The role of AChE activity on proctolin-stimulated stridulation

neurotransmitters, neuromodulators and various neuroactive substances used in pharmacological investigations. The power and the character of the modulation depend strongly on the concentration of the substances. Opposite effects of par-ticular substances on AChE activity at high and low concentrations have been de-scribed. Usually substances that alter the activity of AChE also influence the activ-ity of nAChRs (open time, conductance, open probabilactiv-ity nAChR, inactivation or desensitization). A well-known example for such complex effect on ACh transmis-sion is eserine (physiostigmine, PHY), frequently used to enhance ACh signals by blocking AChE. Eserine is known as AChE inhibitor, but a direct agonistic action on nAChRs has been reported in both vertebrates (Schrattenholz et al., 1993;

Schröder et al., 1994) and inveretebrates (van den Beukel et al., 1998; Jackson et al., 2002). On the other hand, it has been also reported that high concentrations of eserine block nAChRs (Pereira et al., 1993a, b; Schrattenholz et al., 1993;

Schröder et al., 1994; Zheng et al., 1997). This leads to blockade of ACh transmis-sion and ACh evoked responses. Furthermore, prolonged inhibition of AChE leads to desensitization of nAChRs and thus to short-term blockade of ACh transmission.

ACh, depending on its concentration, plays a modulatory function on the AChE activity being alosteric factor as it was reported in biochemical studies on its enzy-matic action. In other words, the modulation of ACh release may affect indirectly the activity of both AChE and nAChRs. In many systems, mAChRs (M2-like) are the main regulator of ACh release. Furthermore, an unusual type AChR in insects, with both nicotinic and muscarinic features, had been described. Finally, as the fine modulation of nAChRs through neuropeptides comes in account, the complexity of this classical transmission goes far beyond our “classical” comprehensions.

In previous work, studying the effect of eserine on muscarine-stimulated stridulation, it has been reported that eserine at different concentrations had

op-posing effects – from inhibition to enhancing the muscarinic responses in Ch.b.

(Hoffmann, 2003).

Experiments examining the effect of eserine (eserinesalicilate, physostigmine, PHY) on both muscarine and proctolin-initiated stridulation were carried out, using protocol 3-3-3. Eserine, co-injected to muscarine and proctolin affected in a com-plex manner both muscarine and proctolin-sustaining stridulations. Inhibition of both muscarine- and proctolin-stimulated stridulation were recorded in response to eserine. But the following proctolin and muscarine stimuli elicited singing longer than 600 sec (~100 times the duration of initial proctolin injection).

These two experiments are presented in detail. Table 3-1 shows the latency and duration of the proctolin-stimulated stridulation before, during and after eserine co-injections.

Table 3-1

Time (min) Protocol 3-3-3

(No of the pulse): Injection of: Latency (in sec) S Duration (in sec)

0 1^st Proctolin 5,1 4,1

3 2^nd Proctolin 5,27 4,96

6 3^rd Proctolin 5,05 4,18

9 1^st Proctolin + eserine 0 0

12 2^nd Proctolin + eserine 72 5

15 3^rd Proctolin + eserine 51 5,5

18 1^st Proctolin 20 >600

Proctolin-stimulated stridulation before eserine application is shown in Fig. 3-33, A1. In response to the 1^st injection proctolin (1mM) and eserine (0,5mM) only active locomotor activity occurred. To the 2^nd and 3^rd pulse proctolin and eserine (Fig. 3-33, B1 and B2) specific stridulatory sequences were accompanied with un-specific movements, consisting stridulatory components.

Fig. 3-33 A1. Proctolin-initiated stridulation before application of eserine (male, Ch.b.).

A1a. The internal structure of proctolin-induced stridulation (chirps containing 4 syllables).

Fig. 3-33 B1. Irregular stridulatory movements in response to the second pulse proctolin (1mM) and eserine (0,5mM). B1a. Single up- and down stridulatory movements accompanied with unspecific movements.

The stridulation elicited by the following proctolin pulse after eserine applica-tion, lasted over 10 min (Fig. 3-33, C). The response began with unspecific move-ments consisting stridulatory-like elemove-ments (Fig. 3-33, C1). Sequences of 1^st order (“burst”-like) were performed regularly every 5-10 sec (Fig. 3-33, C3). Occasionally in between the bursts-like sequences were produced unspecific up- and down-movements with stridulatory elements. The single sequences 1^st order (1-2 sec) were performed within the “bursts” (up position of the hindlegs, 3-4 sec). The inter-nal structure of a single “burst” and sequence 1^st order is shown in Fig. 3-33, C3b.

Fig. 3-33 B2. Specific and unspecific stridulatory movements in response to the 3^rd pulse proctolin/eserine.

Fig. 3-33 C. Prolonged stridulation, stimulated by proctolin after eseine co-application (Ch.b.).

Fig 3-33 C1. Unspecific up- and down- movements, consisting stridulatory-like elements.

Fig. 3-33 C3. „Burst-like“ sequences 1st order performed by the hind legs. They were lifted 3-4 sec and produced 1-2 sec stridulatory movements (C3b). After the stridulatory movements the hindlegs remained “silent” at up position (C3a). Than the hindlegs were bring down suddenly (180-200 msec) preceding by an initial fast lifting (60-80 msec) and were kept in down position 5-10 sec (C3).

Eserine also affected the muscarine-induced stridulation in another male grasshopper Ch.b. in a very similar manner. Table 3-2 presents the latencies and durations of the muscarine-stimulated responses before, during and after eserine co-injections.

Table 3-2:

Time (in min)

Protocol 3-3-3

(No of pulses): Injection of: Latency

(in sec):

S Duration (in sec):

C Duration (in sec):

0 1^st Muscarine 3,36 15,29 26,39

4 2^nd Muscarine 4,12 13,78 25,49

8 3^rd Muscarine 4,34 11,58 19,98

12 1^st Muscarine+ Eserine 20,01 2,54 2,54

16 2^nd Muscarine+ Eserine 16,4 10,14 149,82

18 3^rd Muscarine+ Eserine 15,66 37,91 150,28

22 1^st Muscarine 11,99 <240 <600

Again, the initial muscarine stimulations elicited similar durations of stridula-tion occurring after latencies of approximately 4 sec (Table 2). The latencies in-creased with application of muscarine/eserine mixture, the first of which elicited very short stridulation that increased gradually with third pulse up to 3 times com-pared to the initial muscarine stimulations. The following muscarine application in-duced singing of additionally prolonged durations that started after a medium la-tency. Fig. 3-34 A and B present the singing patterns, stimulated by muscarine before and after co-injection of eserine respectively.

Fig. 3-34, A. Muscarine-induced singing before eserine co-injection (male, Ch.b.). A1. Single sequence 1^st order; A2. Internal structure of the sequence – chirps consisting 4 and 6 syllables

The following to eserine co-injections muscarine pulse stimulated singing af-ter 12 sec latency, consisted of 15 sequences 1^st order, each 3-7 sec long, occur-ring every 5-10 sec. Stridulatory hindleg movements of low (Fig. 3-34, B1) and very high amplitude (Fig. 3-34, B2) were produced.

Similar to proctolin (Fig. 3-33, C3) muscarine produced also “burst”-like movement pattern (Fig. 3-34, B2). Interestingly, in the case of muscarinic “bursts”

Fig. 3-34 B. Muscarine stimulation after eserine co-injections. A prolonged singing for 10 min was performed.

B1. Stridulatory hindleg movements of low amplitude; B2. Stridulatory movements of high amplitude;

B3. “Burst”-like excitation was observed, similar to proctolin, after eserine co-injection (see Fig. 3-33, C3)

the up- position of the hind legs was kept 7-10 sec and the stridulatory movements lasted 5-8 sec (Fig. 3-34, B3). The proctolin-induced “bursts” was respectively 3-4 sec and 1-2 sec (Fig. 3-33, C3b).

During this very high level of excitation triggered by co-injection of eserine, again as it was shown in section 3.4.4 (Fig. 3-24), the muscarine-induced song sequences were 2-3 times longer than the relevant proctolin-induced song se-quences.

The appearance of additional excitation to the muscarine- and proctolin-induced ones, through

inhibi-tion of the AChE, suggests the additional mechanisms in-volved into the maintenance of the singing in Ch.b., be-sides mAChRs and PRs acti-vation. A specific fine-tuned modulation of the AChE activ-ity during the ACh and/or proctolin release may play also a role in the cephalic control of singing in Ch.b.

Fig. 3-34 B3. Structure of the „burst“-like stridulatory hindlegs movements (see the text).

3.5 Intracellular pathways activated by proctolin receptors in the brain