsilateral ganglion, e Only one NOS mR- NA-positive neuron (arrowhead) is observed in a control ganglion. d Some neurons (arrowheads) express NOS mRNA in an ipsilater- al ganglion, e No NPY mRNA-positive neurons are seen in a con- trol ganglion, f Numerous neurons are NPY mRNA-positive in an ipsilateral ganglion, g There are no VIP mRNA-positive neurons in a control ganglion, h Many neurons express VIP mRNA in an ipsilateral trigeminal ganglion, i, 1 Many Y1 mRNA-positive small neuron profiles (arrowheads) are observed in a control ganglion, j, m Fourteen days after axotomy some large neuron profiles contain Y1 receptor mRNA (arrows) in addition to small Y1 receptor mR- NA-positive neuron profiles (arrowheads). k, n Forty-two days af- ter axotomy many large (arrows) and small (arrowheads) neuron profiles contain Y1 receptor mRNA. Scale bar represent 100 ~tm in a (a=b-h) and 50 gm in I (l=m=n)

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Controls

Control hybridizations were carried out with an excess of cold probe (100-fold) together with the labeled probe. None of the mR- NA-positive cells described above were seen after addition of an excess of cold probe to the hybridization cocktail.

Results

21.5_+3.1% (P<0.01) NOS mRNA-positive, 54.3_+1.8% (P<0.01) NPY mRNA-positive, 31.1_+1.0% (P<0.01) VIP mRNA-positive and 28.2_+1.0% (P<0.01) Y1 recep- tor mRNA-positive. After axotomy many large neurons in the ipsilateral trigeminal ganglia expressed Y1 recep- tor mRNA (Fig. 2m,n). These changes in peptide immu- noreactivities and mRNAs were still seen 42 days after axotomy.

Trigeminal ganglia

Control and contralateral trigeminal ganglia

In control and contralateral trigeminal ganglia GAL/GMAP-LIs were seen in several neurons (Fig. la). NOS-LI (Fig. le) and NPY-LI (Fig. lg) were observed in low numbers of neurons. GAL/GMAP and NPY immu- noreactive neuron profiles were small in size (20-32 gm in diameter; longest diameter measured). NOS-L! was observed in small and medium-sized (32-50 gm in diam- eter) neuron profiles. PHI-LI was not detected in these trigeminal ganglia.

The quantitative analysis based on in situ hybridiza- tion (Table 1) revealed that 10.9+1.1% of all counted neuron profiles were weakly GAL mRNA-positive (Fig. 2a), 3.7_+0.5% NOS mRNA-positive (Fig. 2c), 4.8_+0.5% NPY mRNA-positive (Fig. 2e), 7.2_+1.5% VIP mRNA-positive (Fig. 2g) and 18.5_+0.4% Y1 receptor mRNA-positive (Fig. 2i,1) in normal trigeminal ganglia. Y1 receptor mRNA was expressed in small neuron pro- files (Fig. 21).

The distributions of neuropeptide-LIs and -mRNAs, of NOS-LI and -mRNA, and Y1 receptor mRNA in the trigeminal ganglia contralateral to the axotomy were similar to the ones in control trigeminal ganglia.

Ipsilateral trigeminal ganglia

Fourteen days after peripheral axotomy there was a dra- matic increase in the number of GAL/GMAP-positive (Fig. 1b-d) and NPY-positive (Fig. lh,i) cell bodies in the ipsilateral trigeminal ganglia. There was also an in- crease in the number of NOS-positive (Fig. lf) and PHI- positive (Fig. lk) neurons, although this effect was less pronounced than for the two previous peptides. GAL/GMAP-, NOS- and PHI-LIs were mainly observed in small neuron profiles, but also in some medium-sized and large ones, whereas NPY-LI was seen mainly in large neuron profiles (50-75 gm in diameter).

The in situ hybridization analysis of the ipsilateral tri- geminal ganglia (Table 1) revealed parallel changes in mRNA levels 14 days after peripheral axotomy. Both the percentage of positive neurons and the labeling intensity increased. Thus, there was a dramatic increase in levels of GAL (Fig. 2b), NPY (Fig. 2f) and VIP/PHI (Fig. 2h) mRNAs and a less pronounced change in NOS mRNA (Fig. 2d). Two weeks after axotomy 34.2_+3.7% (P<0.01) of all counted neurons were GAL mRNA-positive,

The nodose ganglion

Control and contralateral nodose ganglia

The immunohistochemical analysis of control and con- tralateral nodose ganglia revealed single GAL/GMAP- positive (Fig. 3a) and NPY-positive (Fig. 3i) cell bodies. They represented small neuron profiles with a diameter of 24-32 gm. The control nodose ganglia contained many NOS-positive cells, and these profiles were of both medium (32-37 gm in diameter) and large (37-51 gm in diameter) size (Fig. 3e,f).

In situ hybridization revealed only single GAL/GMAP (Fig. 4a) and NPY (Fig. 4g), many NOS (Fig. 4d) and almost no VIP (Fig. 4j) mRNA-positive neurons in control nodose ganglia. GAL mRNA was ob- served in 3.2-+0.6%, NOS mRNA in 17.1_3.6%, NPY mRNA in 3.4_1.2% and VIP mRNA in 0.6_+0.1% of all counted neurons in normal nodose ganglia.

In the contralateral nodose ganglia, 14 days after pe- ripheral axotomy, there was a slight increase in the num- ber of both GAL/GMAP immunoreactive neurons (Fig. 3d), as well as GAL (Fig. 4c), NPY (Fig. 4i) and particularly VIP (Fig. 41) mRNA-positive neurons. Thus, 2 weeks after axotomy GAL mRNA was expressed in 7.3_+1.3% (P<0.05), NPY mRNA in 8.3+1.9% (P<0.05) and VIP mRNA in 13.6_+4.9% (P<0.05) of all counted neuron profiles. No difference was observed with regard to NOS mRNA-positive neurons in the contralateral no- dose ganglia before and after axotomy. No signal for NPY Y1 receptor mRNA was observed in these nodose ganglia.

Ipsilateral nodose ganglia

In the ipsilateral nodose ganglia there was at 14 days a dramatic increase in the number of GAL/GMAP-positive neurons (Fig. 3b,d) and a somewhat less pronounced in- crease in the number of NPY-positive neurons (Fig. 3j,k). These immunoreactive neurons were now found in all size ranges, but most positive profiles were small or me- dium-sized. There was also a marked increase in the number of NOS-positive neurons (Fig. 3g,h). In particu- lar, there were many more small NOS-positive neuron profiles.

Parallel observations were made with regard to mR- NA levels visualized with in situ hybridization, and some of the effects were more clearly revealed using this

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Fig. 3 Immunofluorescence micrographs of control (white stars) nodose ganglia (a, e, f, i) and of ipsilateral (b, d, g, 11, j , k) and contralateral (open star) (c) nodose ganglia 14 days after unilater- al axotomy, incubated with antiserum to GAL (a, b), GMAP (e, d), NOS (e-h) and NPY (i-k). a A few GAL-immunoreactive (IR) neurons (arrowheads) are seen in a control ganglion, b Many neu- rons are GAL-IR in an ipsilateral ganglion, e Some GMAP-IR neurons are observed in a contralateral ganglion, d High magnifi- cation of ipsilateral ganglion showing many GMAP-IR neurons. Arrow points to weakly fluorescent large neuron, e Some NOS-IR neurons are present in a control ganglion, f High magnification of

NOS-IR neurons in a control ganglion, g Many neurons are NOS- IR after axotomy, and in particular the number of small neuron profiles is increased, h High magnification of an ipsilateral gangli- on showing many NOS-IR small neuron profiles, i A few NPY-IR neurons (arrowheads) are present in a control ganglion, j Some NPY-IR neurons are observed in an ipsilateral ganglion. Arrows point to immunofluorescent large neuron profiles, k High magnifi- cation of ipsilateral ganglion showing many small and two large (arrows) NPY-positive neuron profiles. Scale bar represents 100 gm in a (a=b=e=g=i=j) and 50 gm in d (d=c=f=h=k)

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Fig. 4 Darkfield micrographs of control (white stars) nodose gan- glia (a, d, g, j), and of ipsilateral (b, e, h, k) and contralateral (open stars) (e, f, i, 1) nodose ganglia 14 days after unilateral axo- tomy, hybridized with probes for GAL (a-e), NOS (d-f), NPY (g-i) and VIP (j-l) mRNAs, a No GAL mRNA-positive neurons are detectable in a control ganglion, b Numerous GAL mRNA- positive neurons are observed in an ipsilateral ganglion, e GAL mRNA is expressed in a few neurons in a contralateral ganglion, d Some neurons express NOS mRNA in a control ganglion, e, f More NOS mRNA-positive neurons are seen in an ipsilateral than in a contralateral ganglion, g A few NPY mRNA-positive neurons (arrowheads) are seen in a control ganglion, h Many neurons ex- press NPY mRNA in an ipsilateral ganglion, i Some NPY mRNA- positive neurons are observed in a contralateral ganglion, j There are no VIP mRNA-positive neurons in a control ganglion, k Many neurons express VIP mRNA in an ipsilateral ganglion, l Some VIP mRNA-positive neurons are observed in a contralateral ganglion. Scale bar represents 100 gm; all pictures have the same magnifica- tion

methodology . On the les ion side there was a dramat ic in- crease in the number of G A L (Fig. 4b), VIP (Fig. 4k) and to a lesser extent of N P Y (Fig. 4h) m R N A - p o s i t i v e cells. A modera te increase in the number o f NOS mR- NA-pos i t i ve neurons was obse rved in the ips i la tera l no- dose gangl ia (Fig. 4e). Two weeks after axo tomy 78.1_+3.8% (P<0.01) of all counted neurons were G A L mRNA-pos i t i ve , 22 .9+6.1% (P<0.05) N P Y m R N A - p o s i - rive, 37 .0_3.3% (P<0.01) NOS m R N A - p o s i t i v e and 46.0+2.9% (P<0.01) VIP mRNA-pos i t ive . No N P Y Y1 receptor m R N A could be de tec ted in the ips i la tera l no- dose gang l ia 14 days after axotomy.

Discussion

In the present study we have analyzed to what extent changes in peptide expression known to occur after axo-

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tomy in spinal DRG neurons (for references see Introduc- tion) also take place after this type of lesion in the trigem- inal ganglion (which is another somatosensory neuron) as well as in the nodose ganglion (which contains visceral sensory neurons and has a placodal origin, in contrast to the neurocrest origin of the somatosensory systems) (Pa- intal 1973). Here we have focused only on peptides and an enzyme (NOS) which have been shown to be upregu- lated in DRGs after nerve injury, although it is well known that some peptides and their mRNAs decrease in response to this manipulation (for references see Htkfelt et al. 1994) Also, we have analyzed only two time inter- vals after axotomy: 14 and 42 days. In the DRGs more complete time curves were studied; marked changes were seen already after 3 days, and these effects remained for at least 2 months provided that regeneration was prevent- ed (see, e.g., Villar et al. 1989). It is therefore likely, but not absolutely certain, that the time intervals studied here represent an appropriate experimental paradigm.

The present results strongly suggest that the trigemi- nal sensory neurons react in qualitatively the same way as DRG neurons. Thus there is dramatic upregulation of both GAL/GMAP- and NPY-LI. However, less dramatic changes than in DRGs were observed for VIP/PHI, and the nitric oxide synthesizing enzyme NOS seemed much less affected than observed in the lumbar 4 (L4) and L5 ganglia after transection of the sciatic nerve (Verge et al. 1992; Fiallos-Estrada et al. 1993; Zhang et al. 1993c). Whether these changes are due to the fact that these pep- tides and this enzyme are indeed under less strong regu- lation by axotomy or whether neurons containing these compounds do not project into the submandibular nerve branch, which is the only one transected in the present study, remains to be analyzed. Another apparent quanti- tative difference concerns NPY. Thus NPY mRNA and NPY-LI are observed in a small population of neurons in the control trigeminal ganglia, whereas no NPY mRNA neurons were observed in control DRGs (Wakisaka et al. 1993). The distribution of Y1 receptor mRNA and its regulation after axotomy in trigeminal ganglia are simi- lar to the ones observed in DRGs (Zhang et al. 1994).

The nodose ganglia have been studied in particular by Helke and collaborators in a series of papers and have been shown to contain virtually the same peptides as the DRGs (Katz and Karten 1980; Mantyh and Hunt 1984; Helke and Hill 1988; Helke and Niederer 1990; Helke and Rabchevsky 1991) and also the catecholamine syn- thesizing enzyme tyrosine hydroxylase (Lundberg et al. 1978; Katz et al. 1983; Katz and Black 1986). Interest- ingly, in contrast to the trigeminal ganglion and cervical and lumbar DRGs, a high proportion of the nodose gan- glion neurons (30%) contains NADPH diaphorase (Aimi et al. 1991) and NOS mRNA (Verge et al. 1992). Helke and Rabchevsky (1991) have shown that axotomy of the cervical vagus nerve causes a dramatic increase in VIP- immunoreactive neurons and a reduction in the number of tyrosine hydroxylase positive cells, whereas CGRP and substance P were unaffected by this type of nerve section. Zhuo et al. (1995) have found that arrest of axonal trans-

port by the mitosis inhibitor vinblastine also causes changes in the expression of peptides and enzymes, as has previously been shown for lumbar DRGs (Kashiba et al. 1992a).

In the present study we have confirmed the effect on VIP and demonstrated that there is a dramatic increase also in the number of GAL-positive neurons: after axotomy GAL is present in 80% of all ganglion cells, which is in marked contrast to the small percentage in control ganglia. A slight increase was also seen in the contralateral nodose ganglion. Thus, GAL mRNA is increased about 25-fold in the nodose ganglion compared with the only 3-fold in- crease in the trigeminal ganglion. However, since only one branch from the trigeminal ganglion was lesioned, many neurons escaped axotomy and thus the stimulus for upreg- ulation. Also NPY-LI and NPY mRNA increased substan- tially in many small nodose neurons, which is in contrast to DRG and trigeminal ganglion, where the increase oc- curs mainly in large neurons (Wakisaka et al. 1991, 1992, 1993; Noguchi et al. 1993; Zhang et al. 1993b). With re- gard to NOS-LI and NOS mRNA there was almost a dou- bling of the number of positive cells in the nodose gangli- on after axotomy. The increase was observed especially in the population of small neuron profiles, which were la- beled only a little in the control ganglia.

Taken together the present findings show that the regu- lation of neuropeptides and NOS in response to nerve in- jury seems largely to occur in the same way in the trigem- inal and visceral nodose ganglia as in DRGs, in spite of their different origin. It is therefore likely that the mecha- nism(s) underlying this regulation, as well as the purpose of the regulation, are similar in these three types of senso- ry neurons. Major differences were, however, the high numbers of NOS-positive cells in normal nodose ganglia, and especially the fact that Y1 receptor mRNA was ob- served neither in normal nodose ganglia nor in the ipsilat- eral nodose ganglia after axotomy. Whether or not this is related to the fact that the vagal afferent system does not convey the pain modality but is mainly related to intesti- nal function and behavioral responses such as feeding, anxiety and nausea (Grundy 1992), remains to be eluci- dated. Moreover, we have proposed that at least one pur- pose of the upregulation of GAL in spinal DRG neurons is to prevent pain signals from reaching higher centers (Wiesenfeld-Hallin et al. 1992). In the vagal system the marked upregulation of GAL is thus presumably not re- lated to pain mechanisms but perhaps to counteracting excessive activity in systems mediating other modalities such as those just mentioned.

The mechanisms underlying the dramatic upregula- tion of peptides and enzymes in sensory neurons are not well known. However, neurotrophic factors such as nerve growth factor may play a role (Kessler and Black 1980; Fitzgerald et al. 1985; Lindsay and Harmar 1989; Lind- say et al. 1989; Mulderry 1994; Otten et al. 1980; Verge et al. 1995), and more recently leukemia inhibitory factor (LIF; or cholinergic differentiation factor, CDF) (Fukada 1985; Yamamori et al. 1989) has been shown to be a key molecule in the control of expression of transmitter and

402

peptides not only in sensory but also in sympathetic neu- rons (Nawa and Patterson 1990; Freidin and Kessler 1991; Nawa et al. 1991; Rao et al. 1993, 1992; Shadiack et al. 1993; Bamber et al. 1994; Banner and Patterson 1994; Patterson 1994; Zhang et al. 1995a). To what ex- tent these factors also are important for the expressions and regulations demonstrated in the present study in the trigeminal and nodose systems remains to be shown.

Acknowledgements This work is supported by the Swedish MRC (04X-2887), the Bank of Sweden Tercentenary Foundation, Marianne and Marcus Wallenbergs Stiftelse, Gustav V:s and Drottning Victorias Stiftelse, National Institute of Aging (AG- IDY91-03) and ASTRA Pain Control AB. We thank Prof. S. Moncada, The Wellcome Research Laboratories, Beckenham, UK (NOS), Prof. J. Fahrenkrug, Bispebjerg Hospital, Copenhagen, Denmark (VIP, PHI), Prof. L. Terenius, Karolinska Institute, Stockholm, Sweden (SP) and Dr. J. Walsh, CURE/UCLA, DDC Antibody Core (NIH grant DK 17294) (NPY) for generously sup- plying antisera.

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