Biogenic amines in the nervous system of the cockroach, Periplaneta americana: Association of octopamine with mushroom bodies and dorsal unpaired median (DUM) neurones

Insect Biochem., Vol. 9, pp. 535 to 545. © Pergamon Press Ltd. 1979. Printed in Great Britain.

0020-1700/79/0901-0535

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BIOGENIC AMINES IN THE NERVOUS SYSTEM OF THE COCKROACH, PERIPLANETA AMERICANA: ASSOCIATION OF OCTOPAMINE WITH MUSHROOM BODIES AND DORSAL UNPAIRED MEDIAN (DUM) NEURONES GILLIAN R. DYMOND* and PETER D. EVANS A. R. C. Unit of Invertebrate Chemistry and Physiology, Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, U.K. (Received 7 March 1979)

Abstract--The distribution of dopamine and noradrenaline in the CNS of the cockroach Periplaneta americana is examined using a radioenzymatic assay. Histochemical techniques for amine localisation (neutral red staining and histochemical fluorescence) have also been applied to the cockroach CNS. The results suggest that both the dorsal unpaired median (DUM) neurones in the thoracic and abdominal ganglia and the globuli cells of the mushroom bodies of the cockroach cerebral ganglia are octopaminergic. These results are confirmed by radioenzymatic assays for octopamine on microdissected somata. Key Word Index: Biogenic amines, dopamine, noradrenaline, octopamine, radioenzymatic assay, Periplaneta americana, nervous system, dorsal unpaired medial (DUM) neurones, mushroom bodies

INTRODUCTION

BIOGENICamines may function as neuromodulators (local neurohormones) or neurotransmitters in the insect nervous system. Octopamine is widely distributed in the nervous systems of cockroaches and locusts (ROBERTSON and STEELE, '1974; ROBERTSON, 1976; EVANS, 1978a), and has been shown to be associated specifically with an identified neurone of the dorsal median neurosecretory group in the metathoracic ganglion of the locust (EVANS and O'SHEA, 1977, 1978). This cell modulates the neuromuscular activity of the identified slow motoneurone to the extensor muscle of the tibia of the hindleg. It increases the height of twitch tension, the rate of relaxation of tension and the size of the excitatory junctional potentials produced in the extensor muscle fibres by stimulation of the slow motoneurone. All these effects can also be mimicked by superfusing low concentrations of octopamine over the muscle (EVANS and O'SHEA, 1977; O'SHEA and EVANS, 1979). Dopamine, another biogenic amine present in large quantities in insect nervous tissue (ROBERTSON, 1976), has been proposed as a neurotransmitter in the salivary glands of the cockroach (BLAND et al., 1973; HOUSE et al., 1973; FRY et al., 1974) and of the moth M a n d u c a s e x t a (ROBERTSON, 1975). The regional synthesis ofbiogenic amines has also been examined in the CNS of M a n d u c a s e x t a (MAXWELL et al., 1978). In this paper the distribution of dopamine and noradrenaline in the CNS of Periplaneta americana is examined using a radioenzymatic assay. The neutral * Present address: Department of Biochemistry, University Hospital and Medical School, Clifton Boulevard, Nottingham NG7 2UH, England. 535

red staining technique for the localisation of biogenic amine containing cells (STUART et al., 1974) has been applied to cockroach nervous tissue, together with the histochemical fluorescence technique for the demonstration ofcatecholamines. The results of these combined studies suggest that octopaminergic neurones are located in specific regions of the cockroach nervous system and this is confirmed by radioenzymatic assays for octopamine on microdissected somata. The results are discussed in relation to possible functional roles for biogenic amines in the cockroach nervous system.

MATERIALS AND M E T H O D S Cockroaches (Periplaneta americana) were reared at 25°C on a 12 hr light-dark cycle. They were fed on a diet of crushed dog biscuits and water ad lib. Measurements were made on adult male insects dissected during the first quarter of the light period. (1) Assays (a) Catecholamines. Tissues were dissected under saline (EVANS, 1978a), weighed and transferred to microtubes containing 21)--40/A of 0.1 M perchloric acid with 2.7 mM ethylenediaminetetraacetic acid disodium salt. Samples were alternately frozen and thawed three times and sonicated to disrupt the tissues, and a clear supernatant obtained by centrifugation (2000 g for 5 min at room temp.). The method used for the determination of dopamine and noradrenaline is a modification of the radioenzymatic assay of CUELLOet al. (1973). It is based on the 3-0-methylation of dopamine and noradrenaline by the mammalian enzyme catechol-0-methyl transferase in the presence of [3H-methylS-adenosyl-L-methionine (8.9 Ci/mmol, Amersham Radiochemical Centre, England) as a [3H]-methyl donor. Modifications to the original assay (see DYMOND, 1978) include the use of small reaction volumes (50 pl), the interpolation of a borate buffer wash after the initial organic

536

GILLIAN R. DYMOND AND PETER D. EVANS

extraction of the assay products, and the drying down under vacuum and reconstitution of the samples, all of which procedures reduced the blank values for the assay. The use of thin layer chromatography (TLC) to separate the reaction products reduced the time taken to perform the assay and facilitated the running of large numbers of samples. The TLC solvent systems used were either 17~/oaqueous methylamine/ tertiary amyl alcohol, 1:4 (v/v) (AM/TAA) on cellulose plates (Camlab, CEL 300) or chloroform/ethanol/70~o aqueous ethylamine 16:3:2, (by vol.) (Ch/E/E) on silica plates (Camlab, SIL G). N-Acetyldopamine, a major metabolite of dopamine in insect tissues, will also react to give a labelled methylated product under the assay conditions described above. The latter product is not resolved completely from 3methoxytyramine (the 0-methylated derivative of dopamine) using TLC with Ch/E/E but resolution is almost complete when the AM/TAA system is used. The excellent correlation between the dopamine and noradrenaline values obtained from the two solvent systems suggests, however, that the endogenous level of N-acetyldopamine in cockroach nervous tissue is too low to interfere in the assay. The use of the Ch/E/E system also resolves the O-methylated derivative of adrenaline from those of dopamine and noradrenaline. The modified assay resulted in a twice background sensitivity of 30--40 pg for dopamine and noradrenaline. (b) Octopamine. Tissues for analysis were dissected in saline (EVANS, 1978a) cleaned of adhering fat body, blotted and then transferred to microtubes containing 25 pl 0.01 M

formic acid. Groups of isolated somata were transferred to microtubes using a glass micropipette (EVANS and O'SrrEA, 1978). In each case the number of somata was noted. Tissues were alternately frozen and thawed at least three times in this solution prior to analysis. The method used for the assay of octopamine is based on the procedure introduced by MOLINOFF et al. (1969) as modified by EVANS,(1978a). It resulted in a twice background sensitivity of 10-15 pg for DL-OCtopamine. The authenticity of the assay products formed was checked by chromatography in four different solvent systems for each tissue and cell type analysed (see EVANS, 1978a).

(2) Histochemical techniques (a) Neutral red staining. This technique was used to locate amine-containing cells (STUARX et al., 1974). Tissues were dissected free from the animal and stained intact in freshly prepared solutions of neutral red (0.01 mg/ml) in cockroach saline. The solution was filtered (Whatman No. 1) immediately before use and tissues were incubated for 3 hr at room temperature (approx 20°C) or overnight at 4°C. (b) Catecholamine localisation. The Falck-Hillarp technique (see FALCK and OWMAN, 1965) for the demonstration of catecholamines and indolylalkylamines was applied to cockroach nervous tissues.

Table 1. Dopamine and noradrenaline content of cockroach central nervous tissue

Nervous tissue

Neurohaemal tissue

Cerebral ganglion Optic lobes (per pair) Suboesophageal ganglion Corpora cardiaca (per pair) Corpora allata (per pair) Whole thoracic nerve cord Prothoracic ganglion Mesothoracic ganglion Metathoracic ganglion Whole abdominal nerve cord Abdominal ganglion 1 2

Mean tissue Dopamine (n) Noradrenaline (n) Octopamine Ratio Ratio weight (mg) pmole-+ S.E.M. pmole 4- S.E.M. pmole~[ DA/NA DA/OCT 1.73-+0.07 26.81 -+1.03 (29) 5.53-+0.42 (11) 0.38-+0.06 4.41 -+0.24 (20) 0.36-+0.11" (6)

14.68 7.24

4.84 6.69

1.83 0.33

0.53-+0.11

3.29-+0.27 (20)

0.33-+0.10' (6)

5.60

9.97

0.59

--

0.19_+0.06 (8)

0.11 -+0.02¢ (4)

1.20

1.73

0.16

--

0.02-+0.01 (5)

(4)

0.03

--

0.67

3.80-+0.28

8.27-+0.90 (16)

0.74+0.06 2.61-+0.29 (11)

0.29--+0.08* (4)

5.28

9.00

0.49

0.94-+0.08 2.25-+0.21 (10)

0.23-+0.08* (3)

4.33

9.78

0.52

0.98-+0.08 2.14_+0.33 (13)

0.16-+0.06" (3)

5.32

13.38

0.40

(4) (5) (4) (4) (4) (3)

1.56 1.56 1.56 1.56 1.56 5.26

4.87 4.33 5.67 4.71 6.86 30.60

0.72 0.67 0.65 0.72 0.92 0.29

0.14+0.07:~ (3)

0.07

2.36

4.71

n.d.t

2.25-+0.20 6.69-+0.82 (16) 1.12-+0.21 (4) 1.04-+0.11 (4) 1.02-+0.10

3

--

4

--

1.13_+0.00

5

--

1.44-+0.22

0.53-+0.08

1.53-+0.19

6 Abdominal medial neurohaemal organs (per organ)

0.23+0.10§ 0.24+0.09§ 0.18+0.06§ (3) (3) 0.24+0.08§ 0.21 +0.02§ (3) (11) 0.05+0.02§

---

0.33+0.09 (3)

* Mean NA and DA per piece of tissue + S.E.M. obtained from assays on several pools each containing two pieces of tissue. t Each pool contained ten pairs of glands. Each pool contained twenty to thirty organs. §Each pool contained four pieces of tissue. - - N o t determined, n.d. not detectable. Mean tissue weights ±S.E.M. were obtained from four to eight pieces of tissue. (] Octopamine values reproduced from EVANS, 1978a.

537

B

A t

IIII

..

G

i=

F

Fig. 1. Light micrographs of dorsal surfaces of cockroach thoracic (A-D) and abdominal (E and F) ganglia showing organization of the dorsal median cell group as revealed by neutral red staining. A--prothoracic ganglion; B--mesothoracic ganglion. C and D--two examples of metathoracic ganglia to show variable organization of cells; E--a typical abdominal ganglion; F--sixth abdominal ganglion showing several groups of cells indicating fused segmental origin of this ganglion. In each case the anterior edge of the ganglion is towards the top of the figure. Scale bars 100 ltm.

538

A

"C

S

B

r

C

Fig. 2. Light micrographs of regions of cockroach cerebral ganglion as revealed by neutral red staining. (A) Whole cerebral ganglion viewed from the rear. Note the intense staining of the mushroom bodies (m). S = suboesophageal ganglion; C = circumoesophageal connective. Scale 500/~m. (B) Isolated mushroom body from right half of ganglion showing double calyx viewed from above. Anterior is towards top of picture. (C) Isolated mushroom body from left half of ganglion viewed from the side; P = pedunculus; a = or-lobe and b = /%lobe. Anterior is towards the right hand side of the picture. (D) Cross section through calyx region of mushroom body from right half of ganglion. Note the intense staining of the globuli cell layer (g) on the surface of the calyces (c). Scale for B, C and D is 100 ~m.

539

A

0

J

Fig. 3. Light micrographs of the dorsal surface of the cockroach sixth abdominal ganglion, after processing by the formaldehyde histochemical method for localizing monoamines. (A) Ganglion showing specific catecholamine fluorescence in the neuropile region. Cell bodies in dorsal midline do not fluoresce; sheath autofluorescence is visible at the periphery of the ganglion. (B) Control ganglion, processed without exposure to formaldehyde vapour, only shows non-specific autofluorescence associated with the sheath. (C) Dorsal midline region of a ganglion at higher magnification showing absence of specific fluorescence in dorsal unpaired median or DUM cell somata. The scale bars represent I00 ~m.

Biogenic amines in cockroach CNS RESULTS (1) Biogenic amine distribution The distribution of the catecholamines dopamine and noradrenaline, in the nervous system of the cockroach P. americana is given in Table 1. No adrenaline could be detected in any of the samples analysed. The ratio of dopamine to octopamine is calculated using the values reported previously for octopamine (EVANS, 1978a). It can be seen there is from four to thirteen times as much dopamine as noradrenaline present in most regions of the cockroach nerve cord. In the sixth abdominal ganglion the ratio rises to 30:1. In neurohaemal tissue such as the corpora cardiaca and the abdominal medial neurohaemal organs the ratio is only 2:1. There appears to be more octopamine than dopamine in most areas of the cockroach nervous system. Exceptions are the cerebral ganglion and the abdominal medial neurohaemal organs where the converse is true. There is a considerable difference in the ratio of dopamine to octopamine found in the two neurohaemal tissues studied; the corpora cardiaca having much more octopamine than dopamine compared with the abdominal medial neurohaemal organs. The sixth abdominal ganglion differs from the other ganglia in the abdominal nerve cord in that it has over three times as much octopamine as dopamine, whereas the other ganglia have only about one and a half times as much. (2) Neutral red staining The most notable staining feature of the thoracic and abdominal ganglia of the cockroach nerve cord is the presence of a median group of cells on the dorsal surface of each ganglion (Fig. I A - F ) . These cells in the cockroach correspond presumably to the dorsal unpaired median or D U M neurones of the locust nervous system (CRoSSMAN et al., 1971) which also stain selectively with neutral red (EVANS and O'SrmA, 1977, 1978). In the pro- and meso-thoracic ganglia of the cockroach the cells lie towards the posterior end of the ganglia between the insertions of the connectives (Fig. 1A and B). In both these ganglia a variable number (from five to eight) of cells stained in different preparations. In the metathoracic ganglion a group of eight cells invariably stained in the centre of the dorsal surface and a variable number (from one to four) of cells were stained in a more posterior position in the dorsal midline (Fig. 1C and D). The arrangements of the cells within the central grouping of eight was again variable as had been observed for the corresponding cells in the locust metathoracic ganglion (EVANS ~nd O'SrmA, 1978). In the abdominal ganglia (Fig. 1E) a group of large cells (from two to four) stain in ~he posterior midline together with a variable number of smaller cells in the anterior midline. The presence of several groups of stained cells (Fig. IF; three groups of from eighteen to twenty-five cells) on the dorsal surface of the sixth abdominal ganglion presumably reflects the multisegmental origin of this fgsed ganglionic mass. In the cerebral ganglia (Fig. 2), intense staining with neutral red is seen in the mushroom bodies or corpora pedunculata. In the cockroach each mushroom body

541

has a pair of calyces attached to a common stalk or pedunculus (WEIss, 1974). It can be seen from cross sections of the calyces (Fig. 2D) that the most intense staining is found in the globuli cell body layer around the neuropile of the calyx. The increased thickness of the globuli cell layer within the centre, of the cupshaped calyces is responsible for the double banded appearance of each mushroom body in the intact ganglion after neutral red staining (Fig. 2A). Several large fibres are well stained in the pedunculus of the mushroom body and they can be seen to bifurcate at its base and to send branches into the ~t-and E-lobes (Fig. 2C). (3) Catecholamine histofluorescence The application of the Falck-Hillarp technique to the sixth abdominal ganglion of the cockroach nerve cord did not reveal the presence of any fluorescent cell bodies. The only specific fluorescence detected occurred in areas of neuropile. Figure 3A shows a dorsal view of a sixth abdominal ganglion indicating an intense fluorescence in the neuropile areas. The dorsal median neurones can be seen outlined as nonfluorescent somata (Fig. 3A and C). The same view of an untreated control ganglion is shown in Fig. 3B, where only autofluorescence of the sheath is visible. (4) Octopamine assays The dorsal median neurones of the cockroach nerve cord, although staining selectively with neutral red which indicated their possible aminergic nature, did not fluoresce with the Falck-Hillarp technique for catecholamines. This is similar to the finding for D U M cells in the locust metathoracic ganglion (HoYLE, 1975; EVANS and O'SrmA, 1977, 1978). There are several possible explanations for this observation. It could be that the cell bodies in these systems do not contain enough catecholamines for detection by the Falck-Hillarp technique. Alternatively as has been shown for D U M cells in the locust, the corresponding cells in the cockroach may contain a non-fluorogenic amine such as octopamine.

Table 2. (A) Octopamine content of isolated dorsal, median somata pmole/soma+ S.E.M. (n) Sixth abdominal ganglion Metathoracic ganglion

0.085+0.014 0.140+0.038

16" 6t

(B) Octopamine content of cockroach nervous tissue

Globuli cells ~ + Calyx Globuli cells (from one calyx) Calyx Antennal lobe Tritocerebrum (per half brain)

pmole + S.E.M.

(n)

2.15+1.07

10

1.65 + 0.37 1.16+0.19 3.11 +0.50 3.73 ___0.62

6 6 10 10

* Estimated in groups containing from five to thirty somata. t Estimated in groups containing from seven to ten somata.

542

GILLIAN R. DYMOND AND PETER D. EVANS

To test this latter possibility groups of unidentified somata of dorsal median neurones were dissected from the metathoracic and sixth abdominal ganglia of the cockroach and assayed for their octopamine content. The results are presented in Table 2A. Cell bodies from both regions contain significant amounts of octopamine. The average amount estimated per soma from the metathoracic ganglion was 0.14 pmole, and from the sixth abdominal ganglion, 0.09 pmole. These values are very similar to those obtained for corresponding DUM cell bodies in the locust (EVANS and O'SHEA, 1978). No octopamine could be detected in non-neutral red staining samples of control somata. These included equivalent tissue samples taken from regions of the dorsal surface of the ganglia, lateral to the DUM somata and also pools of unidentified nonneutral red staining cells from the ventral surface of the ganglia. In the cockroach cerebral ganglia, the somata of the globuli cells stained intensely with neutral red, but are reported to be non-fluorescent in the Falck-Hillarp technique in many studies (see KLEMM, 1976 for refs). Again this suggested the possible presence of octopamine, and globuli cell somata were isolated and analysed accordingly. The results of this are presented in Table 2B. Octopamine can be detected both in the neuropile regions of the calyces and in samples of isolated globuli cell somata. This suggests that the reason the globuli cells do not fluoresce in the Falck-Hillarp technique is that they contain octopamine but no, or only low levels, of catecholamines. The presence of octopamine in the neuropile regions of the calyx is presumably due to the fact that the intrinsic fibres of this neuropile area arise from the globuli cells. Octopamine values for other regions of cockroach cerebral ganglia are also included in Table 2B for comparison. Large amounts of octopamine were found in the antennal lobes and tritocerebra but the cellular location of this octopamine has not yet been resolved.

DISCUSSION The results of the present study taken in conjunction with those of EVANS (1978a) indicate that biogenic amines are widely distributed in the cockroach nervous system. In general, the levels of octopamine are higher than those of dopamine, which in turn are higher than those of noradrenaline. An exception is the cerebral ganglion where more dopamine is present than octopamine. In S. gregaria ROaEgTSON (1976) found that the optic lobes had about six times more octopamine than dopamine but that the brain minus optic lobes (equivalent to the cerebral ganglion in present study) had roughly equal amounts of octopamine and dopamine. The values obtained in the present study by radioenzymatic assay of cockroach CNS tissues agree well with those found by fluorimetric determination for both dopamine (FRONTALIand H,g,GGENDAL,1969; KuscH, 1975) and noradrenaline (FRONTALI and H.~GGENDAL, 1969). The use of TLC using the solvent system Ch/E/E which separates the O-methylated products of dopamine, noradrenaline and adrenaline demonstrates an apparent lack of adrenaline in the

cockroach nervous system which is in agreement with previous studies on nervous tissue from a variety of insect species (FRONTALI and H~,GGENDAL, 1969; BJORKLUND et al., 1970; KLEMM and BJSRKLUND, 1971; HIRIVIand ROZSA, 1973; KLEMMand AXELSSON, 1973). Biogeiiie amines and neurohaemal tissue

The association of biogenic amines with neurohaemal tissue has been noted previously (KLEMM, 1976; EVANS, 1978a) but the function is unknown. A similar association of amines with a neurohaemal structure is found in the lobster. Here the pericardial organs contain octopamine (EvAr~S et al., 1976a,b) as well as dopamine and 5-hydroxytryptamine (COOKEand GOLDSTONE,1970), but again the functions of the endogenous amines are unknown. It is possible that in both insects and crustaceans, amines serve as releasing factors for peptide hormones, as has been suggested to be the case in several vertebrate neurosecretory systems (MACLEODand LEH~mYER,1974; BRIDGESet al., 1976). Alternatively the amines might accumulate in these neurohaemal organs as the most efficacious sites for their release into the blood system as circulatory neurohormones (EVANS, 1978a). In the corpora cardiaca there is more octopamine present than dopamine but the converse is true of the median neurohaemal organs of the abdominal nerve cord. The latter organs have also been shown to accumulate radioactively-labelled dopamine, as have several groups of neurones in the midline of cockroach abdominal ganglia although the specificity of the accumulation process in unknown (SMALLEY, 1970). Since octopamine, dopamine and noradrenaline are present in these organs (EVANS, 1978a) and the specificity of the high-affinity uptake systems is quite broad for biogenic amines in cockroach abdominal nerve cord (EvANs, 1978b; DYMOND, 1978) it is not yet possible to identify the cellular locations of each amine. Biogenic amines and the sixth abdominal ganglion The terminal abdominal ganglion of the cockroach is much larger than the other abdominal ganglia; it is produced by the fusion of several ganglia from the last abdominal segments. This ganglion provided one of the earliest preparations for the study of synaptic transmission (PUMPHREYand RAWDON-SMITH,1937). Much of the work, dealing with the transfer of coded information from the cereal afferent fibres to the giant interneurones of the nerve cord in the terminal abdominal ganglion of the cockroach has been reviewed by CALLEC (1974). It seems likely that acetylcholine is the cereal-giant transmitter on the grounds that cercal-giant transmission is more strongly affected by nicotinic cholinerglc ligands than by any other class of pharmacological agents (CALLEC, 1974; SArTELLE, GEPr~R and HALL, in preparation). In this ganglion, neurosecretory terminals have been suggested to be closely associated with the cereal giant fibre synapses, usually adjacent to the cereal sensory endings in the cockroach (FARLEY arid MILBURN, 1969) and in a close association with the medial giant interneurone dendrite in the cricket

Biogenic amines in cockroach CNS Acheta domesticus (O'SI-mAand MURPHEY,1978). The neurosecretory endings contain granules of about 100 nm in diameter, and have been suggested to contain biogenic amines 'such as catecholamines or indoles' (FARLEY and MmBURN, 1969). Degeneration studies suggested that the neurosecretory processes belonged to intraganglionic interneurones, which FARLEYand MILBURN (1969) proposed might 'regulate in some manner the transmission at the cercal-giant fibre synapse'. Recently, similar neurosecretory terminals have been reported to be associated with the processes of giant fibres in the metathoracic ganglion of the cockroach (CASTELet al., 1976). It is interesting to note that a sympathetic adrenergic enhancement of peripheral sensory responsiveness has been demonstrated in the frog (Rana pipiens and R. clamitans) and suggested to be part of a general behavioural arousal system (CHERNETSKI,1964). The dorsal medial neurones of the cockroach sixth abdominal ganglion are selectively stained with the dye neutral red, indicating their possible aminergic nature, and isolated cell bodies from the group have been shown to contain octopamine. A similar selective staining with neutral red has been reported for the corresponding cells in the terminal ganglion of the cricket (O'SrrEA and MURPI-mY, 1978). It has been known for some time that the morphological properties (SEABROOK,1968, 1970) and physiological properties (JEGoet al., 1970; CROSSMANet al., 1971) of the dorsal median neurones of the terminal abdominal ganglia of cockroaches and locusts are similar to those described for the corresponding cells in the metathoracic ganglia (PLOTNIKOVA,1969; CROSSMAN, et al., 1971; EVANSand O'SHEA, 1977, 1978; HOYLE, 1978). Anatomically the terminals of the neurosecretory intraganglionic interneurones described by FARLEYand MILBURN(1969) have many similarities with the octopamine containing neurosecretory terminals in locusts (HOYLE et al., 1974) and lobsters (EVANS et al., 1975; 1976b; SCHAEFFERet al., 1978). This suggests that the former cells might represent the neuropile processes of the octopamine-containing dorsal median neurones in the cockroach sixth abdominal ganglion. Early pharmacological studies on the Cockroach sixth abdominal ganglion (TwARtm and ROEOER, 1957; HODGSON and WRIGHT, 1963) noted the sensitivity of the cercal afferent giant fibre synapse to high concentrations (10 - a_ 10- 4M) of catecholamines such as adrenaline and noradrenaline (TwARoG and ROEDER, 1957). Phenylephrine (neosynephrine or m-synephrine) was tested by HODGSONand WRIGHT (1963) but found to be ineffective. They assumed that the system was activated preferentially by catecholamines and that adrenaline itself was probably the neurologically active substance in insects. However, more recent studies have failed to substantiate the original identification of adrenaline (OSTLUND,1953) in insect nervous tissue. The present study indicates that the sixth abdominal ganglion of the cockroach contains a higher ratio of octopamine to dopamine than any other region of the nerve cord (except for the corpora cardiaca). Unfortunately the early pharmacological studies mentioned above did not test octopamine or p-synephrine which has been shown to be a potent agonist of several other insect I.B. 9/5

H

543

octopamine receptors (EVANS and O'SHEA, 1978; O'SHEA and EVANS, 1979). O'SHEA and MUm'HEY (1978) have recently presented preliminary evidence for the sensitising action of octopamine on the response to sound of the medial giant interneurones in the cricket terminal abdominal ganglion. This effect was blocked by the ct-adrenergic blocking agent phentolamine, as are other effects of octopamine in insects (EVANS and O'SHEA, 1978; O'SHEA and EVANS, 1979). In the terminal abdominal ganglion of P. americana DYMOND(1978) has demonstrated that octopamine (in concentration range 10-a-10-*M) is the most potent of several amines tested, in causing 'ganglionic depolarization' and blocking of the polysynaptic excitatory potentials at the cercal afferent/giant fibre synapses, as measured by the mannitol-gap recording technique (CALLECand SATTELLE,1973). It is thus tempting to speculate that exogenously applied octopamine mimics the action of octopamine released from the terminals of the dorso-medial neurones in this ganglion. Direct evidence for this hypothesis will require studies on physiologically identified individual dorsal median neurones in the terminal abdominal ganglion. Biogenic amines and the corpora pedunculata The distribution of monamine-containing fibres in the corpora pedunculata (or mushroom bodies) of the insect brain varies from species to species (KLEMM, 1976). Histofluorescent studies reveal the presence of catecholamine-containing fibres in the ~t-and fl-lobes of the mushroom bodies, and they are frequently arranged in zones of different fluorescent intensity, as in P. americana (FRONTALI,1968). The location of the cell bodies of these fibres are unknown. The association of biogenic amines with the globuli cells of the calyx which give rise to the intrinsic fibres of the mushroom bodies seems to be a matter of controversy (KLEMM, 1976). Electron microscopical evidence demonstrating the presence of dense cored vesicles in the intrinsic fibres of the mushroom bodies of Acheta domestica (SCHORMANNand KLEMM, 1973) and P. americana (MANCINIand FRONTALI,1970) has been taken as evidence for their aminergic nature. However, histofluorescent techniques in general have not been able to detect any monamines in the globuli cell bodies or in the calyx itself. In S. gregaria the globuli cell bodies always remain non-fluorescent even after in vitro treatment with highly fluorogenic compounds such as ~t-methyl-noradrenaline, 6hydroxytryptamine and L-DOPA suggesting that these cell bodies lack uptake systems for biogenic amines (KLEMM, 1976). Our evidence suggests that the globuli cells are octopaminergic. The globuli cells of P. americana, and S. americana gregaria (EVANS, unpublished) are specifically stained with the dye neutral red. This dye selectively stains amine-containing cells in the leech Hirudo medicinalis (STuART et aL, 1974) and lobster Homarus americanus (WALLACEe t al., 1974; EVANS et al., 1976a). Furthermore, micro-dissected samples of globuli cell bodies, and the calyx itself, have been shown to contain considerable quantities of octopamine. Octopamine is a non-fluorogenic amine in the

544

GILLIAN R. DYMOND AND PETER D. EVANS

Falck-Hillarp technique. It might be expected that the highly fluorogenic compounds mentioned above would be taken up into octoaminergic neurons, as may occur in certain regions of the medulla of the optic lobe of S. gregaria (KLEMM, 1976). However in the latter case the uptake is into areas of neuropile containing axons and it is possible that somata containing octopamine do not possess as many uptake sites as their corresponding axons. The proposed octopaminergic nature of the globuli cells is also consistent with the presence of large dense-cored vesicles in the intrinsic fibres of the m u s h r o o m body since octopamine has also been suggested to be contained in such vesicles (HoYLE and BARKER, 1975; HOVEL et al., 1974; EVANS et al., 1975, 1976a; SCHAEFF~R et al., 1978). The localization of octopamine in globuli cell clusters provides an explanation for the earlier apparently contradictory findings on the aminergic nature of these cells. It is not clear at present if octopamine is evenly distributed between all the globuli cells or confined to a specific subpopulation. The functional role of the intrinsic aminergic fibres of the mushroom body remains a matter of speculation as does that of the m u s h r o o m body itself. In P. americana the m u s h r o o m bodies have been suggested to represent second order antennal sensory processing centres (WEISS, 1974). They contain highly ordered regions of neuropile where the intrinsic fibres from the globuli cells proceed along the pedunculus to the ~-and fl-lobes in parallel bundles which undergo torsion (PEARSON, 1971). The bundles appear to be arranged in a regular relationship to their cell bodies (STRAUSFELD,1970; 1976; HOWSE, 1974, 1975). Along their length the intrinsic fibres synapse with each other and with extrinsic fibres (MANCINI and FRONTALI, 1970). The major input of extrinsic fibres has been reported to come from the antennal lobe in certain insects (WEISS, 1974; STRAUSFELD, 1976). However, recent electro-physiological studies on the m u s h r o o m bodies of the bee, Apis mellifera carnica, reveal that a high percentage of the units recorded give multimodal responses to light, scent, taste and mechanical stimulation (of the antennae) (SuzuKI, et al., 1976; ERBER and MENZEL, 1977; ERBER, 1978). Thus the m u s h r o o m bodies cannot be regarded exclusively as second order integration centres for olfactory input. They should also be considered as association centres where one sensory modality can modulate the response characteristics of a particular neurone to an input from a second modality (ERBER and MENZEL, 1977). The role played by biogenic amines in such an association mechanism requires further investigation. Acknowledgements--We wish to thank Drs. D. B. SATTELLEand M. V. S. SIEGLERfor their critical reading of the manuscript. GI~D was financially supported by a C.A.S.E. award sponsored jointly by the S. R. C. and ICI Plant Protection Limited.

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