Serotonin-immunoreactive neurons in the retrocerebral neuroendocrine complex of the cricket Teleogryllus commodus (Walker) (Orthoptera : Gryllidae) and cockroach Periplaneta americana (L.) (Dictyoptera : Blattidae)

Int. J. InsectMorphol. & Embrvol. Vol. 17. No. 4/5. pp. 3/13-311, 1988 Printed in Great Britain

0020 7322/88$3.00+ .00 © 1988PergamonPressplc

SEROTONIN-IMMUNOREACTIVE NEURONS IN THE RETROCEREBRAL N E U R O E N D O C R I N E COMPLEX OF THE CRICKET T E L E O G R Y L L U S C O M M O D U S (WALKER) (ORTHOPTERA : GRYLLIDAE) AND COCKROACH P E R I P L A N E T A A M E R I C A N A (L.) (DICTYOPTERA : BLATTIDAE) RUDOLPH PIPA D e p a r t m e n t of Entomological Sciences, University of California, Berkeley, California 94720, U.S.A. and

DARRELL MOORE D e p a r t m e n t of Psychology, Life Sciences Centre, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J1

(Accepted 25 February 1988)

Abstract--Retrocerebral glandular complexes of Teleogryllus commodus (Orthoptera : Gryllidae) and Periplaneta americana (Dictyoptera : Blattidae) were examined for 5hydroxytryptamine (serotonin)-immunoreactive (5-HTi) neurons. In Teleogryllus, a p r o m i n e n t tract of 5-HTi axons crosses the ventral surface of the corpus allatum (CA) from nervus corporis allati 1 ( N C A 1), and seems to end at varicosities in N C A 2. Serotoninergic axons within this tract pass cephalad to the corpus cardiacum (CC), which also contains n u m e r o u s , fine 5-HTi branches. 5-HTi axons originate anteriorly, presumably from the pars intercerebralis (PI) and pars lateralis (PL) of the brain. This is suggested by absence of immunoreactivity at the N C A 2-subesophageal ganglion junction, by intense immunofluorescence of the nervi corporis cardiaci (NCC) 1 and 2, by the presence of 5-HTi perikarya in PI and PL, and by previous data obtained by backfilling N C A 1 and 2. In Periplaneta, 5-HTi varicosities are rare in the C A , but abound in the N C A 2, and in N C C 1, 2, and 3. A few 5-HTi fibers project anteriorly from N C A 2 into the cap-like junction of C A and CC, and some traverse the C A to enter the postallatal nerves. Large, 5-HTi axons of N C C 3 ramify within the CC, while others contribute to an anterior b~:anch of N C A 2. A s in Teleogryllus, it is unlikely that 5-HTi fibers in N C A 2 originate from somata in the subesophageal ganglion. W h e n cobalt staining and serotonin immunocytochemistry were combined to stain subesophageal neurons of Periplaneta, 5HTi somata could not be paired with those back-filled via N C A 2. Conspicuous 5-HTi tracts between N C A 2 and the CC of Teleogryllus have no counterpart in Periplaneta. Index descriptors (in addition to those in title): 5-HT immunocytochemistry, neurohemal sites, corpus allatum, corpus cardiacum, endocrine innervation.

INTRODUCTION

THERE is biochemical evidence that 5-hydroxytryptamine (5-HT, serotonin) accumulates in the insect nervous system, where it is thought to be synthesized (Gersch et al., 1961; 303

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C o l h o u n , 1963; H i r i p i a n d R o z s a , 1973; K l e m m a n d A x e l s s o n , 1973; O s b o r n e a n d N e u h o f f , 1974; K u t s c h , 1975; M a x w e l l et al., 1978; Musz~,nska-Pytel a n d C y m b o r o w s k i , 1978; O m a r et al., 1982; S l o l e y a n d D o w n e r , 1984; Sloley et al., 1986). P h y s i o l o g i c a l a n d p h a r m a c o l o g i c a l studies i n d i c a t e t h a t this i n d o l e a l k y l a m i n e has w i d e s p r e a d f u n c t i o n a l significance. It s e e m s to r e g u l a t e visceral m u s c l e c o n t r a c t i o n (e.g. C o o k et al., 1969; Collins a n d M i l l e r , 1977; H u d d a r t a n d O l d f i e l d , 1982) a n d fluid s e c r e t i o n (e.g. M a d d r e l l et al., 1971; B e r r i d g e et al., 1975; M o r g a n a n d M o r d u e , 1984; T r i m m e r , 1985). F u r t h e r m o r e , w h e n 5 - H T is a p p l i e d to the n e r v e c o r d ( N a t h a n s o n a n d G r e e n g a r d , 1974; T a y l o r a n d N e w b u r g h , 1979) o r c o r p o r a c a r d i a c a ( G o l e et al., 1987) cyclic A M P p r o d u c t i o n is i n c r e a s e d . This s u p p o r t s t h e possibility that 5 - H T - s e n s i t i v e a d e n y l a t e cyclase m a y m e d i a t e s y n a p t i c t r a n s m i s s i o n , a n d r e g u l a t e e n d o c r i n e activity in insects. Sensitive i m m u n o c y t o c h e m i c a l m e t h o d s have b e e n used to identify p u t a t i v e s e r o t o n i n e r g i c n e u r a l p a t h w a y s (N~ssal, 1988), a n d o r t h o p t e r o i d insects h a v e f i g u r e d p r o m i n e n t l y as t h e s u b j e c t s of this r e s e a r c h ( B i s h o p a n d O ' S h e a , 1983; K l e m m a n d S u n d l e r , 1983; K l e m m et al., 1984; T a g h e r t a n d G o o d m a n , 1984; T y r e r et al., 1984; D a v i s , 1985, 1987; K l e m m et al., 1986; H u s t e r t a n d T o p e l , 1986). F o r the m o s t p a r t , h o w e v e r , scant a t t e n t i o n has b e e n p a i d to 5 - h y d r o x y t r y p t a m i n e - i m m u n o r e a c t i v e (5H T i ) n e u r o n s in the r e t r o c e r e b r a l n e u r o e n d o c r i n e c o m p l e x of t h o s e species. O u r curiosity a b o u t t h e i n n e r v a t i o n of this r e g i o n in the c o c k r o a c h , Periplaneta americana (L.) ( P i p a , 1978; P i p a a n d N o v a k , 1979; N o v a k and P i p a , 1986) a n d A u s t r a l i a n field c r i c k e t , Teleogryllus c o m m o d u s ( W a l k e r ) ( M o o r e a n d L o h e r , 1988), m o t i v a t e d us to i n v e s t i g a t e a n d c o m p a r e the s e r o t o n i n i m m u n o r e a c t i v i t y f o u n d t h e r e . W e w e r e e s p e c i a l l y i n t e r e s t e d in c o r r e l a t i n g the p r o j e c t i o n s of 5 - H T i n e u r o n s with i n n e r v a t i o n r e v e a l e d p r e v i o u s l y by a p p l y i n g i n t r a c e l l u l a r staining m e t h o d s .

MATERIALS AND METHODS Sexually mature, unmated male and female Teleogryllus commodus and sexually mature, virgin female Periplaneta americana, all more than 10 days past their imaginal molt, were examined. The protocols for rearing the insects, and anesthetizing and dissecting them have been described (Moore and Loher, 1988; Pipa, 1978, 1982). The data reported here were obtained by examining about 15 preparations from each species. Brains, subesophageal ganglia, and retrocerebral complexes were removed and collected in cricket or cockroach saline during a 2-hr period. In some cases 10 3M serotonin hydrochloride (Sigma, St. Louis, MO) was added to these saline solutions. To assure proper orientation, the specimens were pinned onto Sylgardcoated (Dow Corning, Midland, MI) dishes immediately before applying fixative. They were fixed overnight at 4°C in a 4% solution of paraformaldehyde in 0.1M phosphate buffer (pH 7.5). The next morning, they were washed several times in 0.1M phosphate buffer containing 0.1% sodium azide (PBS). Soon thereafter, or following storage for 1-2 days in PBS at 4°C, the specimens were rinsed 4-6 times during 18h in PBST, a solution of 0.3% (V/v) Triton X-100 in PBS. The PBST rinses and all subsequent immersions were at 4°C, in containers mounted on a rocking agitator. The specimens were soaked 4 hr in PBST containing 10% (V/v) non-immune goat serum (Cooper Biomedical, Malvern, PA). This was followed by 18 hr in primary antibody (rabbit antiserotonin antiserum (Immuno Nuclear, Stillwater, MN) diluted 1 : 200 in PBST containing 10% non-immune goat serum). After receiving 6 1-hr rinses in PBST, they were soaked overnight in fluorescein-labelled secondary antibody (goat antirabbit IgG antiserum (Cooper Biomedical) diluted 1 : 50 in PBST). Following 18 hr in several changes of PBST and 2 l-hr rinses in PBS, the specimens were stored for 1-2 days at 4°C in 80% glycerol, diluted with 4raM carbonate buffer (pH 9.4). For microscopic inspection, the specimens were mounted on depression slides in 90% glycerol. This was diluted with PBS (pH 9.0), and contained p-phenylenediamine (0.1% W/v) to reduce photobleaching of the fluorescein dye (Adams and Pringle, 1984). The whole-mounts were examined and photographed with a Zeiss microscope equipped for fluorescent epiiluminatiou (PB 450-490nm; FT 510; LP 520). Specificity of staining was tested by comparing fluorescence intensity seen after the above regimen to that shown by 2 kinds of controls: (1) specimens treated with the primary, but not with the fluorescein-labelled

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secondary antibody; (2) those incubated in primary antibody that had been preabsorbed by adding serotonin hydrochloride (1 mg/ml)) to the antiserum 5 hr previously. This was followed by the usual treatment with secondary antibody. Yellow-green fluorescence was absent in the first kind of control, and significantlyreduced or absent in the second. The likelihood was tested that neuron somata in the subesophageal ganglia of Periplanetacontribute 5-HTi axons to the nervi ccrporis allati II (NCA 2). This was done by identifying such somata after intracellular staining, and determining whether corresponding ones situated contralaterally showed serotonin immunoreactivity. Neurons projecting to NCA 2 on one side of the subesophageal ganglion were revealed by backfilling with 500mM CoCI2solution (Pipa and Novak, 1979). Following silver intensification of the precipitated CoS (Davis, 1982), the ganglia were prepared for immunofluorescentstaining as described by Davis (1987). Six preparations were examined for paired somata, one showing immunoreactivity, the contralateral member staining black.

RESULTS

5-HTi neurons in the retrocerebral complex ofTeleogryllus commodus Within the retrocerebral complex of Teleogryllus (Fig. 13), a dense tract of axons traversing the ventral surface of the corpora allata (CA) shows a strong immunoreactivity to serotonin antiserum (Fig. 1). The stained fibers project from the nervus corporis allati 1 ( N C A 1) into the CA, where they diverge slightly over the surface of the gland (Fig. 2) and then converge to enter the N C A 2. Several fibers typically do not remain in the ventral axon tract, but course through the C A along its dorsal surface or through the central core. Without ultrastructural confirmation, it is uncertain if any of the 5-HTi fibers end within the CA, although a few appear to do so. Most fibers, however, project into the N C A 2 where they probably terminate, as no stained processes continue on into the subesophageal ganglion (Fig. 3). Varicosities, present at all levels, are especially abundant within 1:he N C A 2. Because 5-HTi fibers within N C A 2 cannot be traced into the subesophageal ganglion, it is reasonable ~Io suppose that they arise anterior to the CA. No perikarya in the retrocerebral complex show immunoreactivity to 5-HT, although a few weakly staining somata occur in the hypocerebral ganglion (stomatogastric nervous system). Nickel infusion of the N C A 2 in the anterior direction has shown that axons terminating within the N C A 2 originate from somata in both the contralateral partes intercerebralis (PI) and ipsilateral partes lateralis (PL) of the protocerebrum (Moore and Loher, 1988). Similarly, backfills of the N C A 1 stained somata in both protocerebral loci, but in greater numbers than in the N C A 2 backfills. If, indeed, the immunoreactive fibers shown in the present study correspond to the axons previously revealed by intracellular staining, then those originating from the PI enter the retrocerebral complex via NCC 1 and those axons stemming from the PL pass to the retrocerebral complex through N C C 2. Strong 5-HT immunoreactivity is present in both the N C C 1 and NCC 2 (Fig. 4). In the corpus cardiacum (CC), staining is most intense along the lateral edges, where numerous fine branches occur, and several stained axons run posteriorly along the entire length of the organ (Fig. 5). ]Fibers enter the N C A 1 from the lateral tract. Consistent with the supposition that the 5-HTi axons originate from the brain, 45-50 cell bodies in the PI and 5-10 in each PL are stained (Fig. 6). The axons from these somata could not be traced the entire distance from the dorsal p r o t o c e r e b r u m to the N C C 1 or N C C 2, leaving open the possibility that the 5-HTi fibers within the retrocerebral complex may originate from some other location. H o w e v e r , nickel backfills of the N C C 1 and N C C 2 in Teleogryllus commodus have revealed somata only in the PI, PL, or in a small cluster along the medial

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Fro. 1, 5-HTi tract over ventral surface of CA in T. commodus. Fibers descend from NCA 1 and enter NCA 2. One stained fiber, indicated by arrowheads, lies along dorsal periphery of CA. (x 114). FIG. 2. Ventral surface of CA in T. commodus. NCA 1 at top, NCA 2 at bottom. Serotoninergic tract spreads over surface of gland. Dark elongate shadows are tracheae. (x 184). Fro. 3. 5-HTi fibers with varicosities are present throughout NCA 2 of T. commodus except at ventral-most extremity where nerve inserts onto subesophageal ganglion (arrow). Some glial cells within NCA 2 show faint autofluorescence, distinguishable by color from fluorescein-labelled serotoninergic fibers. (x 184). Fro. 4.5-HTi fibers in NCC 1, NCC 2, and along lateral edge of CC in T. commodus. Dorsal view. (x 288). F1G. 5. Posterior, dorsal view of CC in 72 commodus. (x 184). FIG. 6. Anterior, dorsal protocerebrum of T. commodus showing 5-HTi somata in PI and in both PL. (x 114).

m a r g i n of the t r i t o c e r e b r u m ( M o o r e a n d L o h e r , 1988). T h e tritocerebral cells exhibited n o s e r o t o n i n i m m u n o r e a c t i v i t y in the p r e s e n t study.

Serotonin i m m u n o r e a c t i v i t y in the retrocerebral c o m p l e x o f P e r i p l a n e t a a m e r i c a n a U n l i k e Teleogryllus, Periplaneta lacks a robust tract of 5 - H T i axons that pass from N C A 1 to N C A 2. I n s t e a d , strong s e r o t o n i n i m m u n o r e a c t i v i t y is largely restricted to fibers having n u m e r o u s varicosities within N C A 2 a n d its a n t e r i o r ( A B ) a n d posterior (PB) b r a n c h e s (Fig. 7). A B b e c o m e s tripartite (Fig. 14) n e a r its j u n c t i o n with n e r v u s corporis cardiaci 3 ( N C C 3); the dorsal b r a n c h fuses with N C C 3 (Fig. 10), the m e d i a l b r a n c h j o i n s the r e c u r r e n t n e r v e , a n d the lateral, ventrally directed o n e has an u n d e t e r m i n e d d e s t i n a t i o n . PB joins a n e r v e that can be traced to the p r o t h o r a c i c

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Fro. 7.5-HTi fibers of P. americana. Note varicosities in NCA 2 and its anterior (AB) and posterior (PB) branches. AB displaced posteriorly in this ventral view. (x 114). FIG. 8.5-HTi axons from NCA 2 of P. americana project into allatal cap (ACP). (x 184). FIG. 9. Serotoninergic axons (arrowheads) from NCA 2 of P. americana traverse lateral aspect of CA to enter postallatal nerve. (x 184). FIG. 10. 5-HTi fibers and varicosities in NCC 1, 2 and 3 of P. americana. Note intensely immunofluorescent fiber (arrow) from NCC 3 entering anterior branch (AB) of NCA 2. (x 114). FIG. 11. 5-HTi axons from NCC 3 (arrow, upper left) enter and branch within CC of P. americana. (x 184). FIG. 12. Ventral view of anterior subesophageal ganglion of P. americana, showing somata (arrow) of NCA 2 neurons back-filled with Co2+. Although several paired 5-HTi somata (arrowheads) are evident along ganglion midline (dotted line), none occurs opposite the cobalt-stained group. (x 114).

ganglion (Fraser and Pipa, 1977). In comparison, the N C A 2 of Teleogryllus (Fig. 13) exhibits no b r a n c h e s along its entire extent from C A to subesophageal ganglion. N o t all 5-HTi fibers remain within N C A 2 and its branches. A few conspicuous ones can be traced anteriorly, into the allatal cap ( A C P , Figs 8; 14). In Periplaneta, which differs f r o m Teleogryllus by lacking a well-defined N C A 1, this putative n e u r o h e m a l region joins C C to the C A (Pipa and N o v a k , 1979; N o v a k and Pipa, 1986). M o r e o v e r , several 5-HTi fibers exit N C A 2 posteriorly (Fig. 9), t h r o u g h the lateral aspect of the C A , and into the postallatal nerves (PN, Fig. 14). W e were unable to follow the fine fibers b e y o n d A C P or PN, which suggests that they terminate there, or contain insufficient serotonin to be visible. Results s o m e w h a t like this are o b t a i n e d by infusing Co 2+ anteriorly, t h r o u g h either N C A 2 (Pipa and N o v a k , 1979). F u r t h e r m o r e , intracellularly stained axons that p e n e t r a t e the C A and b r a n c h a m o n g the A C P and PN apparently arise posteriorly, f r o m 7 s o m a t a located on each side of the subesophageal ganglion midline. Similarities in fiber

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Ft~. 13. Schematic diagram of T. commodus retrocerebral complex (dorso-anterior view). Abbreviations as in Fig. 14. FIc. 14. Schematic diagram of P. americana retrocerebral complex (dorsal view). AB = NCA 2 anterior branch; ACP = allatal cap; CA = corpus allatum; CC = corpus cardiacum; EN = esophageal nerve; HG = hypocerebral ganglion; NCA 1, 2 = nervi corporis allati I and 2; NCC 1, 2, 3 = nervi corporis cardiaci 1, 2 and 3; PB = NCA 2 posterior branch; PN = postallatal nerves; RN = recurrent nerve. distribution shown by the 2 m e t h o d s p r o m p t e d us to investigate w h e t h e r the 5-HTi axons originate f r o m those somata. Observations of double-stained preparations suggest that this is unlikely; we never f o u n d 5-HTi s o m a t a that could be m a t c h e d with contralateral h o m o l o g s stained with CoS. This was so even though o t h e r pairs of serotoninergic s o m a t a were evident (Fig. 12). M o r e o v e r , as is the case in Teleogryllus, conspicuous serotoninergic fibers are not f o u n d where N C A 2 joins the subesophageal ganglion. This, too, suggests that the 5-HTi axons within that nerve arise elsewhere. Intense serotonin i m m u n o f l u o r e s c e n c e is evident in the nerves that c o n n e c t the retrocerebral c o m p l e x to the brain. D e n s e meshworks of 5-HTi fibers surround N C C 1, N C C 2, and N C C 3 (Fig. 10). Several conspicuously fluorescent axons within N C C 3 can be traced posteriorly, into the C C and the dorsal limb of A B p r o d u c e d by N C A 2. T h e rami entering the C C branch there (Fig. 11), but a b u n d a n t 5-HTi fibers, like those in Teleogryllus, are not apparent. DISCUSSION O u r results indicate that, in the cricket Teleogryllus commodus, an anatomically distinct axon tract passing over the C A and terminating within the N C A 2 is serotonin i m m u n o r e a c t i v e . This serotoninergic tract has not previously b e e n described in any o t h e r insect. T h e serotoninergic axons originate anteriorly, p r o b a b l y f r o m the pars intercerebralis (PI) and pars lateralis (PL) of the brain, as suggested by the intense staining of the N C C 1 and N C C 2 as well as the presence of 5-HTi perikarya in the PI and PL. H o w e v e r , alternative possibilities cannot as yet be ruled out. F o r instance, the fibers could originate f r o m intrinsic neurons of the retrocerebral complex with cell bodies too small to be visually discriminated from the varicosities that characterize most of the stained processes. A n o t h e r possibility is that the stained fibers arise from s o m a t a external to the retrocerebral complex, but the axons only accumulate e n o u g h stainable material near their terminations within the complex itself. Therefore, matching the

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proper somata with the appropriate nerve terminals may be difficult. Nonetheless, previous nickel backfills of the N C A 2 and NCA 1 in the anterior direction have marked cell bodies in the brain neurosecretory loci, the PI and PL, and nowhere else (Moore and Loher, 1988). In 2 other cricket species, Gryllus bimaculatus and Acheta domesticus, as well as in the locust Schistocerca gregaria, 5-HTi fibers presumably originating from the brain form a dense fiber net over the NCC 1 and NCC 2 (Klemm et al., 1986). These fibers apparently continue on through the N C A 1 and end as single processes within the CA. This is in marked contrast to our results in Teleogryllus commodus in which 5-HTi fibers are arranged in a definite tract that traverses the periphery of the CA and enters N C A 2. Ultrastructural evidence of synaptoids (putative hormonal release sites) in the peripheral layer of the N C A 2 in Acheta (Weber and Gaude, 1971) has implicated this nerve, at least in crickets, as a neurohemal organ. This interpretation is supported by the finding in the cricket Teleogryllus commodus that axons originating from brain neurosecretory loci terminate within the nerve and do not enter the subesophageal ganglion (Moore and Loher, 1988). Therefore, serotonin may be a neurosecretory material that is released from the N C A 2 neurohemal structure. In both species we found 5-HTi varicosities throughout the retrocerebral complex, suggesting that release of serotonin may occur at diverse locations. In Periplaneta, the fine structural organization of ACP, PN NCA 2 and NCC 1 and 2 also implies that they are neurohemal areas (Novak and Pipa, 1986). Whether these data indicate multifunctional use of serotonin, or simply that the entire retrocerebral complex is a single, diffuse neurohemal organ remains to be clarified. With regard to the distribution of 5-HTi fibers in the retrocerebral complex of Periplaneta, we could confirm some of the observations reported by Davis (1985). In our preparations, as in his, serotoninergic varicosities were abundant in N C A 2, and in NCC 1, 2 and 3, but rare in the CA. We found, however, that a small number of 5-HTi fibers from N C A 2 project into the ACP (his NCA 1), while others traverse the lateral aspect of the CA to enter PN. An additional difference is that we could trace large, serotoninergic fibers from NCC 3 into the CC, where they ramify. Other 5-HTi fibers from NCC 3 enter the dorsal branc]a of AB, which is an anterior extension from N C A 2. The occurrence of these robust fibers in the anteriormost limb of NCC 3 implies that they project into the brain. It remain,; to be determined whether their somata are located there. In Periplaneta, as in Teleogryllus, it is unlikely that the abundant serotoninergic varicosities of N C A 2 arise from subesophageal ganglion neurons that enter that nerve directly. This is suggested by the absence of 5-HTi fibers at the NCA 2-subesophageal ganglion junction. It is also supported by the results obtained when cobalt staining and serotonin immunocytochemistry were combined to identify the somata of such neurons in Periplaneta. In such preparations we never found serotoninergic somata that corresponded to those back-filled via NCA 2. The apparent dissimilarity in distribution of the profuse serotoninergic innervation of NCA 2 in these 2 orthopteroids remains enigmatic. For the reasons presented above, we suspect that in Teleogryllus these fibers arise anterior to the CA, perhaps from the brain itself. Because Periplaneta lacks conspicuous 5-HTi tracts passing from the CC, to N C A 1 (allatal cap), through CA and into NCA 2, cephalic innervation via that pathway seems unlikely. An alternative antero-posterior projection, yet to be explored, might be the robust 5-HTi fibers from NCC 3 that enter the anterior branch of N C A 2.

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RUDOLPH PIPA and DARRELLMOORE

Acknowledgements-- We thank Dr R. Lane for use of the epifluorescence microscope and Dr W. Loher for his encouragement. This research was supported by University of California Hatch Project 4238-H (R.P.), by a NIH Postdoctoral Fellowship (D.M.), and by NIH grant HD 03619 to Dr W. Lohcr.

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