ORIGINAL ARTICLES—Laboratory science

Pituitary adenylate cyclase activating polypeptideand nitric oxide synthase are expressed in the ratciliary ganglion

Tor Elsås, Rolf Uddman, Hindrik Mulder, Frank Sundler

AbstractAims—To study the distribution in the ratciliary ganglion of neurons synthesisingand storing the recently discovered neu-ropeptide pituitary adenylate cyclase acti-vating polypeptide (PACAP) and neuronalnitric oxide synthase (NOS), the neuronalmarker of the novel gaseous transmitternitric oxide.Methods—Neurons expressing PACAPand neuronal NOS mRNA were identifiedin the rat ciliary ganglion by in situhybridisation with radiolabelled oligonu-cleotide probes. Immunocytochemistrywas used to demonstrate immunoreactiveneuropeptides and NOS.Results—Immunocytochemistry demon-strated immunoreactivity for PACAP andNOS in a small number of neuronal cellbodies. In situ hybridisation revealed thatNOS and PACAP were expressed innumerous ganglion cell somata. The wellestablished ciliary messengers vasoactiveintestinal peptide and neuropeptide Ywere found in a large number of neuronalcell bodies.Conclusion—These results demonstratethat PACAP and NOS are synthesised andstored in the ciliary ganglion. These find-ings further illustrate the mixed nature ofthe ciliary ganglion and may provide abasis for the understanding of the diversephysiological functions of this ganglion.(Br J Ophthalmol 1997;81:223–227)

The concept of the ciliary ganglion as a purelycholinergic, parasympathetic ganglion hasbeen challenged by the finding of not onlyparasympathetic but also sympathetic and sen-sory transmitters in this ganglion.1–4 Recently, ahost of novel neuronal messengers that over-ride the ‘classic’ taxonomy of sympathetic,parasympathetic, and sensory messengers hasbeen identified. For instance, pituitary ade-nylate cyclase activating polypeptide (PACAP)is a vasoactive intestinal peptide (VIP)-likeneuropeptide, initially isolated from ovinehypothalamus, which is expressed both in thesensory and autonomic nervous system.5–8 Fur-

ther, the identification of nitric oxide (NO) as aneuronal and non-neuronal messenger is ofparticular interest owing to its chemicalidentity, its wide distribution, and its broadspectrum of eVects.9 Because of its highlability, information on the cellular localisationand the site of formation of NO has beenattained by immunocytochemistry and in situhybridisation for nitric oxide synthase(NOS),10 11 or by the histochemical techniquefor demonstration of NADPH diaphorase,which stains neurons containing NOS.11–13

VIP and neuropeptide Y (NPY) are wellestablished as neuropeptides in the ciliary gan-glion and the eye, and seem to exert severalphysiological eVects on ocular structures.2–4 14 15

Given the wide range of suggested physio-logical roles for the ciliary ganglion, we have inthe present study used in situ hybridisation andimmunocytochemistry to investigate whetherrat ciliary neurons express these novelmessengers—that is, PACAP and NOS, andcompared this with the expression of the estab-lished ciliary messengers NPY and VIP.

Materials and methodsTwelve adult female Sprague-Dawley rats(body weight 200–250 g) were used. Theanimals were anaesthetised by chloralose andkilled by exsanguination, the ciliary ganglionwas subsequently dissected. For in situ hybridi-sation, the specimens were rapidly frozen ondry ice and stored at −80°C until they were cuton a cryostat (10 µm). For immunocytochem-istry, the specimens were fixed by immersion ina mixture of 2% formaldehyde and 0.2% picricacid solution in 0.1 M phosphate buVer (pH7.2) overnight, followed by repeated rinsing ina Tyrode solution containing 10% sucrose.They were then frozen on dry ice and stored at−80°C. The specimens were sectioned at 10µm thickness in a cryostat.

IN SITU HYBRIDISATION

The cellular localisation of mRNAs forPACAP, NOS, VIP, NPY, and calcitonin generelated peptide (CGRP) in the rat ciliaryganglion was determined by in situ hybridisa-tion, using oligodeoxyribonucleotide probes.These probes have previously been charac-

British Journal of Ophthalmology 1997;81:223–227 223

Department ofOphthalmology,Trondheim, NorwayT Elsås

Department ofOto-Rhino-Laryngology,Malmö GeneralHospital, Malmö,SwedenR Uddman

Department ofPhysiology andNeuroscience, Sectionfor NeuroendocrineCell Biology,University of Lund,SwedenHMulderF Sundler

Correspondence to:Tor Elsås, Department ofOphthalmology, UniversityHospital of Trondheim,N-7006, Trondheim,Norway.

Accepted for publication25 November 1996

terised.7 8 16 17 To ensure that the probesdisplayed no sequence homology to otherknown mRNAs, a computerised databasesearch was performed; no such homologieswere discovered (Genbank EMBL 3/96). Theprobes were 3'-endtailed by terminal trans-ferase to a specific activity of approximately 2 ×109 cpm/µg as described previously.7

The hybridisation protocol used has beendescribed in detail elsewhere.7 Briefly, the freshfrozen sections were rapidly warmed to roomtemperature and fixed in 4% paraformalde-hyde. The sections were acetylated by 0.25%acetic anhydride in 0.1 M triethanolamine,dehydrated, and cleared in chloroform. Hy-bridisation was carried out overnight in sealedmoist chambers at 37°C using probe concen-trations of approximately 1 pmol/ml. Afterhybridisation, the sections were washed in 1 ×SSC (1 × SSC = 0.15 M NaCl, 0.015 Msodium citrate; 4 × 15 minutes, 55°C +1 × 30minutes, room temperature). The slides weredipped in Ilford K5 emulsion and exposed for2 to 3 weeks.For control purposes, sections were incu-

bated in RNase A (45 µg/ml; Sigma, St Louis,MO, USA) for 30 minutes at 37°C beforehybridisation. As an additional control a100-fold molar excess of unlabelled probe wasadded to the hybridisation buVer.

IMMUNOCYTOCHEMISTRY

For the immunocytochemical demonstrationof PACAP, NOS, VIP, NPY, and CGRPindirect immunofluorescence was used.Detailsof the primary antibodies are given in Table 1.The sections were exposed to the primaryantiserum overnight at 4°C in a moist cham-ber. The site of the antigen–antibody reactionwas revealed by application of fluoresceinisothiocyanate (FITC) labelled antibodiesraised against rabbit immunoglobulin G (Da-kopatts, Copenhagen, Denmark), in a dilutionof 1:320 for 1 hour at room temperature. Con-trol sections were exposed to antiserum thathad been preabsorbed with an excess amountof the antigen (10–100 µg of synthetic peptideper ml diluted antiserum). Additionally, theantiserum was tested for cross reaction withother peptides (10–100 µg of peptide per mldiluted antiserum). No such cross reaction wasfound.Cross reactions with still other peptides or

proteins containing amino acid sequences rec-ognised by the diVerent antisera cannot beexcluded. It is appropriate, therefore, to referto the immunoreactive material as NOS-like,VIP-like, and so on. For brevity, however, the

shorter terms NOS and VIP will be usedhenceforth.

ResultsIN SITU HYBRIDISATION

Almost all neuronal cell bodies displayed somelabelling with an intensity that varied fromweak to strong. When labelled cells werecounted in random sections from the rat ciliaryganglion, approximately 20% of the ganglioncell somata demonstrated strong labelling forPACAP and NOS mRNA as evidenced by ahigh density of autoradiographic grains (Fig1A, B). The corresponding figures for the VIPand NPY probes were 35% (Fig 1C, D). Theintensity of labelling varied between the cells,but was consistently weaker for PACAPmRNA. The NOS and NPY probes showedstrong and uniform labelling, while the probesfor PACAP and VIP mRNA demonstratedvariability in the intensity of labelling of theneuronal cell bodies. Cell bodies harbouringCGRP mRNA were not observed.When the sections were treated with RNase

A before hybridisation or when an excess ofunlabelled probe was added to the hybridisa-tion buVer, autoradiographic labelling of nervecell bodies was abolished. Usually the sectionsdemonstrated probe labelling, but occasionalsections were negative.

IMMUNOCYTOCHEMISTRY

Scattered nerve cell bodies harboured immu-noreactivity for PACAP (Fig 2A) or NOS (Fig2B), which also occurred in varicose nervefibres within the ganglion. When neuronal cellbodies immunoreactive for PACAP and NOSwere counted in random sections of the rat cili-ary ganglion, scattered perikarya containedPACAP or NOS. A rich supply of nerve cellbodies, about 30%, contained VIP or NPY(Fig 2C, D). Immunoreactivity for CGRP wasnot observed in ciliary ganglion cells.The sections exposed to antibodies against

VIP or NPY usually showed some degree oflabelling of ganglion cell somata. Several of theslides treated with antibodies against PACAPor NOS were negative, probably due to the lownumber of cells harbouring immunoreactivityfor PACAP or NOS.

DiscussionThe classic concept of the ciliary ganglion asbeing a pure parasympathetic ganglion hasbeen replaced by the view of it being a mixedganglion comprised of neurons exhibitingparasympathetic, sympathetic, and sensoryneuromessenger phenotypes. Although ciliary

Table 1 Details of the antisera used for immunocytochemistry

Antigen Code Raised against Raised inWorkingdilution Source

PACAP-27 88121-3 Protein conjugated synthetic PACAP-27 Rabbit 1:640 Dr A Arimura, Lousiana, USANOS 9223 Synthetic fragment of rat cerebellar

NOSRabbit 1:2560 Euro-Diagnostica, Malmö, Sweden

VIP 7852 Unconjugated pure porcine VIP Rabbit 1:640 Euro-Diagnostica, Malmö, SwedenNPY E2210 Protein conjugated porcine NPY Sheep 1:800 Dr J Furness, Flinders University,

Bedford Park, AustraliaCGRP 8427 Protein conjugated rat CGRP Rabbit 1:640 Euro-Diagnostica, Malmö, Sweden

224 Elsås, Uddman,Mulder, Sundler

cell bodies exhibit immunoreactivity forcholine acetyltransferase, immunocytochemi-cal studies have revealed that the rat ciliaryganglion, in addition, harbours nerve cell bod-ies containing VIP, NPY, CGRP, andleu-enkephalin.1–4 18 It also contains nerve cellbodies immunoreactive for tyrosine hydroxy-lase and dopamine â hydroxylase.19

Our study demonstrates that PACAP is alsoexpressed in the rat ciliary ganglion; a sparsesupply of nerve cell bodies in the ciliaryganglion harboured immunoreactivity forPACAP, while around 20% of the ciliaryneuronal cell bodies demonstrated stronglabelling for PACAP mRNA. PACAP is arecently discovered neuropeptide with amarked sequence homology with VIP.5 Nervecell bodies containing PACAP mRNA andPACAP immunoreactivity can be seen in thesphenopalatine and otic ganglia, where PACAPcolocalises with VIP.8 Our demonstration ofPACAP expression in ciliary neurons is of par-ticular interest in relation to findings demon-strating the presence of PACAP receptors inocular tissues of the rabbit, and further, that inthe rabbit choroid PACAP is about 100 timesmore potent than VIP as a vasodilator.20 21 Sev-

eral lines of evidence suggest that PACAP inaddition to its autonomic functions also acts asa sensory neuropeptide, since nerve cell bodiesin sensory ganglia contain PACAP-like immu-noreactivity and PACAP mRNA.6 7 Further-more, PACAP depresses a nociceptive spinalreflex.22 Wang et al 23 found a CGRP-like distri-bution of PACAP in the rabbit eye, and thatPACAP probably mediated inflammatoryeVects.Around 20% of the perikarya in the ciliary

ganglion revealed strong labelling for NOSmRNA, while only scattered ganglion cellsomata were immunoreactive for NOS. Theseresults and previous findings of NOS immuno-reactive and NADPH diaphorase stainedciliary neurons makes it highly likely thatciliary neurons employ NO as a neuronalmessenger.11 24 Recently, several functionalstudies have indicated that NO is an importantmediator of inhibitory non-adrenergic, non-cholinergic responses. For instance, there isevidence that neuronal NO, released uponelectrical stimulation, takes part in non-adrenergic, non-cholinergic relaxation ofsmooth muscle in the bronchi and the digestivetract.25–27 Moreover, several studies suggest that

Figure 1 In situ hybridisation with radiolabelled oligonucleotide probes for PACAP mRNA (A),NOS mRNA (B), VIP mRNA (C), and NPY mRNA(D) in the ciliary ganglion of the rat. A moderate number of nerve cell bodies expressing PACAP, albeit at a low level is seen (A; indicated by arrows).NOS mRNA is present in a moderate number of nerve cell bodies (B), while VIP and NPY are seen in greater number of nerve cell bodies (C,D); in thisparticular section labelled for VIP mRNA, all nerve bodies express VIP (C). Bar = 50 µm.

Pituitary adenylate cyclase activating polypeptide and nitric oxide synthase are expressed in the rat ciliary ganglion 225

NO plays an important role in the regulation ofocular blood vessels.28–32

There are conflicting data regarding thenumber of ciliary ganglion cells containingVIP. Suzuki et al 3 reported the presence of VIPin almost all ciliary cell bodies, while Leblanc etal 1 and Stone et al 2 only found VIP in aminority of the ciliary ganglion cells. The dem-onstration of VIP both by in situ hybridisationand immunocytochemistry in numerous ciliaryganglion cells in the present study supports theview that VIP is an important neuropeptide inthe ciliary ganglion.A rich supply of neuronal ciliary cell bodies

expressed NPY.This is in concert with findingsin other studies.2 4 Tyrrell et al 4 found that85% of the rat ciliary ganglion cells containedNPY mRNA. In sympathetic ganglia, such asthe superior cervical ganglion, NPY-containingnerve cell bodies are numerous. In addition, amoderate supply of NPY-containing cell bodiescan be seen in parasympathetic ganglia such asthe otic and sphenopalatine ganglia.3 33 TheNPY fibres repopulating the iris following longterm sympathectomy arise from the ciliaryganglion.34 The ciliary ganglion as well as thesphenopalatine ganglion could well be a sourcefor the NP-containing nerve fibres remaining

in the choroid following removal of thesuperior cervical ganglion.35

One interesting aspect of the present study isthe much higher sensitivity of the mRNAprobes in labelling neuropeptide-containingneuronal cell bodies compared with immuno-cytochemistry. Similar findings were reportedby Tyrrell et al.4 In the rat ciliary ganglion, 25%of the neuronal cell bodies were NPY immuno-reactive and 85% expressed NPY mRNA.Studies on the distribution of PACAP andNOS in cranial ganglia show correspondingfindings with a much higher detection rate within situ hybridisation compared withimmunocytochemistry.8 16 The discrepancy be-tween the sensitivity of immunocytochemistryand in situ hybridisation raises the possibilitythat neuropeptides are present in many ciliaryneuronal cell bodies at levels not detectablewith immunocytochemistry.We were unable to find CGRP-containing

nerve cell bodies in the present study usingeither in situ hybridisation or immunocyto-chemistry. However, Stone et al 2 reportedCGRP immunoreactivity in around 25% of theneuronal cell bodies in the rat ciliary ganglion.In conclusion, the present study demon-

strates expression of the novel neuromessen-

Figure 2 Immunofluorescence for PACAP (A),NOS (B), VIP (C), and NPY (D) in the ciliary ganglion of the rat. A solitary PACAP immunoreactivenerve cell body (A; arrow) is seen as well as a supply of fine varicose PACAP-containing nerve fibres (A; indicated by arrowheads). NOS is harboured ina moderate number of nerve cell bodies (B; indicated by a solitary nerve cell body, arrow), while VIP and NPY occur in numerous nerve cell bodies andfibres in the ciliary ganglion (C,D; fibres indicated by arrows). Bar = 50 µm.

226 Elsås, Uddman,Mulder, Sundler

gers PACAP and NOS, as well as VIP andNPY in the rat ciliary ganglion. This furtherhighlights the complexity of the ganglion, sug-gesting its participation in multiple physiologi-cal functions.

This study was supported by the Swedish Medical ResearchCouncil (project no 12X-4499), The Albert Påhlsson and Cra-foord Foundations, the Faculty of Medicine, Lund University,Nycomed’s grant for Norwegian ophthalmologists, NordicResearch Council, Norwegian Medical Research Council, andthe Ophthalmological Society of Central Norway.

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