Cytological evidence for serotonin-containing fibers in an abdominal neurohemal organ in a hemipteran

Brain Research, 306 (1984) 235-242

235

Elsevier BRE 10204

Cytological Evidence for Serotonin-Containing Fibers in an Abdominal Neurohemal Organ in a Hemipteran THOMAS R. J. FLANAGAN*

Biology Department, WesleyanUniversity,Middletown CT06457 (U.S.A.) (Accepted December 27th, 1983)

Key words: serotonin - - neurohemal - - insect - - histochemistry- - immunocytology- - neurochemistry-- autoradiography

The abdominal neurohemal organs of the hemipteran Rhodnius prolixus contain an extensive serotonin containing arborization. Endogenous serotonin within fibers and terminals in the neurohemal area were detected with histochemical and immunocytological techniques. The abdominal nerves which contain the neurohemal organs selectively sequester exogenous serotonin. Serotonin and its metabolites are biochemically detected within the mesothoracic ganglion, which is a known source of projections into the neurohemal organ. However, the source of the cell bodies which might send serotonergic fibers to the neurohemal organ remains undetermined because no correspondence was found between immunocytologicalmaps of serotonin-containing cells in the ganglion, and projection maps into neurohemal organ (determined by cobalt back-filling). INTRODUCTION Insect nervous systems contain two major neurohemal organs: cephalic corpora cardiaca (c.c.)45, 46 and abdominal neurohemal organs3,42,43. A variety of peptide neurohormonesqA5,20, 37 and biogenic monoamines12,27,2s,37 have been identified in extracts and in cytological preparations of these organs. The presence of monoamines in neurohemal organs known to secrete peptide hormones might be indicative of a hormonal role for these neurohumors, but alternatively might reflect a mechanism for the neural control of the release of secretory material from neuron terminals. Within the cephalic system of insects, for instance, neurons with the ultrastructural characteristics of monoamine-containing cells are known to project to the c.c. 29,45 and pharmacological evidence has been presented that the monoamines may regulate the release of peptide hormones from intrinsic cells or neuron terminals in that structurel7, 38. The presence of diverse neuron types and intrinsic cells in the c.c. complicate the analysis of control mechanisms for secretion. Abdominal neurohemal organs lack intrinsic cells, and thus offer a less complex structure in which to study possible interactions between mono-

amine and peptide systems. A well-characterized abdominal neurohemal organ is found within the hemipteran Rhodnius prolix/gs6,32,33. In this species, neurosecretory fibers arborize beneath the neural sheath of a set of 5 abdominal nerves, and neurosecretory terminals are circumferentially distributed along the lengths of these nerves. These terminals contain and release at least one, and possibly several, neurosecretory productsS,35. The best studied neurosecretory product is the peptidic diuretic hormone (DH)I,2, 24,25. Maddrell has identified NSC within the distal margin of the mesothoracic ganglion (MG) which give rise to the D H secretory terminals in the neurohemal organ 33. These cells have been isolated and individually bioassayed, and show cycle levels of D H activity which parallel a physiologically-induced diuretic cycle 7. As part of a continuing effort to characterize the cellular design of the abdominal neurohemal organ in Rhodnius, this report focuses on the monoaminergic components of this neurohemal organ. Serotonergic fibers comprise the primary, and probably the exclusive, monoaminergic projection into the abdominal neurohemal organ of this species.

*Present address: P.O. Box 100, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY 11724, U.S.A.

236 MATERIALS AND METHODS

Dissections Fifth instar larvae of a laboratorY colony of Rhodnius prolixus Stal of the Cambridge strain were dissected in a physiological saline 34 or a modified Ca 2÷free, Mg2*-rich (25 mM) saline. Physiological saline was used for incubations with radioisotopes, and Ca2+-free, Mg2+-rich salines were used to restrict release of endogenous monoamine prior to histofluorescence or immunocytological analysis. Preparations display patterned electrical activity even after 10-h incubation periods.

Autoradiography Isolated central nervous systems were incubated in either 0.1/aM [1,2-3H]serotonin (28,9 Ci/mM; NEN Co.) with 10-fold excess unlabeled dopamine or in 0.1/zM [7,8-3H]dopamine (43,0 Ci/mM; Amersham Corp.) with 10-fold excess unlabeled serotonin to demonstrate selective uptake 16. Tissues were incubated for 180 rain, briefly washed in saline, and fixed with 4% gluteraldehyde in 0.2 M cacodylate buffer (pH 7.2) for 4 h at room temperature. Fixed tissues were post-washed in 0.2 M cacodylate buffer overnight at 2 °C, dehydrated through butanol and paraffin-embedded. Sections were cut at 5/~m, hydrated, dipped in NBT-2 (Eastman Kodak Co.), dried slowly and then stored over CO 2 in desiccation boxes at -12 °C for 3 weeks. The resultant autoradiographs were developed in D19 and lightly counter-stained with phloxin. Several preparations were held in physiological saline for 8 h after loading to assess retrograde transport into the mesothoracic ganglion.

Histofluorescence Glyoxylic acid (glyoxylate) was used to detect fluorogenic monoamines within wholemounts of the central nervous system of Rhodnius (see ref. 13). Tissues were dissected in CaZ+-free, Mg2+-rich saline and perfused with 5% glyoxylate (Mallinkrodt Co.) in 0.2 M sucrose in saline at either pH 5 or p H 7. To catalyze the second step in the glyoxylate reaction, tissues were thoroughly dried, coated with oil, and flash-heated for 2 min on copper blocks at 85 °C. Fluorescence was visualized with a Leitz dark-field fluorescence microscope equipped with KG1, BG38 and BG3 excitation filters and K49obarrier filters. Freshly

dissected preparations were compared with preparations which were incubated in 0.1/~M dopamine for 20 min prior to histofluorescence analysis. To detect monoamine-sequestering somata within the mesothoracic ganglion, tissues were incubated in 0.1 mM dopamine for comparable times 13.

Immunocytology Methods for the immunocytological identification of serotonergic processes in Rhodnius have been reported elsewhere 13. Tissues were fixed in periodate-lysine-paraformaldehyde fixative using the method of McLean and Nakane 36 for 3-6 days at 2 °C in the dark. Fixed tissues were washed in 0.1 M NaHPO4 at pH 7.2 overnight, permeabilized with 0.3% Triton in phosphate buffer overnight, and incubated with a 1:100 dilution of rabbit anti-5HT antiserum (Immuno Nuclear Co.) for 8-12 h at 2 °C. These tissues were washed in 0.3% Triton buffer for 6 h at 2 °C and then incubated with a 1:20 dilution of goat anti-rabbit IgG fluoresceine conjugate (Boehringer Mannheim Biochem.) for 2-5 h at room temperature. The preparations were washed again for 3-6 h with Triton buffer and cleared in 80% glycerol-4 mM NaHCO3. Fluorescence was examined with a Leitz dark-field fluorescence microscope equipped for histofluorescence illumination. Specificity of immunocytological staining was established by pre-adsorbing anti'5HT antiserum with 5HT-conjugated bovine serum albumin for 16 h at 4 °C and running preadsorbed antiserum in parallel with samples.

Diffusion mapping Cobalt chloride was used to back-fill representative abdominal nerve trunks 4. Freshly prepared 0.6 M CoCI 2 with 1% bovine serum albumin was allowed to diffuse into transected nerve trunks held within suction electrodes. After 8-12 h of incubation at room temperature, cobalt diffusion patterns were developed with ammonium sulfide precipitation. Tissues were dehydrated, and then cleared with methyl salicilate.

Neurochemistry Pooled samples of 15 mesothoracic ganglia were dissected into Ca2+-free, Mg2+-rich saline and homogenized in an H a - a c e t o n e (1:4) extraction bur-

237 fer. Samples were centrifuged at 10,000 rpm for 10 min and the supernatant containing the indole fraction was removed, dried under nitrogen, resuspended in 0.1 M sodium acetate, 6% methanol, and 0.23 mM ascorbic acid buffered to pH 4.7, and injected into a Bioanalytical Systems LC-50 equipped with a 'uBondapack' C-18 column 44. Standards were run in parallel with samples to qualitatively identify serotonin and serotonin metabolites. RESULTS Four pairs of thin (10-30 p m ) , abdominal nerves project towards the viscera from the fused mesothoracic ganglion (MG) in fifth instars of Rhodnius prol/xus (Fig. 1A). A fifth pair of thin, abdominal nerves branch from two large, medial nerve trunks at a variable point proximal to the second abdominal segment. These 5 abdominal nerves contain the abdominal N H O 6,33. Fig. 2. Glyoxylate-treated abdominal nerve trunk which has been loaded with dopamine to reveal monoaminergic fiber morphology.

Fig. 1. A: schematic diagram of the mesothoracic ganglion of a fifth instar Rhodnius prolixus. Nerves with neurohemal organs are indicated by brackets. B: autorad~ograph of a coronal section through the mesothoracic ganglion indicating the selective accumulation of [3H]5HTinto the abdominal nerves which contain neurohemal organs (arrows). Label is not detected within the ganglion, nor within nerve trunks which do not contain the neurohemal organs. Other abdominal nerves which took up serotonin were out of the plane of section.

When M G are incubated in 0.1/~M [3H]5HT with 10-fold excess unlabeled dopamine, label is detected autoradiographically in abdominal nerves which contain the neurohemal organ (Fig. 1B). At these concentrations, label does not penetrate the ganglionic sheath, and does not enter other nerve trunks. Tissues incubated in 0.1/~M [3H]DA with 10-fold excess unlabeled 5HT display no uptake of label. These results demonstrate a selective uptake of 5HT into the nerve trunks which contain the neurohemal organ. When freshly dissected MG are treated with glyoxylate at pH 5, a rapidly fading yellow histofluorescence (characteristic of 5HT) is seen in beaded fibers which branch throughout superficial aspects of only those nerves which contain neurohemal organs. This histofluorescence is very faint, and fades too rapidly for photographic resolution. The morphology of these fibers can be recorded photographically from ganglia which have been preincubated in 0.1/~M dopamine prior to histofluorescent analysis (Fig. 2). The yellow, rapidly fading histofluorescent fibers clearly sequester the more highly fluorogenic monoamine under these non-selective conditions. This non-selective uptake of dopamine is an expected re-

238

iI

taining cellsl3 (in addition to other monoamine-containing and monoamine-sequestering cells; Fig. 5B). The major group of serotonin-containing cells (group c in Fig. 5B) have well-defined projections within the ganglion. Primary neurites, and often finer branches, are resolved histochemically and immunocytologically (see ref. 13). The primary branches of other monoamine-containing cell groups were also visible in most preparations, with the exception of those from groups d and f (Fig. 5B). Despite the ability to detect projections within the ganglion, and the extensive staining of axons and fine processes within the neurohemal organs, serotonergic connections between the ganglionic somata and the intensely staining arborizations of the NHO were not found in any preparations. In addition, retrograde transport of [3H]serotonin taken up into the abdominal nerves is not detected in the MG even after an 8-h incubation

a

b

| 15' Fig. 3. Immunocytological staining of an abdominal nerve trunk to demonstrate the morphology of fibers which contain endogenous stores of serotonin. Preadsorbed antiserum fails to stain these nerve trunks. A: typical morphology of fibers in abdominal nerves. B: recurrent morphology of fibers in neurohemal organ and in non-neurohemal nerve trunk (see arrow). e-

sponse of a serotonergic fiber TM. Antisera raised against 5HT selectively and specifically react with beaded fibers extensively distributed throughout these same nerve trunks (Fig. 3). Serotonin-like immunoreactive fibers correspond with the histochemically identified monoamine-sequestering (and rapidly-fading, serotonin-like) fibers; they are restricted to the thin abdominal nerves, they arborize to similar extents within the neurohemal organ, and they both apparently fail to invade the mesothoracic ganglion. Serotonin-containing fibers in the abdominal neurohemal organ could arise from somata in the mesothoracic ganglion or from peripheral cell bodies. Homogenates of MG contain biochemically detectable levels of serotonin and serotonin metabolites (Fig. 4), and the MG contain immunocytologically and histochemically recognizable serotonin-con-

s~ I

1

\

0J

h

I MINUTE8

fll

i

"

i

'Ill

M~UTES

Fig. 4. High performance fiquid chromatograph of homogenates of mesothoracic ganglia indicating the preacnce of serotonin and serotonin metabolites. HVA, h o r a o v ~ a,,fid; NMS, n-methyl serotonin; 5HIAA, 5-hydroxyindolacetic acid; 5HT, 5-hydrodytryptamine; 5HTP, 5-hydroxytryptophan:

239

/

A

0

B ~4-,- a

"'il::i::

c . - . ~ o .,%

/

d

k o

el 11041 Ill.. d f-.-~o o//) C)~0 o gf/~)

h

cNL,( "

o° "*'e

,,

/,~

/

/

'~¢'

f

. '~

+::.,.

Fig. 5. A: the typical pattern of cobalt-filled somata which project into the most lateral abdominal nerve. Solid cell bodies are ventral. a, anterior set; b, dorsomedial set; c, lesser dorsomedial set; d, ventral band; e, contralateral set; f, outer ipsilateral set; g, ipsilateral set; h, deep pair; i, neurosecretory cell set. B: the distribution of mesothoracic histofluorescent monoamine-handling cells. Solid cell bodies contain endogenous stores of histochemically reactive monoamine and open cell bodies become reactive only after ganglia have been dopamine-loaded, a, flanking cells; b, intensely fluorescent cells; c, serotonin-containing, yellow cells; d, lesser satellite cells; e, dorsomedial cells; f, satellite cells13. period, during which time translocation of label to cell bodies would be expected to occur. Neurochemical and translocation mapping have failed to directly identify monoamine-handling cells which project into the N H O . Cobalt back-fill mapping methods were used to identify somata of any cell type which projects into or through the abdominal neurohemal organ (Fig. 5A). Preparations which satisfied an acceptance criterion (at least 10 cells filled, including one cell from either of two reference sets) were pooled, and cell sets were designated on the basis of cell size, position and coappearance. For example, cells in the contralateral set (see Table I) generally appear as a group of 3 , small (about 7/~m), clustered cells or f a i l to appear at all. The variability in the effectiveness of the cobalt back-fill technique in filling members of these sets is presented in Table I. Of the 9 cell groups which were found to project into the back-filled nerve, 7 showed up in more than 70% of the preparations. It is unlikely, therefore, in view of the large n u m b e r of preparations tested that major projections into the N H O remained undetected by this technique. The morphological resolution of these back-fills was often good enough to allow comparisons with histologically

identified cell types (Fig. 6). For example, the peptide-secreting cells (identified by azan-staining) which project to the N H O and are the only class of cells known to arborize extensively there, were de-

TABLE I Variability in cobalt backfill mapping of cell sets

Cells were assigned to groups based upon somata size, position and coappearance (see text). Preparations were pooled if they contained at least 10 cells and one cell from either group AS or DMS (see asterisk above). AS, anterior set; CS, contralateral set; DMS, dorsomedial set; DP, deep pair; IS, ipsilateral set; LDMS, lesser dorsomediai set; NSC, neurosecretory cells; OIS, outer ipsilateral set; VB, ventral band. Cell set

Backfill failures (n = 45)

Cell count (n) per cell set (x + S.E.M.)

Mode per set

AS* CS DMS* DP IS LDMS NSC OIS VB

27% 2% 2% 20% 47% 29% 27% 84% 4%

1.03 + 0.03 2.52 4- 0.14 1.41 + 0.09 1.75 + 0.07 1.79 + 0.17 2.94 + 0.28 2.88 + 0.23 2.00 + 0.44 6.70 4- 0.42

1 3 1 2 1 4 2 1 7

(33) (44) (44) (36) (24) (32) (33) (7) (43)

240 serotonin-containing fibers are probably the only monoamine-containing fibers which ramify within the NHO. Endogenous fluorogenic catecholamines (i.e. dopamine and noradrenaline) are not histochemically detected within the N H O , and the pattern of monoamine-sequestering fibers which load under non-selective uptake conditions (i.e. all fibers which have monoamine uptake systems) corresponds in form and extent with the pattern of fibers that contain immunoreactive serotonin. No additional monoamine-handling fibers are detectable. For these reasons, the abdominal neurohemal organ of Rhodnius may be a particularly good system in which to study the role of a monoamine in insect neuroendocrine function 14.

8

Ii

w ~,'~1~

¸

Fig. 6. A: lateral view of cobalt-filled neurosecretory cells (LC) in the mesencephalic ganglion. B: azan stain of B1 neurosecretorv cell in a parasagittal section through the same tissue area (after the method of BaudryS).

tected by cobalt back-filling in 73% of the preparations. Comparison of projection maps (based on cobalt back-fills) and monoamine-containing or sequestering cells showed only one case of overlap. A single large dorsal cell is back-filled from the most lateral abdominal nerve. This cell, one of the dorsomedial set (b in Fig. 5A), corresponds in size and position to one of a group of neurons which probably contain octopamine rather than serotonin. The cells of this group do not arborize within the N H O . DISCUSSION This is the first report of 5HT associated with an insect abdominal N H O , although previous reports have demonstrated serotonergic fibers within cephalic N H O (of locust26,31). Serotonergic innervation of this abdominal neurohemal organ is unusual because

The cellular origin of the serotonin-containing fibers in the abdominal N H O remains unknown. Immunocytochemical techniques have proved successful in tracing fine diffuse intraganglionic projection from serotonergic neurons both in this study and in the detailed analysis of Bishop and O'Shea of the central nervous system of cockroaches s. In cockroaches, projections from the central nervous system toward the periphery were only consistently traced in some regions localized to the anterior nervous systemS. In this study of Rhodnius, serotonin-containing cells in the mesothoracic ganglion ('c' in Fig. 5B) do not detectably project into the NHO. Cobalt back-fill methods label mesothoracic neurosecretory cells which do project into the N H O , and additionally stain 6 sets of cells with unidentified functions which also project into the abdominal nerves. The single cell type which both projects to the abdominal nerves with neurohemal organs and sequesters monoamine is probably not a serotonergic neuron. It is possible that additional serotonin-producing cells exist within the ganglia but fail to accumulate detectable stores of serotonin in the somata or proximal axons 2s. However, the recurrent morphology of serotonin-containing fibers in the N H O and the absence of transport of [3H]serotonin from terminals in the N H O into the ganglion argue against this suggestion. Recently, Kuster and Davey 30 have described a cell at the peripheral terminus of the N H O which contains dense core granules; however, this cell does not react detectably with the histochemical or immunocytological methods used in this study. Peripheral serotonin-containing cells in Rhodnius have not been detected during an extensive

241 survey in this study; therefore, any s o m a t a which do give rise to serotonin-containing fibers in the N H O contain u n d e t e c t a b l e somatic stores of serotonin under the conditions of this study. A n additional possibility that must be considered with regard to the origin of the serotonergic fibers in the N H O is that the m o n o a m i n e is contained in (and co-secreted by?) the terminals of peptidergic neurons. M o n o a m i n e s and p e p t i d e s a p p a r e n t l y coexist within a variety of v e r t e b r a t e neuron types t°,t1,19, 22,23,39,40,41, and in some cases the m o n o a m i n e prod-

form population 32. These terminals contain large dense cored granules indistinguishable from those found in the s o m a t a of ganglionic p e p t i d e neurosecretory cells 7. The absence of a second class of terminals which might c o r r e s p o n d to the serotonergic arborization could be explained on the basis of co-secretion of p e p t i d e and serotonin by a single cell type.

ACKNOWLEDGEMENTS

ucts are preferentially accumulated only in the peripheral aspects of these cells 21. In R h o d n i u s , a set of cephalic n e u r o s e c r e t o r y cells conditionally displays catecholamine-like histofluorescence 13, suggesting that this insect contains some neurons which accumulate both m o n o a m i n e and p e p t i d e secretory products. In the a b d o m i n a l N H O , ultrastructural studies reveal secretory terminals which a p p e a r to comprise a uni-

I would like to thank J. R e i n h a r d t and B. Beltz for assistance with high p e r f o r m a n c e liquid c h r o m a t o graphy and immunocytology, respectively. I also thank Dr. B e r t a Scharrer for helpful c o m m e n t s on an earlier draft of this manuscript. Portions of this study were funded by N S F G r a n t PCM-7680236 to A . Berlind (Wesleyan University), and grants in support of scholarship from W e s l e y a n University.

REFERENCES

vous system, Proc. nat. Acad. Sci. U.S.A., 75 (1978) 1582-1586. 12 Evans, P. D., Biogenic amines in the insect nervous system, Adv. Ins. Physiol., 15 (1980) 317-473. 13 Flanagan, T. R. J., Monoaminergic innervation in a hemipteran nervous system: a whole-mount histofluorescence survey. In N. Strausfeld (Ed.), Functional Neuroanatomy, Springer-Verlag, Heidelberg, 1983. 14 Flanagan, T. R. J. and Berlind, A., Serotonin modulation of the release of sequestered [3H]seronotin from nerve terminals in an insect neurohemal organ in vitro, Brain Research, 306 (1984) 243-250. 15 Frontali, N. and Gainer, H., Peptides in invertebrate nervous systems. In H. Gainer (Ed.), Peptides in Neurobiology, Plenum Press, NY, 1977, pp. 259-294. 16 Gaudin-Chazal, G., Segu, L., Seyfritz, N. and Puizillout, J, J., Visualization of serotonin neurons in the nodose ganglion of the cat. An autoradiographic study, Neurosci., 6 (1981) 1127-1137. 17 Gersch, M., Experimentelle untersuchungen zm freisetzungsmechanismus von neurohormonen nach elektrischer reizung der corpora cardiaca von Periplaneta americana in vitro, J. Ins. Physiol., 18 (1972) 2425-2439. 18 Gershon, M. D., Biochemistry and physiology of serotonergic transmission. In E. Kandel (Ed.), Handbook of Physiology, Section L Vol. L Amer. Physiol. Soc., Bethesda, 1977, pp. 573-605. 19 Gibbins, I. L., Amine-containing peptidergic synapses in a parasympathetic ganglion? Cell Tissue Res., 218 (1981) 87-91. 20 Goldsworthy, G. J. and Mordue, W., Neurosecretory hormones in insects, J. Endocrinol., 60 (1974) 529-558. 21 Hakanson, R., Lilja, B. and Owman, C., Properties of a new system of amine-storing cells in the gastric muscosa of the rat, Europ. J. Pharmacol., 1 (1976) 188-199.

1 Aston, R. J., Studies on the diuretic hormone of Rhodnius prolixus. Some observations on the purification and nature of the hormone and the dynamics of its release in vitro, Ins. Biochem., 9 (1979) 163-176. 2 Aston, R. J. and White, A. F., Isolation and purification of the diuretic hormone from Rhodnius prolixus J. Ins. Physiol., 20 (1977) 1673-1682. 3 Awashti, V. B., Contribution to the neurohemal organs in pterygote insects, Z. Mikrosk. Anat. Forsch. (Leipzig), 90 (1976) 48-78. 4 Bacon, J. P. and Altman, J. S., A silver intensification method for cobalt-filled neurons in wholemount preparations, Brain Research, 73 (1977) 359-363. 5 Baudry, N., Etude histologique de la neurosecretion dans la chain nerveuse ventrale de Rhodnius prolixus Stal (Hemiptere), C. R. Acad. Sci. Paris, 267D (1968) 2356-2359. 6 Baudry-Partiaoglou, N., Anatomie et histologie des organs neurohemaux de quelques hemipteres, Int. J. Ins. Morph. Embryol., 7 (1978) 1-31. 7 Berlind, A. and Maddrell, S. H. P., Changes in hormone activity of single neurosecretory cell bodies during a physiological secretion cycle, Brain Research, 161 (1979) 459-467. 8 Bishop, C. A. and O'Shea, M., Serotonin immunoreactive neurons in the central nervous system of an insect (Periplaneta americana), J. Neurobiol. , 14 (1983) 251-269. 9 Bodnaryk, R. P., Structure and function of insect peptides, Adv. Ins. Physiol., 13 (1978) 69-132. 10 Brunstock, G., Do some nerve cells release more than one transmitter? Neurosci., 1 (1976) 239-248. 11 Chan-Palay, V., Jonsson, G. and Palay, S. L., Serotonin and substance P coexist in neurons of the rat's central ner-

242 22 Hokfelt, T., Elfvin, L. G., Elde, R., Schultzberg, M., Goldstein, M. and Luft, R., Occurrence of somatostatinlike immunoreactivity in some peripheral sympathetic noradrenergic neurons, Proc. nat. Acad. Sci. U.S.A., 74 (1977) 3587-3591. 23 Hokfelt, T., Rehfeld, J. F., Skirboll, L., Ivemark, B., Goldstein, M. and Markey, K., Evidence of coexistance of dopamine and CCK in meso-limbic neurons, Nature (Lond.), 285 (1980) 476-478. 24 Hughes, L., High-molecular-weight forms of diuretic hormone from Rhodnius prolixus, Biochem. Soc. Trans., 5 (1977) 1060-1063. 25 Hughes, L., Further investigations of the isolation of diuretic hormone from Rhodnius prolixus, Insect Biochem., 9 (1979) 247-255. 26 Klemm, N. and Falck, B., Monoamines in the pars intercerebralis-corpus cardiaeum complex of locusts, Gen. comp. Endocrinol., 34 (1978) 180-192. 27 Klemm, N., Histochemistry of putative transmitter substances in the insect brain, Prog. Neurobiol., 7 (1976) 99-169. 28 Klemm, N. and Schneider, L., Selective uptake of indoleamine into nervous fibers in the brain of the desert locust, Schistocerca gregaria Forskal (Insecta). A fluorescence and electronmicroscopic investigation, Comp. Biochem. Physiol., 50C (1975) 177-182. 29 Krogh, I. M. and Normann, T. C., The corpus cardiacum neurosecretory cells of Schistocera gregaria. Electron microscopy of resting and secreting cells, Acta Zool. (Stockh.), 58 (1977) 69-78. 30 Kuster, J. E. and Davey, K. G., Fine structure of the abdominal neurosecretory organs of Rhodnius prolixus Stal, Canad. J. Zool., 59 (1980) 765-770. 31 Lafon-Cazal, M and Arluison, M., Localization of monoamines in the corpora cardiaca and the hypocerebral ganglion of locusts, Cell Tissue Res., 172 (1976) 517-527. 32 Maddrell, S. H. P., The site of release of the diuretic hormone in Rhodnius: a new neurohaemal system in insects, J. exp. Biol., 45 (1966) 499-508. 33 Maddrell, S. H. P., Functional design of the neurosecretory system controlling diuresis in Rhodnius prolixus, Amer. Zool., 16 (1976) 131-139. 34 Maddrell, S. H. P. and Gee, J. D., Potassium-induced release of the diuretic hormones of Rhodnius prolixus and

35

36

37 38

39

40

41

42

43

44

45

46

Glossina austeni: Ca dependence, time course and localizaton of neurohemal areas, J. exp. Biol., 61 (1974) 155-171. MaddreU, S. H. P. and Phillips, J. E., Regulation of absorption in insect excretory systems, Perspectives in exp. Biol., 1 (1976) 179-185. McLean, I. W. and Nakane, P. K., Periodate-Lysine-Paraformaldehyde fixative: New fixative for immunoelectron microscopy, J. Histol. Cytol., 22 (1974) 1077. Miller, T. A., Neurosecretion and the control of visceral organs in insects, Ann. Rev. Entomol., 20 (1975) t33-149. Orchard, I. and Loughton, B. G., Is octopamine a transmitter mediating hormone release in insects? J. Neurobiol., 12 (1981) 143-152. Pearse, A. G. E., The cytochemistry and ultratructure of polypeptide hormone-producing cells of the APUD series and the embryologic, physiologic and pathologic implications of the concept, J. Histochem. Cytochem., 17 (1969) 303-313. Polak, J. M., Heitz, P. and Pearse, A. G. E., Differential localization of substance P and motilin, Scand. J. Gastroent., 11, Suppl. 39 (1976) 39-42. Potter, D. D., Furshpan, E. J. and Landis, S. C., Multipletransmitter status and 'Dale's principle', J. exp. Biol.i 89 (1980) 1-9. Raabe, M., Les organes perisympathiques. In P. P. Grasse (Ed.), Traite de Zoologie, Tome VIII, fascicule 3, Masson et Cie, Paris, 1975, pp. 511-533. Raabe, M., Baudry, N., Grillot, J. P. and Provansal, A., The presympathetic organs of insects. In F. Knowles and L. Vollrath (Eds.), Neurosecretion: The Final Neuroendocrine Pathway. Springer-Verlag, New York, 1974, pp. 59-71. Reinhard, Jr., J. F., Moskowitz, M. A., Sved, A. F. and Fernstrom, J. D., A simple, sensitive and reliable assay for serotonin and 5-HIAA in brain tissue using liquid chromatography with electrochemical detection, Life Sci., 27 (1980) 905-911. Scharrer, B., Neurosecretion XIII. The ultrastructure of corpus cardiacium of the insect Leucophaea maderae, Z. Zellforsch. M ikroskop. Anat., 60 (1963) 761-796. Scharrer, B. and Scharrer, E., Neurosecretion VI. A. comparison between the intercerebralis-cardiacium-allatum system of the insects and the hypothalamo-hypophyseai system of the vertebrates, Biol. Bull., 87 (1944) 242-251.