Tachykinin-related neuropeptides in the central nervous system of the snail Helix pomatia: an immunocytochemical study

BRAIN RESEARCH ELSEVIER

Brain Research 661 (1994) 223-236

Research report

Tachykinin-related neuropeptides in the central nervous system of the snail Helix pomatia: an immunocytochemical study Kfiroly Elekes a,,, Dick R. N~issel b a

Department of Experimental Zoology, Balaton Limnological Research h~stitute of the Hungarian Academy c>fSciences, PO Box 35, H-8237 Tihany, Hungary b Department of Zoology, Stockholm Unit'ersity, Stockholm, Sweden Accepted 26 July 1994

Abstract The distribution of neurons reacting with an antibody raised against an insect neuropeptide, locustatachykinin I, was investigated in the CNS of the snail Helix pomatia. The localization of the neurons was compared with that of the substance P-like immunoreactive (SPLI) neurons in the different ganglia. Altogether, there arc ~ 800-1000 Iocustatachykinin-like immunoreactive (LomTKLI) neurons in the Helix CNS, occurring with an overwhelming dominancy (83.5%) in the cerebral ganglia. Within the cerebral ganglia, the majority of LomTKLI neurons were localized in the procerebrum. The number of SPLI neurons was high; ~ 2000 SPLI nerve cells were found in the Helix CNS. The majority (44.5%) of SPLI neurons was also found in the cerebral ganglia and they were also concentrated in the procerebrum. The neuropils of all ganglia were densely innervated by both LomTKLI and SPLI fibers except the medullary mass of the procerebrum where only SPLI elements form an extremely dense innervation. In addition to the neuropil processes, LomTKLI neurons sent axon processes to the peripheral nerves. SPLI fibers also formed a dense network of varicose fibers in the connective tissue sheath around the ganglia where they innervated the blood vessel walls too. Immunolabeling on alternating cryostat sections revealed that LomTKLI and SPLI neurons are localized near each other in most cases; co-localization of the two immunoreactive materials could be seen in a very small number of neurons of the pedal and pleural ganglia. The present results show that the Helix CNS possesses distinct neuronal populations using different tachykinin-related peptides. It is suggested that the differential distribution of these neuropeptides also implies a diversity in their central and peripheral functions.

Keywords: Tachykinin-related peptide; Locustatachykinin: Substance P; Immunocytochemistry; Invertebrate; Helix pomatia L.; Mollusc

I. Introduction T h e n u m b e r of r e g u l a t o r y p e p t i d e s a n d t h e i r diversity is very high in b o t h v e r t e b r a t e s a n d i n v e r t e b r a t e s . T h e tachykinins a r e a m o n g t h e b e s t - k n o w n n e u r o p e p tides. V o n E u l e r a n d G a d d u m [40] first i d e n t i f i e d a tachykinin, s u b s t a n c e P (SP), in m a m m a l s , w h o s e a m i n o acid s e q u e n c e b e c a m e k n o w n early on [4]. Subsequently, several o t h e r tachykinins have b e e n d e s c r i b e d in a r a n g e of v e r t e b r a t e groups, i n c l u d i n g t h e n e u rokinins f r o m m a m m a l s [22,20,24], while p h y s a l a e m i n , kassinin, p h y l l o m e d u s i n , u p e r o l e i n , scyliorhinin, n e u -

* Corresponding author. Fax: (36) (86) 348006. E-mail: H8981 Ele~'huella.bitnet. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 ( / 6 - 8 9 9 3 ( 9 4 ) 0 0 9 2 8 - 7

rokinin A - r e l a t e d and s u b s t a n c e P - r e l a t e d p e p t i d e s have b e e n f o u n d in lower v e r t e b r a t e s [7,13,41]. T h e first t a c h y k i n i n i d e n t i f i e d in i n v e r t e b r a t e s was eledoisin, i s o l a t e d from t h e c e p h a l o p o d Eledone [14]. Novel t a c h y k i n i n s have r e c e n t l y b e e n d e s c r i b e d in the e c h i u r o i d w o r m Urechis unicinctus [21] a n d in the f r e s h w a t e r bivalve Anodonta cygnea [15]. In the last few years, several m y o t r o p i c n e u r o p e p t i d e s have b e e n i s o l a t e d from exctracts of b r a i n a n d c o r p o r a c a r d i a c a o f t h e insect Locusta migratoria by m e a n s of a sensitive c o c k r o a c h h i n d g u t c o n t r a c t i o n bioassay. T h e s e p e p t i d e s s h o w e d s o m e a m i n o acid h o m o l o g i e s with v e r t e b r a t e tachykinins and, thus, w e r e n a m e d locustat a c h y k i n i n s I - I V ( L o m T K I - I V ) [9,19,34,35]. Similar to v e r t e b r a t e tachykinins, the i n v e r t e b r a t e tachykinins all have m y o t r o p i c a c t i o n on visceral muscle; the locusta-

224

t,2 l'21ekes'. D.R. Ndsscl / Brain Res'earch 66l (1~)94~ 223 23t~

tachykinins, for example, act on muscle of hindgut, foregut and oviduct, and modulate action of certain skeletal motor

neurons

[36]. F u r t h e r

myotropic

neu-

ropeptides, the leucokinins l-VIII (LK l-VIII), have also been identified from the cockroach brain and c o r p o r a c a r d i a c a [18,19] b u t t h e s e o n l y h a v e a s i n g l e amino acid homology to the tachykinins. In t h e g a s t r o p o d n e r v o u s s y s t e m , t h e p r e s e n c e o f tachykinin-related peptides has been demonstrated imm u n o c y t o c h e m i c a l l y [2,24,33,37,38]. I n H e l i x a s p e r s a , the CNS seems to contain a single tachykinin similar to m a m m a l i a n S P [24]. The CNS of Helix pomatia was also shown to contain many neurons which reacted to an antiserum r a i s e d a g a i n s t t h e i n s e c t m y o t r o p i c p e p t i d e , L K I [12]. Application of two antibodies to study the possible co-localization of SPLI and LKLI showed only a few groups of neurons reacted to both antibodies. We now describe the distribution of locustatachykinin-like imm u n o r e a c t i v e ( L o m T K L I ) n e u r o n s a n d its r e l a t i o n s h i p to the distribution of SPLI neurons. Since Boyd eta[. [2] o n l y d e s c r i b e d t h e S P L I n e u r o n s in t h e v i s c e r o parietal-pleural ganglion complex, we also provide a detailed immunocytochemical analysis of the localizat i o n o f S P L I n e u r o n s in t h e c e r e b r a l a n d p e d a l g a n g l i a . F i n a l l y , a b r i e f c o m p a r i s o n is m a d e w i t h t h e L K L I neurons to have a complete view of the distribution of t h e t h r e e t a c h y k i n i n - r e l a t e d n e u r o p e p t i d e s in t h e s n a i l brain.

2. Materials and methods 2.1. Animals Adult specimens of the snail 11. pomatia were used for the experiments. The animals were collected in the Tihany peninsula, and then kept under natural conditions in the institute's garden from spring to early autumn. During winter, the animals hibernated but

were brought into laboratory conditions and kept at room tempcn~ture for ~ 2 weeks before use. 2.2. Antisera The production and characterization of the Ix)mTK-1 and LK-I antisera have been described elsewhere [25,26,28,30.31]. For SP immuncytochemistry, we used a rat monoclonal antibody (Accurate Chemicals, Long Island, NY) [8]. An antiserum to the mollu~an tachykinin, eledoisin, was also applied [39] but gave no positive immunostaining. 2.3. lmmuno~ytochemist~ Following a quick dissection, the circumesophageal ganglion rings were pinned out on Sylgard and fixed in 4% paraformaldehyde diluted in 0.1 M phosphate buffer (PB; pH 7.2) overnight at 4°C. The buccal ganglia were not examined in this study. Ganglionic rings were then washed in several changes of PB for 24 h, infiltrated with 20% sucrose in 0.1 M PB for 24 h and then sectioned on a cryostat. 20-~zm-thick, serial sections were placed on chrom-alum-gelatincoated slides and processed for immunocytochemistry. LomTK-I and LK-I antisera were diluted 1:1000 and SP antiserum 1:250 in phosphate-buffered saline (PBS) containing 0.25% Triton X-100 (TX) and 0.25% bovine serum albumin (BSA). Cryostat sections were incubated in the primary antiserum either for 24 h at room temperature or 48 h at 8°C. The peroxidase-antiperoxidase (PAP) three-step method was performed for the LK-I and LomTK-I immunolabeling whereas an HRP-conjugated anti-rat secondary lgG was used for the SP-antiserum. The investigation of possible co-localization of LomTK and SPL immunoreactivities was performed on 10-/zm-thick alternating cryostat sections, placed on chrom-alum-gelatin-coated slides and processed for PAP method. Preabsorbtion of SP antiserum with synthetic SP (Peninsula; 20 ~ M / m l 1:1000 diluted antiserum completely abolished the immunoreactivity. Preabsorbtion of LomTK-I antiserum with synthetic peptide (20 tzM/ml 1 : 1000 diluted antiserum) resulted in a significant decrease of immunoreactivity and a total abolition of the immunoreactivity could be achieved by a preabsorption with 40/zM synthetic LomTK-I. Preabsorbtion of LomTK-I antiserum with synthetic SP (20/zM/ml 1 : 1000 diluted antiserum) did not decrease the intensity of immunoreactivity. Method controls were performed in two ways: (1) omission of primary antibody and (2) substitution of primary antibody with 1% normal rabbit (LomTK-I) or 1% normal

Table 1 Number and distrubution of SPLI, LomTKLI and LKLI neurons in the CNS of H. pomatia (n = 5) Ganglia

SPLI No.

Cerebral Procerebral Rest Pedal Pleural Parietal Right Left Visceral Total

924-1066 732- 832 * 192- 204 795- 821 300- 393 28- 47 12- 35 35- 63 2094-2445

LTKLI

LKLI

%

No.

%

No.

%

44 78 21 38 14.5

714-836 602-672 112-164 76- 92 16- 41)

88.25 84.3 15.7 9.4 2.0

298-346 8- 12 290-344 104-124 108-128

54 2.7 ** 97.3 18.9 19.6

1.3 0.6 1.7

1 2 809-971

0.1 0.2 -

27- 36 14- 17

4.9 2.5

551-657

* The number of procerebral (gtobulus cell) neurons strongly varied, probably due to seasonal differences. During autumn, significantly less ( ~ 40 in each procerebrum) SPLI globulus cells could be detected. ** This number represents here only the labeled globulus cells.

K. Elekes, D.R. Ndssel / Brain Research 661 (1994) 223-236

225

cpdc

rat (SP) serum. No immunolabeling could be observed after these control experiments.

3. Results

3.1. Distribution of L o m T K L I neurons Altogether, 8-1000 (809-971) LomTKLI neurons were found in the circumesophageal ganglion ring of H. pomatia. However, the distribution of the labeled neurons was highly uneven (Table 1, Figs. lb, 2b, 3b). The vast majority (88.25%) of labeled neurons were localized in the cerebral ganglia, an additional 9.0% of immunolabeled neurons was found in the pedal ganglia. The entire viscero-parietal-pleural complex contained only 2.3% of the total LomTKLI neuron population. Within the cerebral ganglia, the procerebri are the major location of LomTKLI neurons where 84% of on

MC~~ 'C.~,.'";.""" Inn .:~.~' 7::.:.:

la ~ / ~

"~c~ pdc

c Fig. 1. Distribution of SPLI (a), LomTKLI(b) and LKLI neurons (c) in the cerebral ganglia. Right, dorsal surface; left, ventral surface. PC, procerebrum; MC, mesocerebrum; ME, metacerebrum; PO, postcerebrum; cc, cerebral commissure; on, olfactory nerve; Inn, lip nerves; cplc, cerebro-pleural connective;cpdc, cerebro-pedal connective. Filled circles, dorsal surface; open circles, ventral surface.

C Fig. 2. Distribution of SPLI (a), LomTKL1 (b) and LKLI neurons (c) in the pedal ganglia, cpdc, cerebro-pedal connective: pc, pedal commissure. Filled circles, dorsal surface; open circles, ventral surface.

LomTKLI neurons were found (Table 2). The population of labeled neurons in the procerebrum can be divided into four groups (Figs. lb, 12a; Table 2): (1) globulus cells within the main body of the procerebrum cortex; (2) globulus cells at the base of the procercbrum; (3) neurons in the posterior medial marginal cell group at the border of the meso-, meta and procerebri; and (4) neurons in the posterior lateral marginal cell group below the globulus cells [3]. Further groups of LomTKLI neurons consist of small numbers of cells and are found in the pleural and pedal lobes of the postcerebrum as well as on its dorsal surface where a series of small groups are localized in a rostro-caudal direction between the meta- and postcerebri. The medullary mass neuropil contained a few LomTKL1 fibers, mostly at the base of the procerebrum (Fig. 12a). In the pedal ganglia, four small groups of LomTKLI neurons were found, three of them in the lateral cortex and one rostro-medially, near the origin of the cerebro-pleural connective (Figs. 2b, 14a). This latter group could be followed from the dorsal to the ventral surface. The remaining LomTKL1 neurons were in small group in each pleural ganglion, at the origin of the cerebro-pleural connective (Figs. 3b, 17), while the left and right parietal ganglion contained one and two

226

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K. E/ekes, D,R, Niissel / Brain Research O01 (1994) 223-236

,o

LomTKLI axons (Figs. 4, 6a). In contrast, the neuronal cell body layer and the connective tissue sheath were free of immunoreactive elements.

'."-':::'.

3.2. Distribution of SPLI neurons

Ipn 1/\ \ in

rpnn

an

In the CNS of H. aspersa, the presence of SPL1 material has been demonstrated [2,33] and Boyd et al. [2] mapped SPLI neurons on the dorsal surface of the viscero-parietal-pleural ganglion complex. Special emphasis will, therefore, be placed on the distribution of SPLI neurons in the cerebral and pedal ganglia. In the CNS of H. pomatia (buccal ganglia not included), > 2000 (2094-2445) SPLI neurons were found. The cerebral ganglia contained about half (44%) of the labeled neurons, an additional 38% being found in the pedal ganglia (Table 1). The viscero-parietal-pteural complex contained the rest of SPLI neurons where the majority of labeled neurons was localized in the pleural ganglia (14.5%, Table 1). 3.3. Cerebral ganglia

"

~3o

"o.

!: ,e"

Fig. 3. Distribution of SPLI (a), LomTKLI (b) and LKLI neurons (c) in the viscero-parietal-pleural ganglion complex. V, visceral ganglion; RPa, right parietal ganglion; LPa, left parietal ganglion; RPI, right pleural ganglion; LPI, left pleural ganglion; lpn, left pallial nerve; rpnn, right pallial nerves; in, intestinal nerve; an, anal nerve. Filled circles, dorsal surface; open circles, ventral surface.

large LomTKLI neurons, respectively (Fig. 5). The visceral ganglion did not contain any immunolabeled soma. With a few exceptions, the entire population of LomTKLI neurons consisted of small-size (12-30/xm) neurons; the diameter of the labeled globulus cells reached only 5-10 izm. A single medium-size (40-50 /xm) neuron was found at the base of the procerebri (Fig. 4) and in the pedal lobe of the postcerebrum. The neuropil of all ganglia, except the medulary mass, was richly supplied with immunoreactive fibers and also all important peripheral nerves contained

Altogether, 11 different SPLI cell groups could be distinguished in the right and 12 in the left cerebral ganglion (Table 2). ~ 30% of the total SPLI neuronal population was found in the procerebrum (Table 1). The dorsal surface of the postcerebrum also displayed a significant number of SPLI neurons (Fig. la, Table 2). The only asymmetrical group of SPLI neurons was found in the left mesocerebrum, however, these mesocerebral neurons were not labeled when a FITC conjugated secondary IgG was employed [12]. The density of innervation of the neuropil regions was striking in the cerebral ganglion. Consequently, only the initial projections of labeled cell groups could be followed. The sole exception was the procerebrum where the projection of several groups of SPLI ceils could be traced for a longer distance: (1) small globulus ceils localized along the inner marginal surface of the globulus cell layer projected in a thin bundle to the olfactory nerve (Fig. 6b); (2) small globulus cells projected among the mass of unlabeled globulus cells to the medullary neuropil (Fig. 7); (3) dusters of neurons in the posterior lateral marginal cell group projected to three directions: (a) to the axon bundle on the outer marginal surface (Fig. 6a); (b) to the lateral medullary neuropil Fig. (6b, 8); and (c) toward other neuropil regions of the cerebral ganglion (6b, 8). The SPLI innervation of the medullary neuropil showed differences. The entire terminal medullary mass appeared as a homogenously immunostained region whereas the base of this neuropil could be divided into two parts: the lateral medullary mass contained a network of varicose fibers and the innermost region (facing the mesocerebrum) was free of immunolabeling (Fig. 6b).

K. Elekes, D.R. Ndssel / Brain Research 661 (1994)223-236

Near to the dorsal surface of the procerebrum a thick bundle of SPLI fibers was found and traced to the olfactory nerve (Fig. 6a). This bundle originated mainly from the postcerebral cell groups 8 and 9.

227

3.4. Pedal ganglia The distribution of SPLI neurons glia c o n s i s t e d

of two

distinct

in t h e p e d a l g a n -

populations

(Figs.

3a,

i i~iiiii!~i

iiiiiiiiiiii(~iiii!iiiiiii~ ~ i i i~~!! ~:i~¸¸¸

%

k

Fig. 4. LomTKLI elements in the procerebrum. A single medium-size ( ~ 40 txm) neuron (arrow) projects to a bundle of immunoreactive fibers. Small globulus cells at the base of the procerebrum (small arrows) and neurons belonging to the posterior marginal cell group (arrowhead) are also labeled. Note lack of immunoractivity in the medullary mass (me). × 250. Fig. 5. A large (90 tzm) LomTKLI neuron (N) in the left parietal ganglion (see also Fig. lc). × 250.

K. Eh'kes, D.R. Ndasel/ Brain Research 661 (1994) 223-230

228

Table 2 Localization of major SPLI and LomTKL1 cell groups in the cerebral ganglion (one 'hemisphere') (n = 5) No. of neurons No. and location of cell group Procerebrum 1 Globulus cells 2 Globulus cells (base) * 3 P.l.m. cells * 4 P.m.m. cells Mesocerebrum 5 Ventral 6 Left Uetacerebrum 7 Dorsal Postcerebrum 8 Dorsal I 9 Dorsal II 10 Pedal lobe 11 Commissural lobe 12 Pleural lobe I 13 Pleural lobe II (cplc)

SPLI

LTKLI

210-230 (40- 42) 120-164 (170-186)

166-185 (58- 64) 104-125 (175-185)

43- 55 (15- 25)

20- 24 (10)

29- 32 12- 15

3.5. lnnert:ation of connective tissue sheath hy SPLI fibers T h e connective tissue s h e a t h a r o u n d t h e ganglia a n d p e r i h e r a l nerves was heavily i n n e r v a t e d by S P L I fibers (Fig. 10). V a r i c o s e fibers o r i g i n a t i n g from relative thick p r o c e s s e s w e r e p r e s e n t all over in t h e s h e a t h and f o r m e d b o t h n e t w o r k - l i k e a r r a n g e m e n t s and bundles. In a d d i t i o n , t h e wall o f the m a j o r vascular e l e m e n t s ( a o r t a a n d a r t e r i e s ) a n d t h e i r b r a n c h i n g s e m b e d d e d in the s h e a t h w e r e also richly s u p p l i e d with l a b e l e d fibers (Fig. 11).

3.6. Co-localization o f L o m T K L and SPL immunoreactivities

18- 29 75128131413-

79 15 10 18 30 27

20- 22 6-

8

9-1l 4-- 5

* The exact anatomical distinction between the two labeled cell groups was not possible. 9a,b): (1) four clusters o f small-size n e u r o n s localized in the r o s t r a l a n d l a t e r a l cell b o d y layer; a n d (2) a large n u m b e r of 4 0 - 5 0 Izm n e u r o n s w e r e f o u n d in t h e caud a l - c a u d o l a t e r a l region, lining u p along t h e innerm a r g i n a l surface to t h e r o s t r a l c o m m i s s u r e . This l a t t e r p o p u l a t i o n of S P L I n e u r o n s c o u l d b e t r a c e d t h r o u g h o u t t h e e n t i r e p e d a l g a n g l i o n f r o m t h e d o r s a l to the v e n t r a l surface. Single a n d p a i r e d g i a n t S P L I n e u r o n s w e r e also f o u n d in this r e g i o n l o c a l i z e d s y m m e t r i c a l l y in b o t h p e d a l ganglia. A d d i t i o n a l l y , two large ( 8 0 / z m ) n e u r o n s a n d an a s y m e t r i c a l l y l o c a t e d g i a n t n e u r o n in the left g a n g l i o n w e r e f o u n d just c a u d a l l y to t h e r o s t r a l c o m m i s s u r e (Fig. 2a). Cell clusters o f g r o u p 1 f o r m e d smaller neurite bundles and projected throughout the n e u r o p i l to t h e c e r e b r o - p e d a l c o n n e c t i v e o r to the rostral commissure. The mass of caudal SPLI neurons p r o j e c t e d p a r a l l e l with t h e i r p r i m a r y n e u r i t e s t o w a r d t h e n e u r o p i l , however, f u r t h e r t r a c i n g was n o t possible b e c a u s e o f the d e n s e a r b o r i z a t i o n in t h e n e u r o p i l .

Co-localization of LomTKLI and SPLI materials was not typical in t h e Helix CNS. It was d e m o n s t r a t e d in a few n e u r o n s o f the cell clusters o f t h e l a t e r a l a n d r o s t r a l cell b o d y l a y e r of the p e d a l g a n g l i a a n d in the p l e u r a l ganglia n e a r b y t h e origin o f t h e c e r e b r o - p l e u r a l c o n n e c t i v e (Figs. 14, 17). A l s o p a r t i a l c o - l o c a l i z a t i o n c a n n o t be e x c l u d e d within the p o p u l a t i o n o f the globulus cells (Fig. 12). H o w e v e r , t h e i r very small d i a m e t e r ( ~ 5 p,m) m a d e it i m p o s s i b l e to resolve this question unequivocally. In the m a j o r i t y of cases, serial a l t e r n a t ing sectioning r e v e a l e d t h a t the L o m T K L I a n d S P L I cell g r o u p s w e r e localized a d j a c e n t l y but w i t h o u t showing c o - l o c a l i z a t i o n (Figs. 13, 15).

3. 7. Distribution of L K L I neurons T h e d e t a i l e d d i s t r i b u t i o n o f L K L I n e u r o n s has b e e n d e s c r i b e d e a r l i e r [12]. F o r c o m p a r i s o n , we p r e s e n t h e r e only t h e s u m m a r y m a p s a n d n u m b e r s o f i m m u n o r e a c tive n e u r o n s in t h e C N S (buccal g a n g l i a not included; T a b l e 1, Figs. lc, 2c, 3c).

4. Discussion O u r i m m u n o c y t o c h e m i c a l o b s e r v a t i o n s clearly show t h a t a high n u m b e r o f n e u r o n s o f t h e Helix C N S a r e reactive to a n t i s e r a r a i s e d to t a c h y k i n i n - r e l a t e d p e p tides. T h e d i s t r i b u t i o n o f L T K L I a n d S P L I n e u r o n s

Fig. 6. SPLI elements in the cerebral ganglion. Two sections of different level taken from the right (a) and left procerebrum (b). a: dorsal surface of the procerebrum, showing labeled axon bundle (thick arrow) projecting to the olfactory nerve (on). SPLI neurons are present below the globulus cells (GC) and in the posterior lateral marginal cell group (arrows). Note the homogenous immunolabeling of the terminal medullary mass (me). b: SPLI elements at the medial level of procerebrum. Labeled globulus cells (arrow) are seen along the outer marginal surface of the globulus cell layer, projecting to a fine axon bundle. To the same bundle projects SPLI neurons of the posterior lateral cell group (double arrow). Note the differently labeled neuropil areas: terminal medullary mass (me) with homegenous labeling; internal medullary mass (open arrow) without labeling; fine varicose network in the lateral neuropil (star). a,b × 200. Fig. 7. Higher magnification of single SPLI globulus cells (arrows) with varicose processes. GC, globulus cells, x 500. Fig. 8. Enlarged view of a SPLI neuron (arrow) in the posterior lateral marginal cell group, showing its first branching (long arrow) with further processes to both rostral and caudal directions (arrowhead). x350

| 91

f

~w

1

t~

23{)

1~ Elekes, D.R. Niissel / Brain Research 601 (1994) 223-236

d e m o n s t r a t e that the two neuron populations are present with minor neuronal overlap in the CNS of H. pomatia. Even when groups of i m m u n o l a b e l e d cell

bodies are found in adjacent areas of a ganglion, lew cells are double-labeled with LomTKLI and SPLI material. This type of distribution is similar to that we

Fig. 9. Distribution of SPLI neurons in the pedal ganglion, a: an overview of the right pedal ganglion, showing the localization ofaU important groups of SPLI neurons. Note giant SPLI neurons (arrows) in the caudal and latero-caudal part. Small arrows indicate clusters of small-size SPLI neurons in the rostro-lateral and rostral celt body layer. The neuropil (np) is densely innervated by immunoreactive fibers, cpdc, cerebro-pedal connective, x 125. b: higher magnification view of the rostro-lateral cell groups (arrows). Two of them project to a bundle (thick arrow) leading to the rostral commissure, x 250. Fig. 10. Innervation of the neural sheath by SPLI elements (arrows) on the ventral surface of the pedal ganglia. SPLI fibers appear in network-like arrangement and also form bundle in a septum of the sheath, running between nerve cell bodies (NC). x 250. Fig. 11. SPLI fibers in the aorta wall (arrows). Note entrance of labeled processes (arrowheads) from the neighboring connective tissue sheath, lu, aorta lumen, x 175.

K. Elekes, D.R. Niissel / Brain Research 661 (1994) 223-236

found previously, when comparing co-localization of SPL and LTKL immunoreactivities in the Helix CNS [12]. It appears that at least three, mostly independent, tachykininergic-leucokininergic systems may function in the snail CNS. On the other hand, the partial reduction of LTKLI following preabsorbtion with the

231

lower concentration of the antigen (20 /xM) indicates that the primary structure of the snail tachykinin-like peptide partially differs from locustatachykinin I. Recently, a tachykinin neuropeptide has been isolated and sequenced from the CNS of the freshwater bivalve Anodonta cygnea [15] which shares partly its amino acid

Fig. 12. Localization of L o m T K L I (a) and SPLI elements (b) in the procerebrum on alternating cryostat sections. A large n u m b e r of globulus cells are reactive, showing, however, different distribution. LTKLI neurons are mainly localized at the base (arrow) of the globulus cells (GC) while SPLI neurons are distributed more evenly, Because of the very small size of globulus cells, co-localization at the base of the procerebrum is possible. Note also differential immunostaining in the terminal medullary mass (me) which contains only a few varicose LTKLI fibers (small arrows) whereas it is homogenously stained for SP. a,b x 250. Fig. 13. Group of L o m T K L I (a) and SPLI neurons (b) (arrows) occur nearby in the pedal lobe of left cerebral ganglion but show no co-localization. Stars indicate the unlabeled cell group immunoreactive on the other section. Alternating cryostat sections, a,b × 500.

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K Lh,kes. D.R. Niissel / Brain Research 601 (1994) 223-236

s e q u e n c e w i t h t h a t o f L o m T K - I , i n c l u d i n g an i d e n t i c a l C-terminus ending (Anodonta-tachykinin: pQYGFH A V R - N H 2 ; L o m T K I: G P S G F Y G V R - N H 2 ) . A p p l y ing t h e L o m T K - I a n t i b o d y u s e d in t h e p r e s e n t s t u d y on

c r y o s t a t s e c t i o n s o f A n o d o n t a g a n g l i a , d i s t i n c t immunolabeling of a number of neurons and neuropil f i b e r s c o u l d b e d e m o n s t r a t e d ( E l e k e s a n d Niissel, in prep.). H e n c e , it s e e m s h i g h l y p r o b a b l e t h a t o u r anti-

Figs. 14, 15. Differential localization of LTKLI and SPLI neurons in the pedal ganglion. Alternating cryostat sections. Fig. 14. Comparison of the localization of the three rostro-lateral celt groups (arrows) in the left pedal ganglion, a: LomTKLI elements; b: SPLI elements. In two groups, the neurons are labeled with both antibodies (arrows). Star in a indicates the cell location of the group which is SPLI in b. a,b × 250. Fig. 15. LomTKLI (a) and SPLI cell groups (b) on the ventral surface of the right pedal ganglion. LomTKLI neurons are localized between (arrows) the two SPLI groups, a,b x 500.

K. Elekes, D.R. Nhssel / Brain Research 661 (1994) 223-236

body recognized related tachykinin neuropeptide(s) in the Helix CNS. Differences in the distribution and number of I_~mTKLI and SPLI (and LKLI) neurons in the CNS of Helix are obvious. The cerebral ganglia contain most of the immunoreactive neurons. The significant number of LomTKLI neurons is especially striking. They represent 86.5% of the total LomTKLI neuron

233

population of the CNS and this is about twice the percentage of SPLI and LKLI neurons in the cerebral ganglia. Characteristic differences can be established for the distribution of the three tachykinin-like immunoreactive substances within the cerebral ganglion, with respect to two main anatomical regions: pro- and mesocerebri. The procerebrum is the major site of localization of LomTKLI neurons. A significant num-

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,? L$

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bl Fig. 16. LomTKLI (a) and SPLI elements (b) in the viscero-parietal ganglion complex. Note the almost identical distribution of immunoreactivity in the neuropil (np) whereas labeled cell bodies (arrows) are only seen in the visceral ganglion (V) stained for SP. Alternating cryostat sections. LPa, left parietal ganglion; RPa, right parietal ganglion; lpn, left pallial nerve, a,b x 100. Fig. 17. Group of LTKLI (a) and SPLI neurons (b) show partial co-localization of immunoreactivities in the right pleural ganglion, a,b x500.

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K. Elekes, D.R. Ndssel / Brain Research 661 (1994) 223-230

ber of SPLI ceils are also represented by the globulus cells and the procerebral lateral and marginal neurons. In contrast, only a small part of the globulus cells react to the leucokinin-antiserum. In case of the mesocerebrum, the situation is inverse. Namely, whereas almost the entire population of mesocerebral neurons was found to be LKL-immunoreactive [12], this is the only region in the cerebral ganglion free of LomTKLI and SPLI neurons. (The visualization of a few SPLI neurons in the left mesocerebrum by the PAP method is rather questionable, since in earlier immunofluorescence experiments [12] these neurons were not labeled in this region.) In the viscero-parietal ganglion complex, LomTKLI neurons are almost completely missing. However, the neuropil of these ganglia, like that of the others, except for the medullary mass of the procerebrum, is densely innervated by immunoreactive fibers of all the three types. It seems that the three tachykininergic systems are equally involved in central signalling events, probably overlapping identical postsynaptic target areas. For the exact resolution of this question, ultrastructural investigations will, however, be necessary. The differential immunostaining of tachykinin-refated peptides in the procerebrum is striking. The procerebrum is considered to be a highly sophisticated integration-associative center of the snail brain and is compared, on the basis of its structure, to the mushroom body of arthropods [43]. In terrestrial snails, the procerebum was shown to participate in processing chemo- and mechano sensory informations [5,6] and to be a potential site of odor memory formation [16]. Also 5-HT, dopamine (DA) and SCP B were demonstrated to increase its cAMP level and so may modulate protein phosphorylation in the procerebrum [41]. The presence of several neurotransmitters/neuropeptides has also been demonstrated immunocytochemically in the procerebrum, such as SCP~ [42] (and also K. Elekes and D.R. Nassel, unpubl, data), FMRFamide [10], met-enkephalin [11], DA [17], 5-HT (K. Elekes, unpubl, data). The three tachykinin-related antisera applied (LomTKI, SP, this study and LK-I [12]) gave the following immunolabeling: LomTK-I - a few varicose fibers, SP - homogeneous staining in the terminal mass and no staining in the internal mass, LK-I - network of varicose fibers. In addition, many globulus cells are stained with SP and LomTK-I antisera but only a minimal number of them with LK-I antiserum. It suggests that tachykinin-related substances may participate in different integrative processes in the procerebrum, connected to both intrinsic and extrinsic elements. For substance P, the question arises: Could a special and central role be attributed to this neuropeptide in senspry processes? The homogenous SPL immunostaining of the whole terminal mass may indicate an 'overall' role for substance P in central sensory processes in the

procerebrum. The physiological role of SP in sensory. processes has widely been demonstrated [1]. We have also analysed the projections of labeled cells to their targets, including neuronal perikarya, ganglion connective tissue sheath and blood vessel walls. This revealed further differerences between these tachykinin-related peptides. Only LKLI fibers innervate neuronal perikarya and then only in certain regions of the CNS [12]; neither SPLI nor LomTKLI elements can be found in the cell body region of any ganglion. Substance P is the only tachykinin-related peptide of this investigation which densely innervate the connective tissue sheath around the CNS and peripheral nerves of H. pomatia. Innervation of the neural sheath by SPLI fibers has also been described in the nervous system of Drosophila [32]. In Helix, we have also found SPLI innervation in the blood vessel walls. In contrast, neither LomTKLI nor LKLI neurons appear to innervate this region. Hence, substance P would be the only tachykinin involved in neurohormonal regulatory processes in the snail whereas all t h e three tachykinin-related peptides take part in regulation of peripheral targets through the peripheral nerves themselves. An additional role for substance P may also be suggested in regulating blood flow by affecting the blood vessels themselves.

Acknowledgements

The skillful technical assistance of Zs. Nagy, M. Szelier, A. Karsson and B. Bal~izs is much appreciated. Thanks are due to C.J.H. Elliott for correcting the English text. This work was supported by O T K A Grant 2477 (to K. Elekes) and by the Swedish Natural Science Research Council Grant B-BU-t820-308 (to D,R. N~issel).

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