Localization and release of FMRFamide-like immunoreactivity in the cerebral neuroendocrine system of Manduca sexta

Neuropeptides secreted by insect cerebral neurosecretory cells (NSCs) regulate a wide range of processes including physiology, development and behaviour. In a few cases, the source NSCs for these insect neurohormones have been identified (Agui, Granger, Gilbert & Bollenbacher, 1979; Copenhaver & Truman, 1985). Recently, insect NSCs and neurohaemal organs have been shown to be immunoreactive to antisera directed against vertebrate and invertebrate neuropeptides (reviewed by Kramer, 1985). Thus, the insect homologues of these neuropeptides may serve a neurohormonal function. A possible neuroendocrine role in insects for the tetrapeptide Phe-Met-Arg-Phe-NH₂ (FMRFamide) (Price & Greenberg, 1977a, b) or analogues thereof was suggested by immunocytochemical staining of neurohaemal corpora cardiaca (Boer, Schot, Veenstra & Reichelt, 1980; Veenstra & Schooneveld, 1984). In addition, NSCs in the suboesophageal ganglion of the Colorado potato beetle contain FMRFamide-like immunoreactivity (FLI) (Veenstra, 1984; Veenstra & Schooneveld, 1984). The current study was undertaken to further clarify the role of FLI in insect neuroendocrine systems.

The protocerebral neuroendocrine system of the moth, Manduca sexta, has been the subject of anatomical, biochemical and physiological studies. This neuroendocrine system comprises two bilaterally symmetrical clusters of identified NSCs, one in the pars intercerebralis and the other in the pars lateralis (Fig. 1). The axons of the NSCs leave each brain hemisphere via the fused nervi corporis cardiaci I and II (NCCI + II) and terminate in either of the two cerebral neurohaemal organs, the corpus cardiacum (CC) or the corpus allatum (CA) (Nijhout, 1975; Carrow, Calabrese & Williams, 1984). The cells in each cluster have been classified according to the size and location of their somata as well as the architecture of their neurites, axons and terminal ramifications in the neurohaemal organs (Nijhout, 1975; Buys & Gibbs, 1981; Carrow et al. 1984; Copenhaver & Truman, 1984).

We now describe the distribution of FLI in the pupal brain and cerebral neurohaemal organs of Manduca. FLI was further localized to identified cerebral NSCs by combining intracellular dye injection and indirect immunofluorescence. Finally, calcium-dependent release of FLI was evoked from isolated neurohaemal organs with high potassium depolarization. A preliminary report of these findings has been presented (Carroll & Carrow, 1985).

Larvae of Manduca sexta were reared at 25 °C on an artificial diet under a 17-h light/7-h dark photoperiod (Bell & Joachim, 1976). Tissues from pupae, 1 or 2 days after ecdysis, were used for immunocytochemical analysis because of the facility with which the brains could be desheathed. Tissues from premetamorphic larvae, 3-4 days prior to pupal ecdysis, were used for extraction and release of FLI since the lower fragility of tissues at this stage facilitated collection and manipulation.

Compositions of the experimental salines as modified from Carrow (1983) are given in Table 1. The osmolarity and pH were the same for all salines.

For immunocytochemistry, brain-retrocerebral complexes (brain with attached CC and CA) along with attached tissue and tracheae were dissected from unanaesthetized pupae. The tissues were pinned to a Sylgard-coated Petri dish under normal saline. Brains were then desheathed (Carrow et al. 1984) to facilitate the entry of antisera.

Organs intended for extraction or release of FLI were dissected from unanaesthetized larvae immersed in normal saline.

Immunocytochemistry was performed on whole-mount preparations (Li & Calabrese, 1985). Tissues were fixed at 10°C for 18-24 h in 4 % paraformaldehyde in 120 mmol 1^-1 sodium phosphate buffer, pH 7·4 or for 4h in 1 % paraformaldehyde in 150 mmol 1^-1 sodium cacodylate buffer and 5 mmol 1^-1 CaC1₂, pH 7·4. The rabbit polyclonal antiserum used was raised against synthetic FMRFamide conjugated to succinylated bovine thyroglobulin (no. 232, O’Donohue et al. 1984) and was a gift from Thomas O’Donohue. This primary antiserum was used at a dilution of between 1:300 and 1: 500. Binding of the anti-FMRFamide antiserum was visualized with rhodamine conjugated to goat anti-rabbit IgG (Cappel) at a final dilution of 1:100.

A check for specificity of immunolabelling was made by incubating tissues with anti-FMRFamide antiserum that had been preabsorbed for 2-4 h with 100μgml^-1 of synthetic FMRFamide (Peninsula Laboratories).

To identify immunoreactive NSCs, intracellular injection of the dye Lucifer Yellow was combined with indirect immunofluorescence (Reaves & Hayward, 1979). NSC somata can be observed under a dissecting microscope due to their reflective opalescence. Thus, NSCs were identified on the basis of soma size and position and were iontophoretically injected with Lucifer Yellow CH (Sigma) using hyperpolarizing current (3-5 nA) for 10—30s (Carrow et al. 1984). Brains with injected cells were subsequently incubated in saline at 10°C for 1–4h. Finally, tissues were fixed and processed for immunocytochemistry. Labelled cells were visualized with a fluorescence photomicroscope (Zeiss or Leitz).

Brains or neurohaemal organs were collected from premetamorphic larvae and washed three times in calcium-free saline; the number of organs in each sample varied in accordance with the organ type (see Table 2). The organs were homogenized in 100μl of an acidified methanol solution (90% methanol, 9% acetic acid, 1 % water). Homogenates were sonicated (20 kHz) and then centrifuged (15 000 g) for 1 min. The supernatant was withdrawn and the pellet was resuspended in 100 μl of acidified methanol and centrifuged again for 1 min. This step was repeated. The supernatants were pooled, dried in a Savant Speed Vac concentrator, and stored at —20°C until assayed.

For each experiment, approximately 30 isolated retrocerebral complexes from premetamorphic larvae were washed three times in calcium-free saline. The organs were transferred to a Linbro culture well and incubated for 1-2 h at 25 °C in 50 μl of either high potassium, calcium-free saline or normal saline. After this preincubation, tissues were transferred to another culture well and incubated in high potassium, calcium-containing saline for 1-2 h. In two experiments, the preincubation period was omitted and organs were incubated immediately in high potassium, calcium-containing saline. After incubation, tissues were discarded and the incubation saline was transferred to a microcentrifuge tube. Samples were then sonicated and extracted as described above.

The amount of released or extracted FLI was measured using the radioimmunoassay (RIA) protocol of Price (1982) as modified by Li (1985). The tracer used for RIA was ¹²⁵I-Tyr-Met-Arg-Phe-NH₂, iodinated by a modified Chloramine T method (Greenwood, Hunter & Glover, 1963). The anti-FMRFamide antiserum was the same as that used for immunocytochemistry. Each assay tube contained synthetic FMRFamide or sample diluted in 100μl of RIA buffer (0·1% bovine serum albumin and 0·02% sodium azide in 0·1 mmol 1^-1 sodium phosphate buffer, pH 7·5). The antiserum (final dilution between 1:50000 and 1:100000) and the tracer (6000c.p.m.; specific activity 50-150 Ci mmol^-1), each diluted in 50μl of RIA buffer, were added to the assay tubes. After vortexing, the tubes were incubated at 4°C for 18-24h. Unbound peptides were then precipitated by the addition of 100μl of a 2% dextran-coated charcoal solution (5% goat serum, 0·02% sodium azide, in 0·1 mmol 1^-1 sodium phosphate buffer, pH 7·5). Finally, the assay tubes were vortexed and centrifuged (15 000g) for 10 min. Pellet and supernatant fractions were counted in a Packard gamma counter.

The sensitivity range of the RIA was between 10 and 300fmol of FMRFamide. The logit transformed binding curve for synthetic FMRFamide was log linear (r ≥ 0·97) and was used to quantify FLI. Salts did not alter the slope of the standard curve; however, they decreased the sensitivity of the RIA, resulting in possible underestimations of FLI.

Approximately 25 bilaterally symmetrical pairs of somata in the pupal protocerebrum showed FLI (Fig. 2). A subset of these cells was located in the two NSC clusters: in the pars intercerebralis, two pairs of bilaterally symmetrical cells stained intensely; in the pars lateralis, 4-7 pairs of bilaterally symmetrical cells showed FLI, and two of these pairs stained intensely (Figs IB, 2). Immunoreactive neurones in the protocerebrum outside these two clusters are likely to be non-endocrine since they do not stain with paraldehyde fuchsin (Nijhout, 1975).

FLI was observed throughout the retrocerebral complexes as shown in Fig. 3. Immunoreactivity was visible in terminals in the CC and CA as well as in axons in the nervi corporis cardiaci and nervi corporis allati. FLI was also evident in neurites in the protocerebrum and tritocerebrum (Fig. 4B,D,F). Similar immunolabelling was observed in the brain-retrocerebral complexes of premetamorphic larvae.

Preabsorption of the anti-FMRFamide antiserum with synthetic FMRFamide eliminated immunoreactive staining in the brain-retrocerebral complexes of pupae and larvae.

FLI was localized to a subset of protocerebral NSCs by double labelling. In the pars lateralis, FLI was observed in groups Ila (Fig. 4A,B) and lib (Fig. 4C,D). When both group Ila cells in one hemisphere were injected with Lucifer Yellow (N = 4), they both showed FLI, indicating that all cells of this group exhibit FLI. Of the 23 group lib cells that were injected, 19 (83 %) showed FLI indicating that most, but not all, of the cells in this group are immunoreactive. Among the immunoreactive group lib NSCs are the two intensely immunolabelled cells in the pars lateralis, one of which is shown double labelled in Fig. 4C,D. The intensely immunoreactive cells in the pars intercerebralis were group lb cells (Fig. 4E,F). Thus, only two of the approximately five pairs of group lb cells showed FLI. None of the group la cells that were injected with Lucifer Yellow (N = 12) exhibited FLI.

To determine whether the RIA could be used to quantify FLI, an analysis was made of the cross-reactivity of brain extracts and synthetic FMRFamide. Tracer binding to antiserum was inhibited with serial dilutions of these antigens. The regression lines for the binding curves were parallel (the mean ratio of the slopes was 1·03 ±0·07, TV =3) indicating complete cross-reactivity of the antigens with this antiserum.

The amount of FLI extracted from brains and neurohaemal organs is shown in Table 2. In order to obtain detectable quantities of FLI, organs were pooled for extraction and RIA. The paired neurohaemal organs contained 23 % of the immunoreactivity extracted from the entire brain-retrocerebral complex. Within the retrocerebral complex, the CC yielded 2·5 times the amount of FLI extracted from the CA.

Localization of FLI to protocerebral NSCs and the neurohaemal organs in Manduca suggested that the immunoreactive substances might function as neurohormones or neurotransmitters. Therefore, we attempted to demonstrate release of FLI. Release was evoked by high potassium depolarization of retrocerebral complexes in vitro and was measured by RIA of the bathing medium (Carrow, Calabrese & Williams, 1981).

As shown in Fig. 5, retrocerebral complexes preincubated in high potassium, calcium-free saline released little FLI. By contrast, there was more than three times as much FLI released upon transferring the same organs to high potassium, calciumcontaining saline (P≤ 0·01). Thus, an amount of FLI equivalent to about 16% of that extracted from the retrocerebral complex was released in a calcium-dependent manner. When preincubated in normal saline, the neurohaemal organs spontaneously released a basal amount of FLI significantly greater than that released from organs preincubated in high potassium, calcium-free saline (P< 0·01). Nevertheless, these same organs released twicè the basal level when transferred from normal saline to high potassium, calcium-containing saline (P<0·01). Regardless of the preincubation treatment, or omission thereof, organs released a similar amount of FLI upon transfer to high potassium, calcium-containing saline (P< 0·01).

FMRFamide-like immunoreactivity was localized to endocrine and non-endocrine neurones in the brain of Manduca as well as to terminals in the CC and CA.

Moreover, relative amounts of FLI in extracts of brain and neurohaemal organs were measured by RIA. Finally, calcium-dependent release of FLI was evoked from the retrocerebral complexes by high potassium depolarization in vitro.

Among the NSCs in each hemisphere, FLI was observed in both group IIa cells, most of the group lib cells, and two cells of group lb (see Fig. 1). Four of these NSCs showed intense immunoreactivity: the two in group lb and two of those in group lib. Although the location of these four cells resembled that of the FLI cells in the protocerebrum of the Colorado potato beetle (Veenstra & Schooneveld, 1984), in the latter case the neurones were not paraldehyde fuchsin positive and hence are probably not neurosecretory. The other FLI-containing NSCs in the pars lateralis stained less intensely. These included the pair of group IIa cells thought to be the principle source of prothoracicotropic hormone, a neuropeptide regulator of the prothoracic, or ecdysial, glands (Aguiet al. 1979). Thus, the FLI detected here may be co-localized with other neuropeptides.

The FLI detected here may be distinct from peptides of the pancreatic polypeptide family. In some cases in insects, FLI cannot be distinguished from bovine pancreatic polypeptide (BPP)-like immunoreactivity; various anti-FMRFamide antisera cross-react with BPP and preabsorption of some anti-BPP antisera with FMRFamide blocks immunoreactive staining (Veenstra, 1984; Veenstra & Schooneveld, 1984; Myers & Evans, 1985a). However, other anti-FMRFamide antisera do not cross-react with BPP in immunocytochemistry (Myers & Evans, 1985b; Verhaert, Grimmelikhuijzen & De Loof, 1985). Furthermore, the anti-FMRFamide antiserum used in the present study does not significantly cross-react with BPP in RIA (O’Donohue et al. 1984). Finally, BPP-like immunoreactivity observed in the protocerebrum of adult Manduca was not located in the pars intercerebralis or pars lateralis (El-Salhy, Falkmer, Kramer & Speirs, 1983) whereas FLI has been observed in neurones in the pars intercerebralis of the adult brain (Homberg, Hoskins & Hildebrand, 1985). Consequently, FLI VAManduca may not be related to BPP-like substances.

Nagle (1981, 1982) previously provided evidence suggesting that FMRFamide is a neurotransmitter or neurohormone in the clam, Macrocallista nimbosa, by evoking calcium-dependent release of immunoreactive FMRFamide in vitro, extracting immunoreactivity from the haemolymph, and detecting high levels of immunoreactive FMRFamide in fractions of homogenized ganglia enriched for neurosecretory granules. Similarly, we evoked calcium-dependent release of FLI from retrocerebral complexes; the amount released was approximately 16% of the yield from extracts of complexes. For comparison, in vitro release of prothoracicotropic hormone is also calcium-dependent; the amount released from depolarized CA was equivalent to about 35 % of the extracted bioactivity (Carrow et al. 1981).

The physiological role of FLI in Manduca remains to be investigated. FMRF-amide was originally identified as a cardioexcitatory neuropeptide in molluscs (Price & Greenberg, 1977a, b). FLI could be cardioactive in Manduca since cardioactive peptides are found throughout the central nervous system, including the brain-retrocerebral complex (Platt & Reynolds, 1985; Tublitz & Truman, 1985). However, the two cardioacceleratory peptides isolated from the CC of the cockroach, Periplaneta americana, bear no sequence homology with FMRFamide (Scarborough et al. 1984). A non-cardioactive function for FLI in Manduca is conceivable given the multifunctional nature of FLI in other species, including modulatory effects of FMRFamide on locust jump muscle (Walther, Schiebe & Voigt, 1984) and crab motor neurones (Hooper & Marder, 1984).

The data presented here show that FLI, as with other neurosecretory products, is stored in cerebral NSCs and can be released by depolarizing terminals in the neurohaemal organs. Therefore, the FLI detected here may have a neurohormonal or neuromodulatory function. In addition, the FLI might act as a neurotransmitter in the brain since neurites and non-endocrine neurones were also immunoreactive. In summary, the FMRFamide-like substances in the cerebral neuroendocrine system of Manduca may have multiple roles. The accessible system of identified FLI neurones described here should facilitate clarification of the functions of this class of neuropeptides in insects.

We thank T. O’Donohue for his generous gift of anti-FMRFamide antiserum and C. Li for her excellent immunochemistry protocols and comments on an earlier version of this manuscript. We also thank M. Weidner, E. Arbas, J. Kuhlman and B. Norris for many helpful suggestions and discussions, and C. M. Williams and E. Marder for critical reading of the manuscript. This research was supported by NSF grant PCM-8215638 to C. M. Williams and RLC, NIH grant RO1-NS21232 to RLC, and NIH fellowship 1-F32-HD06739 to GMC.

Recently, the sequence of a neuropeptide inhibitor of visceral muscle that was isolated from heads of the cockroach, Leucophaea maderae, was reported to be pGlu-Asp-Val-Asp-His-Val-Phe-Leu-Arg-Phe-NH2 (Holman, Cook & Nachman, 1986). This is the first report of a purified neuropeptide in insects that has the C-terminal sequence recognized by the anti-FMRFamide antiserum used in the work presented here.