Histamine

To examine the involvement of histamine-gated Cl^- channels in the control of stridulation, histamine was either co-applied to proctolin, using stimulation protocol 3-3-3, or injected, during proctolin-stimulated song sequences.

Proctolin-stimulated songs were altered by co-application of histamine (Fig. 3-75, A). The latency increased from 33,94% (SD=9,23 %) before up to 53,91% (SD=51,44%, n=3, P>0,05) with histamine and subsequently decreased to 10,36% (SD=4,05%) after histamine co-injections. The S Duration changed respectively from 72,87% (SD=0,1%), down to 40,80% (SD=11,06%) and back to 63,68% (SD=23,57%). The C Duration was altered from 65,52%

(SD=12,21%), to 14,5% (SD=6,7%) with histamine and did not fully recover during the experiment but remained at 23,35% (SD=10,42%) (Fig. 3-75, B). The differential changes in S Duration and C Duration was also a property of glycine-induced effects (Fig. 3-75, B) and could serve as an indication of changes of the internal time courses of the song sequences – the duration of the particular sequences and/or the pauses in between. One typical experiment is presented in the next figure (Fig. 3-76, A, B, C).

In response to the first proctolin injection the grasshopper performed long stridulatory activity (~40 sec) starting after a latency of 75 sec (Fig. 3-76, A1).

The second pulse 3 min later elicited faster response after 12 sec and weaker late response (after 70 sec) (Fig. 3-76, A2). The 3^rd proctolin injection stimulated

Fig. 3-75 Alteration of proctolin-stimulated stridulation by co-application of histamine (males, Ch.b., n=3) A. Protocol 3-3-3: 0,3,6, and 18,21,24 min– responses to proctolin; 9,12,15 min – responses to proctolin and histamine. B. Increase of the latency and decrease of durations during and after histamine co-injections.

The C duration was in higher degree affected.

only the earlier response, which became stronger and the later sequences disappeared (after 70 sec) (Fig. 3-76, A3). Interestingly, the silent period in between the earlier and the later song sequences was in the range of already demonstrated in previous experiments with glutamate (Fig. 3-68) and desensitization experiments (Fig. 3-17) ~ 30-60 sec.

Fig. 3-76 A. Proctolin-induced responses in the initial phase of stimulation before the application of histamine (male, Ch.b.) (see the text)

In response to the following co-injections of proctolin with histamine early non-specific movements and late song sequences (120 sec) were performed

(Fig. 3-76, B1 and B2). The amplitude of the stridulatory movements decreased gradually to the 3^rd proctolin/histamine co-application (Fig. 3-76, D1, D2), while the duration of the stridulation

remained very similar. The following proctolin pulses after proctolin/histamine co-injections stimulated only the early (8 sec) song sequences (Fig. 3-76, C).

Fig. 3-76 B, C. The responses to proctolin injections before and after histamine co-injections were early and strong (A, C). In response to histamine co-injection late song sequences (85 sec) were performed and the amplitude of the stridulatory movements was two times smaller (B)

Fig. 3-76 D. The amplitude of the stridulatory HLss movements differed in response to proctolin and proctolin and histamine, respectively.

The excitation was clear shifted from late responses to earlier ones throughout a silent phase ~30-60 sec. Similar silent period between singing activity was shown in the previous section, examining the role of glutamate.

Another effect of histamine in some experiments was shortening of the duration of the song sequences, as it is shown in Fig. 3-77 A, B.

When histamine was co-injected during proctolin-stimulated stridulation, 15 sec after the proctolin pulse, the duration of stridulation decreased. Fig. 3-78 A, B presents the results from three experiment of this kind. Every 3 min two injections were applied at a 15 sec interval, the first being always proctolin and the second being either proctolin (control) or proctolin/histamine mixture (test) (Fig. 3-78, A).

Histamine co-injection significantly decreased the duration (S Duration) of proctolin-induced singing from 97,87% (SD=2,72) before, down to 67,55%

(SD=0,12%, P>0,05, n=3) with and to 81,43% (SD=8,3%) after the co-application. The values for C Duration were respectively 84,08% (SD=12,33);

45,63% (SD=14,47%) and 54,79% (SD=23,33%). The latency of the

proctolin-Fig. 3-77 Shortening of the song sequences stimulated by proctolin through co-application proctolin with histamine (male, Ch.b.). A. Song sequences stimulated by proctolin; B. Song sequences stimulated by co-injection of proctolin with histamine

induced singing during the experiment increased from 61% (SD=2,91%) up to the 83,97% (SD=4,14%) after the first co-injection of histamine (Fig. 3-78, A).

The effect of histamine was due to the termination of stridulation, approximately 1,5–2 sec after application of the proctolin plus histamine mixture was injected (Fig.

3-79, A2 and B2). This was similar in two different preparations.

The prematurely termination of the sequences occurred with latency of more than 1 sec (Fig. 3-79), which is

Fig. 3-78 Significant decrease of the durations of proctolin-triggered songs induced by co-injection of histamine (males, Ch.b., n=3). A. Protocol 2-2-2; B. Significant decrease (P>0,05) of the durations

Fig. 3-79 Injection of proctolin and histamine during proctolin-induced stridulation in two different males (Ch.b.). A1 and B1 control proctolin pulses, during the stridulation; A2 and B2 co-injection of proctolin and histamin (in red). The latency of histamine effect was longer than 1 sec.

unlikely to be mediated by fast hyperpolarizing Cl^- channels, locating on the same neurons that drive stridulation upon proctolin stimulation. The time course of histamine effect may suggest that either metabotropic receptors (mHisRs).

In other experiment histamine co-injections during the proctolin-stimulated singing induced shorter song sequences (Fig. 3-80, A, B).

The main problem using natural transmitter within the stimulations is that the mode of action can be triggered by both excitatory and inhibitory, fast and slow mechanisms, which might be strict coupled, producing complex responses.

All substances tested in these series experiments glycine, glutamate, histamine and GABA activate not only Cl^- channels but gated also metabotropic receptors, coupled to different second messengers cascades. Glycine and glutamate can be ligands of ionotropic cation channels, triggering fast depolarization.

In order to examine the general role of only Cl^- channel activation in the control circuits for singing behavior an activator of these channels, the insecticide ivermectine, was tested.

Fig. 3-80 Histamine co-injected with proctolin during the proctolin-stimulated stridulation decreased the durations of the song sequences of 1^st order (male, Ch.b.) A. Song sequences stimulated by two pulses proctolin with 10 sec interval in between (control); B.

Shorter song sequences of 1^st order induced by co-injection of histamine with proctolin during ongoing singing