Neuroscience Vol. 39, No. 3, pp. 743-759, 1990 Printed in Great Britain

03064522/90 $3.00 + 0.00 Pergamon Press plc

0 1990 IBRO

NEUROFILAMENT PROTEIN-TRIPLET IMMUNOREACTIVITY IN DISTINCT SUBPOPULATIONS

OF PEPTIZE-CONTAINING NEURONS IN THE

GUINEA-PIG COELIAC GANGLION

J. C. VICKERS,* M. COSTA,*~ M. VITADELLO,~ D. DAHL§ and C. A. MAROTTA(/

*Centre for Neuroscience, Departmem of Physiology, Flinders University of South Australia, Bedford Park, SA 5042, Australia

3C.N.R. Istituto di Fisologia dei Centri Nervosi, 20131 Milano, Italy $Research Service, Veterans Administration Medical Centre, Boston, MA 02132, U.S.A.

IlMailman Research Centre, McLean Hospital, Belmont, MA 02178, U.S.A.

Abstract-A battery of polyclonal and monoclonal antibodies raised against the triplet of identified neurofilament protein subunits was used to investigate neurofilament protein immunoreactivity in neurons of the guinea-pig coeliac ganglion. Using optimal conditions of fixation and tissue processing for each antibody we found that only 20% of the postgangIionic sympathetic neurons in the guinea-pig coeliac ganglion contain neuro~ament protein-t~plet immunor~ctivity. Double labelling with neuro~ament protein-t~plet antibodies raised in different species demonstrate that all of these antibodies labelled the same population of neurons. Double labelling using mouse monocional antibodies against neurofilament proteins in combination with rabbit polyclonals to neuronal markers showed that neurotilament protein-triplet immunoreactivity is restricted to specific chemically coded subpopulations of noradrenergic neurons. Approximately 52% of neurons in the ganglion contain neuropeptide Y and are presumed vasomotor neurons projecting to blood vessels in the submucosa of the small intestine. Virtually none of the neuropeptide Y-containing neurons were labelled with neurofilament protein-triplet antibodies. Neurons that contain somatostatin (21%) project to the submucous ganglia of the small intestine. Approximately two-thirds of neurons containing somatostatin are immunoreactive for the neurofllament protein-triplet. The other postganglionic neurons in the ganglion (27%) project to the myenteric plexus of the small intestine and do not contain either neuropeptide Y or somatostatin. Approximately a quarter of these neurons were labelled with neurofilament protein-triplet antibodies.

These results suggest that the neucofilament protein-triplet may not be an instrinsic component of the cytoskeleton of all neurons. Furthermore the idea of a chemical coding of neurons should be extended to cytoskefetal proteins. The finding that these neuro~ament proteins are confined to specific neuronal subpopulat~ons has im~rtant implications for the search for a role of the neurofilament protein-t~plet in neurons, for the inte~retation of classical neurohistolo~~l silver imprecation techniques which appear to stain only neurofilament protein-t~plet~ontaining neurons, as well as for neuropathological conditions that may involve these proteins in disease processes.

Neurofilaments are neuron-specific intermediate filaments that appear to consist of a triplet of poly- peptide subunits of approximate molecular weights 68,000 (NF-L), 160,000 (NF-M) and 200,000 (NF-H) in mammals.38~4’~“~55 It is widely accepted that the neurofilament protein (NFP)-triplet is an integral component of the cytos~eleton of most mammalian neurons.y*17*34 However, a number of investigators using antibodies raised against these different NFP subunits have noted that not all neurons show NFP- triplet immunoreactivity. For example, only a pro- portion of nerve cell bodies in the mammalian dorsal root ganglia,‘*3’~52~58 trigeminal ganglion,57 spiral ganglion,4~53 enteric ganglia,6s7 retina23339 and cerebral cortex10,47*62 have been described as being labelled

tTo whom correspondence should be addressed. Abbreoiurions: DMSO, dirne~vls~~hoxi~~ NA. noradren-

aline; NIP, ne~o~~t protein; ~Y,-ne~~~tide Y, PBS, phosp~t~buffe~ solution; TH, tyrosine hydroxyl- ase; VIP, vasoactive intestinal peptide.

with NFP-triplet antibodies. Some antibodies are known to recognize only specific NFP subunits,2~22~M to distinguish phosphorylated and non-phosphorylated forms of the NFP-triplet6’ or to be immunoreactive only under particular tissue fixation conditions.35*51

It is important to establish whether the absence of NFP-~plet immunoreactivity in some neurons indicates that not all neurons contain these proteins or that the antibodies used simply do not recognize the form of the NFP present. In the present study we have examined the conditions under which NFP immunoreactivity can be detected, using a battery of antibodies raised against the NFP subunit proteins from different parts of the nervous systems of a variety of species, and by using a number of fixation procedures to optimize immunoreactivity for each of the NFP antibodies.

It would also be important to establish whether NFP-triplet ~~or~ctive neurons belong to specific groups of neurons. Sub~pulations of neurons have been distinguished on the basis of their chemical

743

744 J. C. VICKERS et d.

coding determined by the co-existence of multiple chemical messengers, including transmitter sub- stances.15.2g,37 The chemical coding of the guinea-pig coeliac ganglion is well estabhshed42~43*4s which makes it an ideal neuronal tissue to examine NFP-triplet i~unoreacti~ty in specific neuronal subpopulations, both in the cell bodies of principal neurons and their projections to the small intestine via the mesenteric nerve. In addition, a specific subpopulation of peptide- containing neurons in the myenteric plexus of the small intestine projects to sympathetic neurons in the coeliac ganglion. 13*‘5*28*43,45

In the present study we have investigated both the distribution of NFP-triplet immunoreactivity in the neurons of the coeliac ganglion of the guinea-pig and the relationship between NFP-triplet immunoreactive neurons and that of chemically coded subpopulations of neurons, and their respective projections to the small intestine. Preliminary observations of this work were communicated previously.64

EXPERIMENTAL PROCEDURES

Twenty male guinea-pigs were used (200-300 g). Animals were killed by stunning and severing the carotid arteries.

Fixation and tissue processing

The coeliac ganglion (composed of right and left ganglionic masses or lobes), the mesenteric nerve with the accompanying blood vessels, and segments of the small intestine, were removed and processed as follows before incubation with antibodies.

The coehac ganglion was processed in one of the following ways.

Placed in Zamboni fixative (2% formaldehyde in 0.1 M phosphate buffer and 15% pi&c acid, pH 7.0) for either 1 h, 6-8 h or 24 h at 4°C. The tissue was cleared with dimethyl- sulphoxide (DMSO), washed in a 0.01 M phosphate-buffered solution (PBS, pH 7.2) and placed in PBS-30% sucrose solution-0.1% sodium azide overnight. Cryostat sections (15 pm) of the coeliac ganglion cut in the frontal plane were mounted across a series of chrome-alum-gelatine-coated slides in sequential order, with at least 10 sections separating each section on individual slides.

Unfixed ganglia were frozen in embedding fluid (Tissue- Tek) in a cryostat mold. Cryostat sections (15 pm) were

collected as above and were either placed in ice-cold acetone for 10 min followed by air-drying, or incubated directly with antibodies.

Segments of control and extrinsically denervated intestine (see below) were processed to prepare whole mounts of the myenteric ganglia and submucosa.‘4 Briefly, the segments of intestine were opened out flat, stretched and pinned out over balsa and fixed in Zamboni’s fixative for 1 h. Tissue was cleared using DMSO followed by PBS. The layers of the intestine were dissected to prepare flat pieces of longitudinal muscle with the myenteric plexus attached, and pieces of submucosa containing the submucous plexus and blood vessels.

Branches of the mesenteric artery with a~ompan~ng nerve bundles were prepared as whole mounts. Small re- gions of the mesenteric branches, dissected out with the surrounding mesentery, were gently stretched and pinned over a piece of balsa wood. Tissue was placed in Zamboni’s fixative for 1 h and subsequently cleared in DMSO. After washing in PBS the mesenteric whole mount was com- pressed between two glass slides in PBS for 24 h before being processed for immunohistochemistry.

Immunohistochemistry We used single and double labelling fluorescence immuno-

histochemistry with a battery of antibodies raised against the NFP subunits, as well as polyclonal antibodies raised against peptides and enzyme markers (see Table 1). Mouse monoclonal antibodies were visualized either with a goat anti-mouse IgG secondary antibody conjugated to lluorescein isothiocyanate (Cappel; 1: 80), or with a horse biotinylated anti-mouse IgG (Vector; 1: 50) followed by streptavidin Texas Red (Amersham; 1: 100). Rabbit polyclonal antibodies were visualized with a sheep secondary anti-rabbit IgG conjugated to fluorescein isothiocyanate (Wellcome; 1: 160) or with a goat biotinylated anti-rabbit IgG (Vector; 1: 200) followed by strep~vi~n Texas Red (Amersham; 1: 100). In double labelling experiments the second antibodies used were free of undue cross-reactivities. For other controls, tissue was processed by using either non-immune serum, or by leaving out the primary antibody, before applying the second antibodies. All preparations were examined in a Leitz Grtholux fluorescence microscope equipped with Ploem epiilluminator with the L3 filter and N2 filter for the selective visuali~tion of fluorescein iso~i~yanate and Texas Red, respectively. In double labelling experiments the preparations were viewed with both the L3 and N2 filters to establish whether immtmoreactivity to two antigens could be localized in the same nerve cell body or fibre. Two antigens present in the same nerve cell body or fibre will be referred

Code Raised aeainst

Table 1.

Dilution Immunoreactivitv Source

Neurohlament antibodies: Monoclonals:

iC8 Rat spinal cord BM68 Porcine spinal cord BM160 Porcine spinal cord BM200 Porcine spinal cord RT97 Rat brain

Polyclonals: R-39 Chicken brain Rab68 Chicken brain Rab200 Rat/human brain

Other antibodies used: 1012 TH R264 NPY 7913 VIP 18522 Somatostatin

1: 2000 I:32 I:64 I:32 I:800

1:800 I:200 1:soo

I:200 1:800*/1:1600 1:1600*/1:3200 I:2000

Phos. ind. NF-M/NF-H Phos. ind. NF-L Phos. ind. NF-M Phos. dep. NF-I-I Phos. dep. NF-M/NF-H

NF-L/NF-M/NF-H NF-L NF-H

Th-like NPY-like VIP-like Somatostatin-like

Vitadello”s Boehringer6**’ Boehringefis22 Boehringer”” Wood’@

Dahl’* Hollenbeck Marotta4(’

Eugene-Tech Jarrott and Maccarrone4 Walshz7 Vandesande6’

*Dilution for whole mount tissue. Phos. ind., phosphorylation independent; Phos. dep., phosphorylation dependent.

Neurofilament protein-triplet in the coeliac ganglion 14s

to as being co-localized. Photographs were taken using Kodak TMAX 400 black and white him.

Interruption of nerve pathways Guinea-pigs were anaesthetized with Nembutal (Abbott

Laboratories; pentobarbitone sodium; dose 15 mg/kg body weight), followed by Hypnorm Vet {Jansen-Cilag; fentanyl citrate 0.3 15 m&ml, fluanisone 10 m&n+& dose 0.5 ml/kg body weight), and the following nerve pathways were interrupted.

rnterrup~~on of ~~estino~~~ia~ gunglia proration. Jn order to determine whether vasoactive intestinal peptide DIP)-containing tibres in the mesenteric nerves projecting from the intestine to the coeliac ganglionr3 contained NFP- triplet immunomactivity, the mesenteric nerves were crushed by compression with blunt forceps. The animals were either killed 24 h later to allow VIP immunoreactivity to amumulate in the interrupted fibre, or 14 days later to examine regrowing VIP immunoreactive fibres.

Extrinsic denervation of the intestine. In order to establish the presence of NFP-triplet-containing neurons projecting from the coeliac ganglion to the intestine, the mesenteric nerve trunks were interrupted by multiple crushes in the distal branches of the mesenteric nervesJ6 The animals were killed 14 days after the operation to allow for the degeneration of severed nerve fibres.

RESULTS

Neurofilament protein-triplet immunoreactivity in the coeliac ganglion

All antibodies against the NFP-triplet demonstrated immunoreactivity in neurons in the coeliac ganglion and initial segments of the splachnic and mesenteric nerve trunks (Figs l-4). Only neuronal structures showed NFP-triplet immunoreactivity, Some anti- bodies (BM68, iC8, Rab68, R-39; see Table 1) visual- ized both nerve cell bodies and neuronal processes, while other anti~dies (BM160, BM200, RT97, Rab200) showed neuronal processes preferentially. The monoclonal antibodies RT97 and BM200, and polyclonal antibody Rab200, produced similar patterns of immunoreactivity to BM160, but the nerve fibres in the nerve trunks were more intensely immunoreactive than nerve fibres within the ganglion. In addition, BM200, RT97 and Rab200, but not RM160, showed immunoreactivity in a few nerve cell bodies in each section.

The extent and intensity of NFP-triplet immuno- reactivity obtained using many NFP antibodies de- pended on tissue fixation. The immunoreactivity with BM68, Rab68 and R-39 antibodies was visualized most effectively in cell bodies and ftbres in unfixed and acetone-fixed sections than in Zamboni’s fixed material. The Rab68 and R-39 antibodies produced a fluorescence consisting of a very fine granular appear- ance in some cell bodies using unfixed or acetone- fixed tissue. Incubation with a rabbit non-immune serum produced a similar background fluorescence following application of the secondary antibody, indicating that this fine granular appearance of the cytoplasm was due to non-specific binding of rabbit sera. Using these antibodies with tissue that had been fixed for a short period in Zamboni’s solution elimi- nated most of this background fluorescence, but also

reduced specific immunofluorescence in cell bodies. This procedure did not affect immunofluorescence in nerve fibres. After 6-8 h fixation in Zamboni’s sol- ution, cell body immunoreactivity with the BM68, Rab68 and R-39 antibodies was absent, and immuno- reactivity in most fibres with these antibodies was dim to absent. The iC8 antibody demonstrated the same degree of immunoreactivity in both cell bodies and fibres over all tissue preparation conditions, with cell body immuno~a~tivity being reduced after approxi- mately 12 h of fixation in Zamboni’s solution. The BM 160 antibody showed strong immunoreactivity in all conditions up to 24 h Zamboni fixation. The BM200, RT97 and Rab200 antibodies demonstrated the same pattern of immunoreactivity in fibres over all fixation conditions up to 24 h Zamboni fixation, with cell bodies being immunoreactive only in unfixed or acetone-fixed tissue.

Double labelling using every combination of mono- clonal and polyclonal antibody to the NFP subunits, over all fixation conditions, demonstrate that all of these antibodies showed i~~o~ctivity in the same neurons, whether they visualized both cell bodies and fibres (BM68, iC8, R-39, Rab68) or mainly just fibres (BM160, BM200, RT97, Rab200) (Figs 5-10). Some fibres immunoreactive for Rab68 or R-39 in the ganglionic mass were very dimly labelled for RT97 and BM200. Some fibres (less than 5%) immuno- reactive for R-39 and Rab68 in the ganglionic mass and mesenteric nerves did not show immunoreactivity for BMl60.

Regional retribution of neuro~~a~nt protein-triplet immunoreactivity in the ganglion

The following description combines obervations of immunoreactivity for all the NFP-triplet antibodies in the eoeliac ganglion under optimal fixation conditions.

The coeliac ganglion is composed of two similar ganglionic masses, or lobes, which are joined medi- ally. 4s NFP-triplet immunoreactivity was not distri- buted evenly in the coeliac ganglion. In the medial regions of each lobe there were many NFP-triplet immunoreactive cell bodies and processes, whereas in the lateral regions of the ganglion there were compar- atively fewer i~uno~ctive cell bodies and processes (Figs l-4). Most of the NFP-triplet immunoreactive cell bodies appeared to be binucleated with more intense immunoreactivity in the perinuclear region. In most NFP-triplet immunoreactive nerve cell bodies, the immunoreactivity appeared as a fine lattice of filaments that usually did not fill out the whole cytoplasm. Some cell bodies were intensely fluorescent with the immunoreactivity extending into the initial segments of the dendritic processes.

Many more immunoreactive nerve flbres were observed in the medial regions of each lobe than in the lateral regions. NFP-triplet immunorea~tive tibres ranged from thick (approximately 10 pm) to very thin throughout the ganglia. In rare cases nerve fibres

746 J. C. VICKEKS H trl

Figs i-4. Microphotographs showing NFP-triplet immunoreactive nerve cell bodies and hbres from different regions of the ganglion.

Fig. I. Section from the medial regions of the ganglion. Note that there are many NFP-triplet immuno- reactive nerve cell bodies and fibres. Arrowhead shows region where there are non-immunoreactive nerve

cell bodies. Scale bar = 50 pm.

Fig. 2. Section from the lateral regions of the ganglion. Most nerve cell bodies are non-immunoreactive for the NFP-triplet. Arrow shows rare NFP-triplet immunoreactive nerve cell body in this region.

Scale bar = 50 ltm.

Fig. 3. Higher magnification of medial region of the ganglion While many nerve cell bodies are NFP- triplet immunore~t~ve (arrow) there are nerve cell bodies which are non-immunoreactive (arrowheads),

Scale bar = 25 pm.

Fig. 4. Higher magnification of lateral region of the ganglion. Note that there are comparatively fewer labelled fibres and no immunoreactive nerve cell bodies (arrowheads). Scale bar = 25 ym.

Neurofilament protein-triplet in the coeliac ganglion 747

Figs 5-10. Pairs of ~crophoto~aphs showing co-localization of NPP-triplet immune cell bodies and fibres in the coeliac ganglion and associated nerve tru

Fig. 5. Nerve cells labelled with iC8 (a) are also labelled with Rab68 (b). Scale

Fig. 6. Nerve cells labelled with BM68 (a) are also labelled with R-39 (b). Scale

Fig. 7. Nerve frbres in the lateral regions of the ganghon labelled with BM 160 (a) are R-39 (b). Scale bar = 25 pm.

Fig. 8. Nerve libres in the lumbar splachnic nerve bundle as it enters the lateral regic labelled with BM200 (a) are also labelled with Rab200 (b). Scale bar = :

Fig. 9. Nerve fibres in the lateral regions of the ganglion labelled with RT97 (a) are R-39 (b). Scale bar = 25 pm.

maactivity in nerve nks.

bar = 25 pm.

: bar = 25 pm.

: also iabelled with

Ins of the ganglion 50 pm.

also labelled with

Fig. 10. Nerve fibres in the lumbar splachnic nerve labelied with RT97 (a) are also Iabelled with Rab200 (b). Scale bar = 25 pm.

748 J. C. WCKERS er al.

Neurofilament protein-triplet in the coeliac ganglion 749

Figs 13 and 14. Double iabelled pairs of microphotographs of coeliac ganglion sections showing that some of the somatostatin immunoreactive nerve cell bodies (Figs I3a, 14a) are also NFP-triplet immunoreactive (Figs 13b, 14b). Arrowhead in Fig. I4a shows a somatostatin ~m~oreactive nerve cell body without

NFP-triplet immunoreactivity (Fig. 14b). Scale bar = 50 pm. (Pig. 13); 25 pm (Fig. 14).

Figs 11 and 12. Double labelled pairs of microphotograph with NPY {a) and NFP-triplet (b) antibodies in the coeliac ganglion.

Fig. 11. The NPY ~mmu~orea~tive cell bodies {a) do not show NFP-tripiet immunoreacti~ty (b). Conversely the NFP-triplet immunoreactive nerve cell bodies seen in b do not show immunoreactivity for

NPY (a). Scale bar = 50pm.

Fig. 12. Higher power showing NPY immunoreactive cell bodies (a) without NFP-triplet immuno- reactivity (b) and NFP-triplet immunoreactive nerve cell bodies (arrowhead in b) without NPY

immunoreacti~ty (arrowhead in a). Scale bar i= 25 pm.

750 J. C. VICKERS et al.

Figs 15-18. Double labelled pairs of microphotographs of sections of coehac ganglion showing VIP immunoreactivity (a) and NFP-triplet immunoreactivity (b).

Fig. IS. VIP immunoreactive nerve fibres (a) surround the NFP-triplet immunoreactive nerve cell bodies (b). Note that in the area in which there are no VIP terminals (left side of a) there are no NFP-triplet

immunoreactive nerve cell bodies (b). Scale bar = 50pm.

Fig. 16. High power magnification of VIP immunoreactive nerve fibres (a) surrounding both NFP-triplet immunoreactive nerve cell bodies (arrowheads in a) and non-immunoreactive nerve cell bodies (b). None of the VIP immunoreactive varicose fibres co-localize with NFP-triplet immunoreactivity. Scale bar = 25 pm.

Fig. 17. An example of a rare VIP immunoreactive nerve cell body (a) showing NFP-triplet immunoreactivity (b) (arrows). Scale bar = 25 urn.

Fig. 18. An example of a rare VIP immunoreactive nerve cell body (a) with no NFP-triplet immunoreactivity (b) (arrowheads). Scale bar = 25 pm.

7SI Capfiom overleaif

752 J. C. VICKERS et ol.

were seen to bifurcate within the ganglion. Many fibres had spindle-like swellings of NFP-t~plet immuno- reactivity. In addition, small ring-like structures (3-4 pm) of NFP-triplet immunoreactive material, resembling terminal boutons, were found throughout the ganglion and were strongly immunoreactive with most antibodies. Nerve fibres had a general orientation along the long axis of each lobe of the ganglion (i.e. medial-lateral). Many NFP-triplet immunoreactive fibres were observed in both the nerve bundles enter- ing and leaving the ganglion (e.g. mesenteric and splachnic nerves).

Co-exisfeence of the neuro~l~ment ~roiein-triplet and other neuron~~ markers in the coeliac ganglion

A combination of the mouse monoclonal antibodies iC8 and BM160 provided the best labelling of NFP- triplet immunoreactive neurons in fixed tissue and thus was used in combination with rabbit polyclonal antibodies for other neuronal markers to determine NFP-triplet immunoreactivity in specific neuronal subpopulations in the ganglion.

Neurojiiament protein-triplet and neuropeptide Y. The majority of cell bodies in the more lateral regions of each lobe of the ganglion were immunoreactive for neuropept~de Y (NPY) and virtually none of these were immunoreactive for the NFP-t~plet (Figs 11 and 12). Only a very small proportion (1.4%; S.D. = 0.7) of NPY immunoreactive cell bodies (n = 4365 cells from five animals) were also NFP-triplet immuno- reactive. NPY immunoreactive fibres, generally medium to large in calibre, were seen in the ganglion, in the mesenteric nerves and in other nerve bundles, and did not show NFP-triplet immunoreactivity.

The NPY immunoreactive fibres in the perivascular plexus of blood vessels associated with the ganglion were not NFP-triplet immunoreactive. Fine-calibre NFP-triplet immunoreactive fibres without NPY immunoreactivity were sometimes seen in small nerve bundles associated with blood vessels.

Neuro~iament protein-triplet and ~om~tostatin. Somatostatin immunoreactive cell bodies were com-

monly found in clusters in the more medial regions of the ganglion. A large proportion of these somato- statin immunoreactive cell bodies were NFP-triplet immunoreactive (Figs 13 and 14). Of all NFP-triplet immunoreactive cell bodies 77.8% (SD. = 8.5; n = 2010 cells from five animals) were immunoreactive for somatostatin, and 68.6% (S.D. = 6.1) of all somato- statin immunoreactive cell bodies (n = 2268 cells from five animals) were also NFP-triplet immuno- reactive. There was no obvious morphological char- acteristic that distinguished between somatostatin immunoreactive nerve cell bodies with or without NFP-triplet immunoreactivity. Most of the somato- statin immunoreactive fibres seen in the ganglia were also NFP-triplet immunoreactive.

Ne~ro~~ament protein -triplet and zjusoactive intestinal peptide. VIP immunoreactivity was found in varicose fibres forming baskets around cell bodies in the more medial regions of the ganglion. Double staining immunohistochemistry demonstrated that virtually all NFP-triplet immunoreactive cell bodies were surrounded by these baskets of VIP immuno- reactive fibres, although some nerve cell bodies without NFP-triplet immunoreactivity were also surrounded by such baskets (Figs 15 and 16). The VIP immunoreactive varicose fibres themselves did not show NFP-triplet immunoreacti~ty. Smooth VIP immunoreactive fibres found in the ganglion, in the mesenteric nerves and around blood vessels showed no immunoreactivity for the NFP-triplet.

A small number of VIP immunoreactive neurons were present in the ganglion. These correspond to a small subpopulation (less than 1%) of neurons in the ganglia that do not contain noradrenaline (NA) but are immunoreactive for dynorphin, NPY and VIP.45 These VIP immunoreactive neurons were often found in the central and lateral regions of the ganglia and approximately half of these were NFP-triplet immuno- reactive (Figs 17 and 18). The cell bodies immuno- reactive for VIP or for both VIP and the NFP-triplet were usually not associated with VIP immunoreactive terminals.

Figs 19-22. Double labelled pairs of microphotographs showing TH immunoreactivity (a) and NFP-triplet immunoreactivity (b).

Fig. 19. Section of the medial region of the coeliac ganglion showing that TH immunoreactivity is present in most nerve cell bodies (a) and that NFP-triplet immunoreactivity is only present in some of the TH immunoreactive nerve cell bodies (b). Arrowheads point to TH immunoreactive cells with no NFP-triplet

immunoreactivity. Scale bar = 50 pm.

Fig. 20. Section of the lateral region of the coeliac ganglion showing TH immunor~ctivity in nerve cell bodies and fibres (arrowheads in a) with no NFP-triplet immunor~ctivity (b). Conversely NFP-triplet

immunoreactive fibres (arrows in b) do not show TH immunoreacti~ty (a). Scale bar = 50 pm.

Fig. 21. Whole mount preparation showing a myenteric ganglion from the small intestine. Some of the TH immunoreactive varicose nerve fibres which supply the ganglia (a) also show NFP-triplet immuno- reactivity (b) (arrows). Note that there are many NFP-triplet immunoreactive fibres with no TH

immunoreactivity. Scale bar = 25 pm.

Fig. 22. Whole mount preparation of a submucous ganglion from the small intestine showing that some of the TH immunoreactive varicose fibres supplying the ganglia (arrow in a) also show NFP-triplet

immunoreactivity (arrow in b). Scale bar = 75 pm.

Neurofilament protein-triplet in the coehae ganghon 753

Neurofilament protein-triplet and tyrosine hydroxyl- ase. Antibodies to the enzyme tyrosine hydroxylase (TH) were used to show NA-containing neurons in the ganglia. Under fixation conditions suitable for visualizing NFP immunoreactivity in cell bodies TH immunoreactivity was reduced to absent in the cell bodies of the central regions of ganglion sections. TH immuno~a~tive cell bodies and fibres were found t~ou~out other regions of the ganglion and nerve trunks and represented the vast majority of neurons. Double labelhng with NFP-triplet and TH antibodies confirmed that a large population of cell bodies in the lateral region of the ganglia, and approximately half of the neurons in the more medial regions of the ganglia, did not show NFP-triplet immunoreactivity (Figs 19 and 20).

Small groups of extramedullary chromaffin cells found in clusters were immunoreactive for TH and NPY and were, with only rare exceptions, non- imm~noreactive for the NFP-triplet. Many NFP- triplet immunoreactive fibres, that were not ~mmunoreactive for any of the other neuronal markers used, surrounded the chromaffin cells.

In the mesenteric nerve trunk attached to the ganglia many fibres appeared to be immunoreactive for both TH and the NFP-triplet. Other nerve trunks in more lateral regions of the ganglia consisted mostly of TH immunoreactive fibres, a minor proportion of which were also NFP-triplet immunoreactive. In these bundles there were also a few NFP-triplet immuno- reactive fibres that were not TH immunoreactive. In the splachnic nerves TH immunoreactive fibres were rare and were not co-localized with NFP-triplet immunorea~tivity. TH immunorea~tive fibres closely associated with arterioles in ganglion sections were not NFP-triplet immunoreactive (Fig. 23).

Small intestine

The presence of NFP-triplet immunoreactivity in cell bodies of the coeliac ganglion suggested that the axons of these neurons, that project to the small intestine, may also be NFP-triplet immunoreactive.

In whole mount myenteric plexus preparations from normal tissue, numerous smooth NFP-triplet immuno- reactive fibres were seen both in the internodal strands and coursing through the ganglia. Some of these fibres were also TH immunoreactive. Some of the TH immunoreactive varicosities present in these ganglia showed co-localization with small (3-4pm) NFP- triplet i~unoreactive rings (Fig. 21). Approximately 70% of these NFP-t~plet immunoreactive rings were also TH immunoreactive. However, most TH immunoreactive fibres and varicosities were not NFP- triplet immunoreactive. In the submucous plexus TH and NFP-triplet immunoreactivity was co-localized in many fibres and varicosities (Fig. 22). The TH and the NPY immunoreactive fibres associated with the arterioles of the submucosa were not NFP-triplet immunoreactive (Figs 25 and 26).

Extrinsic denervation of the small intestine elimi- nated most TH immunoreactive nerve fibres and NFP-triplet immunoreactive rings in the myenteric plexus. A large proportion of NFP-triplet immuno- reactive fibres remained in the extrinsically denervated segment, and therefore originated from intrinsic enteric neurons.‘6 Extrinsic denervation also removed all TH immunoreactivity in the ganglia and blood vessels of the submucosa, with a decrease in the number of NFP-triplet immunoreactive fibres in the ganglia. A few NFP-triplet immunoreactive rings and fibres were still present in the submucous ganglia along with infrequent NFP-triplet immunoreactive paravascular fibres.

In mesenteric nerve whole mounts many nerve fibre bundles of different sizes contained NFP-triplet, TH and NPY immunoreactive fibres. The majority of TH immunoreactive fibres appeared to be NFP-triplet immunoreactive in the large bundles, but there were many TH immunoreactive fibres with no NFP-triplet immunoreactivity and a few NFP-triplet immuno- reactive fibres with no TH immunoreactivity. In most medium- to small-sized nerve bundles, the majority of

Plate overleaf

Figs 23-26. Double Iabelied pairs of microphotographs showing TH immunorea~tivity (a) and No-tripiet immunoreactiv~ty (b).

Pig. 23. Cross-section of a small artery near the coeliac ganglion showing that none of the numerous TH immunoreactive fibres in the adventitial perivascular pIexus [arrows in a) shows immunoreactivity for the

NFP-triplet (arrow in b). Scale bar = SOhm.

Fig. 24. Whole mount preparation of a branch of the mesenteric artery (A) showing that the perivascular TH immunoreactive nerve fibres (arrow in a) do not show NFP-triplet immunoreactivity (arrow in b). The separate TH and NFP-triplet labelled fibres run in close association in the paravascular small nerve

bundles. Scale bar = 50 pm.

Fig. 25. Whole mount preparation of arterioles of the submucosa showing that the TH immunoreactive perivascular fibres (arrows in a) do not show NFP-triplet immunoreactivity (arrows in b). Note rare NFP-triplet labeled fibre in the paravascular nerves which is not immunoreactive for TH. Scale

bar = 50 pm.

Fig. 26. Higher magnifi~tion of similar preparation as in Fig. 25 showing TH immunoreactive fibres (arrows in a) surrounding an arteriole (A) with no NFP-triplet immuno~a~tivity (arrows in b). Scale

bar = 25 pm.

Neurofilament protein-triplet in the coeliac ganglion 755

fibres were immunoreactive for TH or NPY but not the NFP-triplet. Small paravascular fibre bundles contained almost exclusively TH or NPY immuno- reactive fibres and had only one or two NFP-triplet immunoreactive fibres. These NFP-triplet immuno- reactive flbres were not immunoreactive for TH or NPY (Fig. 24). In addition, a rich perivascular plexus of TH or NPY immunoreactive fibres was associated with the mesenteric arteries. None of these varicose fibres showed NFP-triplet immunoreactivity. Single, thin and smooth NFP-triplet immunoreactive fibres were seen around the artery. These were not immuno- reactive for TH or NPY.

In mesenteric nerve whole mounts VIP immuno- reactivity was seen in some large fibre bundles and occasionally in paravascular and perivascular fibres. These fibres appear not to contain NFP-triplet immunoreactivity. After a period of 24 h following crushing of the mesenteric nerves VIP immurio- reactivity had a~umulated in fibres distal to the crush and none of these fibres showed NFP-triplet immunoreactivity. After 14 days, thick regenerating VIP immunoreactive fibres were only observed on the distal side of the mesenteric nerve crush, indicating their intestinal origin. None of these were NFP-triplet immunoreactive. After 14 days very few NFP-triplet immunoreactive fibres were observed on the distal side of the crush in comparison with the numbers of fibres observed on the proximal side, indicating that they mainly projected to the intestine. VIP immuno- reactive fibres were only observed in the perivascular plexus proximal to the crush, indicating that these fibres did not originate in the intestine. These fibres were not NFP-triplet immunoreactive.

DISCUSSION

NeuroJTlament protein-triplet immunoreactivity is present in only some neurons

The present results strongly suggest that only some of the neurons in the guinea-pig coeliac ganglion contain the NFP-triplet. Only approximately one- fifth of the nerve cell bodies in this ganglion showed immunoreactivity with any of the antibodies used. Double labelling immunohistochemist~ using mono- clonal and polyclonal anti~dies against the NFP- triplet showed that the same neurons stained with each of the rabbit antibodies also stained with each of the mouse antibodies. These results indicate that a large proportion of neurons in the coeliac ganglion may not contain the NFP-triplet.

Not all NFP-triplet antibodies labelled equally well the different parts of the neurons. The rabbit anti- bodies R-39 and Rab68, and the mouse antibodies iC8 and BM68, showed immunoreactivity both in the nerve cell bodies and in the axons, while the mouse antibodies BM160, BM200 and RT97 and rabbit antibody Rab200 stained axons more intensely. Only the more proximal regions of the dendrites were seen with any of these antibodies. These differences in

NSC 39,3--H

intraneuronal staining patterns could be due to differ- ences in the distribution of the NFP subunits within the neurons,‘9336 to the degree of phosphorylation of the NFP’1,61 and to the susceptibility of NFP immuno- reactivity to the fixation procedures.35%5’ The BM200 and RT97 antibodies, which recognize phosphorylated epitopes of the NF-H subu~t,z~,~,~ visualized axons in the nerve bundles more intensely than they visualized fibres and nerve terminals in the coeliac ganglion. This difference in the distribution of phosphorylated epitopes within neurons has been well described.“~6’ It has been suggestedZo that antibodies directed against the NF-H subunit or phosphorylated forms of the NF-M and NF-H subunits are axon-specific. The antibody BM 160 did not label a small proportion of fine nerve fibres which were labelled by the rabbit polyclonal antibodies. This suggests that either a small proportion of NFP-triplet immunoreactive neurons lacks epitopes recognized by BM160, or that the epitopes recognized by the BM160 antibody are not present in the most distal part of the axon.

The fixation procedures also affected the degree of immunoreactivity with the different antibodies. Thus, the best condition for labelling nerve cell bodies with the R-39, Rab68 and BM68 antibodies was with unfixed tissue. The labelling of nerve cell bodies and fibres with the iC8 antibody was good in either un- fixed or fixed tissue and the intensity of the immuno- reactivity was only slightly diminished by longer fixations. Thus antibodies raised against the NF-L subunit appear to be more susceptible to the masking effects of fixation. Previous studies have shown that various fixatives mask specific NFP-t~plet epitopes in cell bodies, possibly by cross-linking of proteins, while leaving other epitopes in the nerve fibres relatively unaffected.35*51

Given that we have used both monoclonal and polyclonal antibodies against the NFP subunits, antibodies that recognize both phosphorylated and non-phosphorylated NFP epitopes, and we have used unfixed and fixed tissue, we propose that the consist- ent lack of labelling in specific neuronal populations is likely to be due to the absence of the NFP-triplet to which these antibodies are raised.

Chemjc~l coding of ~uro~~~ent protean-triplet imm~oreactive neurons

The regional distribution of NFP-triplet immuno- reactive neurons within the ganglion, with the positive neurons being preferentially located in the medial part of the ganglion, and the negative neurons located laterally, suggests that they are related to specific subclasses of neurons. Indeed, there appears to be a correlation between NFP-triplet immunoreactivity and the chemical coding of the postganglionic sym- pathetic neurons described previously.“2,43”S The three main classes of postganglionic sympathetic neurons in these ganglia include noradrenergic neurons that contain NPY (NA/NPY neurons) projecting to arterioles to the intestinal submucosa, noradrenergic

756 J. C. VICKEXS et al.

Intestinal wall

NA/NFF-triplet

MG

NA

NA/SOM/NFP-triplet

NA,‘SOM

SMG

Fig. 27. Main populations of coeliac ganglion neurons on the basis of their chemical coding. The percentages of each population are based on the proportions of NFP-triplet immunoreactive neurons, established in this work, in relation to the three main subpopulations of postganghonic sympathetic noradrenergic neurons described by Macrae et a1.45 Estimates were derived for NPY and somatostatin- containing neurons from double labelling with NFP-triplet and peptide antibodies. Proportion of NA/- neurons containing the NFP-triplet was derived indirectly by comparing the proportion of neurons containing the NFP-trinket. but not NPY or somatostatin, with the known total proportion of NA/- neurons in the ganglion. MA, mesenteric artery; MG, myenteric ganglia; SMA, submucosal arterioles;

SMG, submucous ganglia.

neurons that contain somatostatin (NA/somatostatin neurons) projecting to the submucous ganglia and noradrenergic neurons with no other recognizable marker (NA/ -) that project to the myenteric ganglia. Our results show that the NFP-triplet is confined to some of the NA/somatostatin and, by extrapolation from Macrae et a1.,4s NA/ - neurons while they are virtually absent from the NA/NPY sympathetic vaso- motor neurons. Indeed, as expected, none of the TH or NPY immunoreactive perivascular fibres in the mesentery or in the submucosa showed NFP-triplet immunoreactivity. The main populations of neurons in the coeliac ganglion coded by the NFP-triplet and other markers are summarized in Fig. 27.

Small cells found in clusters within the ganglia are catecholamine-containing extramedullary chromafhn cells. Like similar clusters of cells in the other guinea- pig prevertebral sympathetic ganglia’* as well as the coeliac ganglion45 these chromaffin cells were immuno- reactive for NPY. Virtually none of these chromaffin cells were immunoreactive for the NFP-triplet. NFP-triplet immunoreactive nerve fibres were found throughout these clusters of cells. It remains to be established whether they correspond to either pre- ganglionic axons that innervate chromaffin cells in

several vertebrate species,2s or whether they are sensory nerve fibres.

The intestinofugal neurons that project from the intestine to the coeliac ganglion, visualized in this work by their VIP immunoreactivity, do not appear to contain NFP-triplet immunoreactivity. These neurons also contain immunoreactivity for cholecystokinin, dynorphin, enkephalin and gastrin-releasing peptide and are probably part of the intestinointestinal reflex that acts on the noradrenergic neurons controlling intestina1 motility and secretion.45

The majority of NFP-triplet immunoreactive fibres in the ganglion running amongst ganglion nerve cell bodies are likely to be extrinsic to the ganglion and probably represent preganglionic and/or sensory fibres.

Implications for neurohistology

Several authors have proposed that the density of neural intermediate filaments is related to the size and length of axons.24’39 Thus, the presence of the NFP- triplet in some coeliac ganglia neurons, but not in others, could be related to the size of nerve fibres. However, in the same mesenteric nerve trunks there were large calibre fibres with NFP-tripfet immuno-

Neurofilament protein-triplet in the coeliac ganglion 151

reactivity and large calibre TH and NPY fibres with- out NFP-triplet immunoreactivity. Furthermore, there were many fine-calibre NFP-triplet immunoreactive fibres. Both the NFP-triplet positive neurons and the NFP-triplet negative neurons project to the intestine and have a similar length. Therefore, it is unlikely that the presence or absence of the NFP-triplet is related to the size of neurons in these ganglia. Indeed, our results do not demonstrate any obvious morphological differences between neurons in the coeliac ganglion which could be correlated with NFP-triplet content.

The recent discovery of new classes of intermediate filament proteins in neurons40v49s66 raises the possibility that they may be present in the neurons in the coeliac ganglion that do not appear to contain immuno- reactivity for antibodies raised against the NFP-triplet.

The lack of staining in some neurons with the NFP-triplet antibodies may provide a new interpre- tation on the use of classical silver impregnation methods based on Bielschowsky.5 While many others attributed the lack of staining in neurons to the capricious nature of the method, ultrastructural evidence indicates that silver staining may be specific for neural intermediate filaments.32*33 Furthermore, a modification of this silver impregnation method selectivity stains the NFP-triplet in chromatography gels.),)O Silver impregnation in the mammalian coeliac ganglia* has shown that there are neurons that do not stain at any stage during development, and were interpreted to represent immature neurons. However, it is likely that the absence of silver impregnation of some of these neurons reflects the absence of the NFP-triplet. If these silver impregnation methods are only staining NFP-triplet-containing neurons, then this raises the possibility that entire specific classes of

neurons not stained by silver impregnation methods have remained unstudied since the beginning of the century. Conversely, this may point to a modern interpretation of silver impregnation as a tool to define neurochemically specified classes of neurons in the central and peripheral nervous systems.

The finding that not all neurons contain the NFP- triplet also has important implications for neuropatho- logical investigations of diseases such as Alzheimer’s disease where it has been shown that silver impreg- nation methods stain the paired helical filaments in intraneuronal neurofibrillary tangles% and that there is a selective loss of immunoreactivity for NFP- triplet antibodies in particular cortical neurons, with their pathological replacement with neurons contain- ing neurofibrillary tangles.47 NFP-triplet-containing neurons may mark those neurons that are vulnerable to develop neurofibrillary tangles.

The significance of the presence or absence of the NFP-triplet in different populations of neurons remains to be established. These observations, how- ever, suggest that the NFP-triplet is not an intrinsic component of the cytoskeleton of all neurons. Inves- tigations on the differences between neurons with and without the NFP-triplet may thus help to elucidate the role of these neuronal proteins.

Acknowledgemenrs-This work was funded by the National Health and Medical Research Council (NH&MRC) of Australia. Antibodies to the NFP-triplet were generously supplied by P. Hollenbeck and J. Wood. Antibodies to pep- tides were generously supplied by B. Jarrott, C. Maccarrone, F. Vandesande and J. Walsh. We are grateful to Judy Morris and Penny Steele for their valued comments on the manuscript. We would like to thank Tania Neville for skilled technical assistance and Joy Davis for help in preparing the manuscript.

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(Accepted 18 June 1990)