Bulgarian Chemical Communications, Volume 41, Number 2 (pp. 104-109) 2009

* To whom all correspondence should be sent:

Received July 20, 2008; Revised September 30, 2008

The acoustic communication of Chorthippus biguttulus (Ch.b.) is a suitable behavioural model system to explore the physiological effects and underlying molecular mechanisms of identified neuropeptides. Neuropentapeptide proctolin was shown to play a modulatory role in a brain neuronal circuit that controls the acoustic, respectively sexual behaviour of males Ch.b. Proctolin receptors activation triggered courtship singing, the second level of excitation in the sexual behaviour preceded by calling singing, triggered by mAChRs activation. Pharmacological studies showed that PLC pathway is involved in courtship singing since neomycine and Li⁺ showed strong inhibitory effect on the proctolin-stimulated singing. In addition, the phorbol ester, injected in proctolin sensitive sites in the brain, elicited stridulation alone. The latter showed that PKC could mediate the effects of PLC activation. The observed results suggest possible molecular mechanisms that are involved in the decision-making brain center controlling the sexual behaviour – what (by altering the context), when (by controlling the initiation) and how long (by increasing the basal excitation) should the male sing.

Key words: neuropeptide, proctolin, PKC, acoustic communication, courtship, Chorthippus biguttulus.

INTRODUCTION

Acoustic communication in orthopteran insects has become one of the favourite subjects for investigations on the neuronal basis of invertebrate behaviour. The stereotyped stridulation patterns, the relative ease of their elicitation, and the simplicity of the neuromuscular organization, which permits electrophysiological and pharmacological work in freely-moving animals, favour both an ethological and a neurophysiological approach [1]. Proctolin (RYLPT) was the first neuropeptide to be isolated and sequenced from insects [2] and was subse-quently found to have wide distribution throughout the arthropods [3]. First proctolin has been reported [4] as a “gut factor”, which caused slow graded contractions of proctodeum longitudinal muscles in the cockroach Periplaneta americana. On the other side, a G-protein coupled receptor (encoded by CG6986) for proctolin in Drosophila melanogaster has been identified and characterized [5]. Proctolin receptor immunosignals have been found in the hindgut, heart and in distinct neuronal populations of the CNS.

Previous studies have demonstrated a role for acetylcholine (ACh) and both nicotinic acetyl-choline receptors (nAChRs) and muscarinic acetyl-choline receptors (mAChRs) in the cephalic control of the singing behaviour of various grasshopper species [6, 7]. Injections of proctolin into the proto-cerebrum can elicit species-specific stridulation in both male and female grasshoppers of the species Ch.b. The stimulated behaviour is similar to the natural stridulation with respect both to the temporal structure and patterns of stridulatory movements of the hindlegs [8]. This study presents the role of intracellular signal transduction coupled to proc-tolin-stimulated singing behaviour.

EXPERIMENTAL

Adult specimen of the gomphocerine grass-hopper Chorthippus biguttulus (L. 1758) (Ch.b.) were caught in the vicinity of Göttingen, Germany, and kept in the laboratory for up to several weeks. Additional Ch. biguttulus were reared from eggs that were collected in the previous summer and kept at 4°C for > 4 months. The nymphs hatched after

Small amounts (< 10 nl) of neuroactive sub-stances were pressure-injected via microcapillaries inserted dorsally into the brain. By the use of double-barrel electrodes two different substances were administered to the same location within the brain. Therefore, sequential injections of identical volumes of the same or of two different substances (excitatory, inhibitory or modulatory) could in principle influence the same set of neurons. To monitor the stridulatory behaviour, the hind leg movements and the produced sound were recorded with two opto-electronic cameras and a microphone. The animal is attached to a holder. The head is opened and the brain is exposed. The micro-capillary is attached to the mechanic micromani-pulator and is connected to the pressure-injection device through pressure resistant tubes. A three-way stopcock allows the pressed air to flow in two directions, respectively into one of the two barrels of the micro-capillary. The pump is connected to a system that continuously supplies compressed air. Two optic-electronic devices according to von Helversen [7] record the stridulatory movements of the hind legs and transform them into a voltage signal. The latter is amplified and visualized on the computer screen. The signal is stored in data format (*.dat) by the program Turbo Lab 4.2 or 4.3 for DOS.

The neuroactive substances were usually dis-solved in grasshopper saline [9] to give concen-trations of 1 mM. Muscarine, proctolin, neomycin obtained from Sigma-Aldrich; SQ 22536 obtained from Calbiochem; phorbol-12-myristate-13-acetate (PhE, porbol ester), thapsigargin, TMB-8, ryano-dine, LiCl, purchased from Sigma-Aldrich were studied using the upper-described basic scheme. Stock solutions of proctolin (10 mM concentration in d H₂O) were preserved at –20°C. Water-soluble substances were dissolved in grasshopper saline to give concentrations of 1 to 0.1 mM. Different ions (Ba²⁺, Ni²⁺) were added to the saline to give 1 to 5 mM. Substances soluble in DMSO (thapsigargin, TMB-8, ryanodyne, obtained from Sigma-Aldrich) were dissolved in saline to give a 5% final content of DMSO. All solutions were preserved at –4°C, not longer than 1 day.

St. R. Vezenkov et al.: The role of brain neuropentapeptide proctolin in acoustic communication

RESULTS AND DISCUSSION

Injections of proctolin into the frontal part of the central complex and an adjacent neuropil anterior and dorsal to it elicited a species-specific singing behaviour in males of Ch.b (Fig. 1). Both proctolin and muscarine stimulated stridulation in a region that includes the anterior portion of the central body and the area between the central body and Proto-cerebral Bridge. However, the responses to both drugs differed in latency, duration (Fig. 2), also in internal time courses of the hind legs movements. The temporal structure of muscarine- and proctolin-stimulated stridulation were likely to respectively calling song and courtship song which correlate to two different excitation levels [8]. The proctolin-elicited singing might be the second level of excitation because the courtship singing in the natural sexual behaviour appears after the calling singing. The male produces courtship song when duets together with the responding female that shows copulatory readiness. Thus, proctolin might trigger the courtship singing of the male, respectively the higher, behavioural different, level of excitation in comparison to muscarine-stimulated calling singing.

St. R. Vezenkov et al.: The role of brain neuropentapeptide proctolin in acoustic communication

The robust appearance of the inhibitory effect of neomycin and Li⁺ on proctolin-stimulated singing behaviour suggested a main role for the PLC pathway mediating proctolinergic excitation in the brain of Ch.b.

Diacylglycerol (DAG) is a second messenger generated through PLC signaling and activates the proteinkinase C which phosphorilates proteins, ion channels, receptors, etc. and alters the neuronal activity. In various insect preparations, DAG has been shown to contribute to proctolin receptor-triggered excitation [11, 12].

Fig. 1. Proctolin, injected inbetween the central body and Protocerebral Bridge of the brain, alone elicits

courtship singing in male Ch.b. (RHL – right hindleg).

Fig. 2. Comparison between proctolin- and muscarine-stimulated stridulation induced at the same spots in the protocerebrum of males Ch.b. A. The latency of proctolin-induced stridulation was shorter (P < 0.05);

B. The duration of the proctolin produced singing was much shorter than muscarinic (P < 0.01).

St. R. Vezenkov et al.: The role of brain neuropentapeptide proctolin in acoustic communication

Fig. 3. Effects of the PLC inhibitors neomycin (A) and Li⁺ (B) on proctolin-stimulated singing (males, Ch.b.). A. Two phases of reversible inhibition were observed: the first one from 4^th to 8^th min and the second – 12^th to 20^th min (n = 10, P < 0.05); B. Li⁺ co-injected with proctolin reduced both S and C Duration of proctolin-induced singing (n = 9, P<0.05).

Fig. 4. Male singing (Ch.b.) induced through proctolin (A) and phorbol-12,13-dibutirate (phorbol ester, PhE) (B),

injected at the same spot in the brain.

Fig. 5. S and D durations of proctolin- and phorbol ester-induced singing (males, Ch.b., n = 4).

A. 0, 3, 6 min and 18, 21, 24 min – responses to proctolin; 9, 12, 15 min – responses to PhE;
B. Average values of the S and D durations (P < 0.05).