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First published online September 19, 2006
Journal of Experimental Biology 209, 3898-3912 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02432

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Perfusion with cGMP analogue adapts the action potential response of pheromone-sensitive sensilla trichoidea of the hawkmoth Manduca sexta in a daytime-dependent manner

Christian Flecke, Jan Dolzer, Steffi Krannich and Monika Stengl^*

Biologie, Tierphysiologie, Philipps-Universität Marburg, Karl-von-Frisch-Straße, D-35032 Marburg, Germany

View larger version (21K): [in a new window]   Fig. 1. The pheromone response is characterized by six parameters. (A) A non-filtered (DC) response to a 50 ms stimulus of 1 µg bombykal (BAL). Action potentials are superimposed on the negative deflection of the transepithelial potential, the sensillar potential (SP) response. The maximal SP amplitude (SPA) is measured between the baseline before the response and the negative peak of the SP. The half-time of the rising phase (trise) is determined between the onset of the SP and the time the potential has reached 50% of the SPA (SPA). The second portion of the rising phase is described by an exponential fit of first order, using only the time constant (). For the analysis of all parameters describing the SP, the responses were low-pass filtered at 50 Hz. (B) The initial phase of the response at an enlarged time scale. The initial slope is determined by dividing SPA by trise. The AP latency is measured between the onset of the sensillar potential and the peak of the first action potential. (C) For the analysis of action potentials, the low-pass filtered response is subtracted from the original trace, yielding a straight baseline. The initial action potential frequency (AP frequency) is computed over the first five interspike intervals. (D) Pseudo-high-pass filtered (AC) response to a 50 ms stimulus of 10 µg BAL. The amplitudes of the large action potentials are reduced after strong BAL stimuli and regain their original amplitude in the course of several seconds (broken line). A spontaneous action potential of the non-BAL cell occurred before (filled arrow) the response. APs of the non-BAL cell can be separated from the BAL-APs by their lower and steady amplitude after stimulation with BAL. (E) Non-adapting stimulus-protocol: 50 ms long stimuli of 10 µg BAL per filter paper were applied with an interstimulus-interval (ISTI) of 5 min over a period of 180 min. 8bcGMP at 10 mmol l-1 was applied by perfusion over the recording electrode with the beginning of the recording [modified after (Dolzer, 2003)].

View larger version (16K): [in a new window]   Fig. 2. Sensillum lymph perfusion with 10 mmol l-1 8bcGMP at ZT 8-11 reduced the action potential (AP; top recordings) response, but left the sensillar potential (SP; bottom recordings) response unaltered. (A) At the beginning of the recording, the AP frequency in response to bombykal stimulation was 245 Hz. After 158 min (B) the SP was unaltered, but the AP frequency was reduced to 147 Hz. Also, the latency to occurrence of the first AP (AP latency) was prolonged. The effect on the AP latency was characterized by high variability between recordings. AC, pseudo-high-pass filtered signal; DC, non-filtered signal.

View larger version (21K): [in a new window]   Fig. 3. Example time course of sensillar potential (A) and action potential (B) parameters for one recording under control conditions at ZT 8-11. The pheromone responses remained virtually constant during 3 h of repetitive stimulation. A bombykal stimulus of 10 µg dose and 50 ms duration was applied every 5 min.

View larger version (20K): [in a new window]   Fig. 4. Time course of sensillar potential (SP; A) and action potential (AP; B) parameters under the influence of 8bcGMP perfusion at ZT 8-11. Shown is one representative recording. During sensillum perfusion with 10 mmol l-1 8bcGMP the AP frequency continuously decreased and the AP latency increased, while the parameters that describe the SP remained virtually constant.

View larger version (50K): [in a new window]   Fig. 5. The normalized and binned sensillar potential (SP) amplitude (A,C) and action potential (AP) frequency (B,D) parameters over 185 min (1 bin=5 min) for recordings from ZT 1-4 (A,B) and ZT 8-11 (C,D). Values are means + s.e.m. (B,D) Under the influence of 8bcGMP (top recordings) a significant decrease of the AP frequency can be recognized for both ZTs when compared to the respective controls (bottom recordings) (Student's t-test for independent samples, P<0.01) and when comparing the three intervals within each time course (separated by dotted lines). Different lower case letters denote significant differences between tested groups of means (ANOVA and Tukey HSD post-hoc test; =0.01, P<0.01). The means in the second (b) and third time interval (c) are not significantly different (b,c) for both ZTs. The decrease of the action potential frequency at ZT8-11 is 12% stronger than in recordings from ZT1-4. (A,C) In contrast, the SP amplitude remained unchanged.

View larger version (36K): [in a new window]   Fig. 6. Action potential (AP) distribution in responses to stimulation with 10 µg bombykal. Post-stimulus-time histograms (binwidth= 10 ms) for recordings from ZT 1-4 for the beginning (A; 0-20 min), middle (B; 80-100 min), and end of the recordings (C; 160-180 min). In the control recordings (left) the numbers of APs in the first part of the phasic response decreased. Also, the number of APs over the first 100 ms of the responses (insert in C) showed a slight but significant decline at the end of the recording duration. Under the influence of 8bcGMP (right) no changes are recognizable. Values in inserts are means + s.e.m. Since the sample sizes differed for the different time windows all y-axes were scaled to y=nx2 (dotted line=100 ms after the onset of the sensillar potential). Different lower case letters denote significant differences between tested groups of means (separated by dotted lines in the insert) (ANOVA and Tukey HSD post-hoc test; =0.01, P<0.01).

View larger version (37K): [in a new window]   Fig. 7. Action potential (AP) distribution in responses to stimulation with 10 µg bombykal. Post-stimulus-time histograms (binwidth=10 ms) for recordings from ZT 8-11 for the beginning (A; 0-20 min), middle (B; 80-100 min) and end of the recordings (C; 160-180 min). Both in the controls (left) as well as under the influence of 8bcGMP (right) the first phasic part of the AP response declined, leading to a more tonic response. Also, the numbers of APs over the first 100 ms (insert in C) showed a significant decrease both with 8bcGMP and in the controls. This decline was stronger in the presence of 8bcGMP. Since the sample sizes differed for the different time windows all y-axes were scaled to y=nx1.6 (dotted line=100 ms after the onset of the sensillar potential). Different lower case letters in the inserts denote significant differences between tested groups of means, which are separated by dotted lines (ANOVA and Tukey HSD post-hoc-test; =0.01, P<0.01).

View larger version (28K): [in a new window]   Fig. 8. Analysis of the amplitude reduction of action potentials (APs). (A) The 8bcGMP-dependent AP amplitude reduction for the beginning (i), the middle (ii) and the end (iii) of one recording at ZT 8-11. Plots show the mean of the positive amplitude of APs that occurred during an interval of 1 min before to 2 min after the stimulation. Arrows indicate the time of stimulation. Each plot consists of the binned (binwidth=10 ms) and averaged AP amplitudes of three consecutive responses to a stimulus of 10 µg BAL. For the first three responses of the recording the AP amplitudes were strongly reduced. The APs returned to its pre-stimulus amplitude not until about 2 min after the stimuli were applied. After about 90 min the slow portion of the recovering phase disappeared. Also, the amplitude reduction showed a slight decrease. At the end of the recording a strong 8bcGMP-dependent decrease in the amplitude reduction was observed. The amplitude reduction was only weak and transient. In the control recordings no or only weak fluctuations in the reduction of positive APs were found. In contrast to the recording with 8bcGMP the slowly recovering phase is even more prominent at the end of the recording. (Due to the non-linear change of the BAL-AP amplitude in the post-stimulus portion BAL- and non-BAL APs could not be distinguished). (B) Normalized ratio between the minimal and maximal positive AP amplitude of a response as a mean of the strength of the amplitude reduction. Each recording was normalized to the first value. Values >1 represent a decrease in the reduction of the AP amplitude. In recordings from ZT 1-4 with 10 mmol l-1 8bcGMP diluted in the sensillum lymph ringer (top right), two populations of recordings can be observed. One population showed a very strong increase from the beginning on, the other one resembled the time course of the reduction in control recordings. For control recordings from ZT 1-4 (top left), most of the data points showed a cumulative composition at the beginning followed by an increasing variance leading to a continuous broadening in the distribution later in the recordings. Under the influence of 8bcGMP all of the recordings from ZT 8-11 (bottom right) showed an increase, whereas in the associated controls (bottom left) most data points were located in a relatively distinct band around 1.

View larger version (46K): [in a new window]   Fig. 9. The shape of both action potential (AP) classes was influenced by the injection of 8-bromo cyclic GMP (8bcGMP). (A) The waveform of large APs was averaged for intervals of 10 min and plotted versus the time of the recording. After the injection of 8bcGMP at t=182 min (transparent wall) both positive and negative phases of the APs increased, indicating an increase in the resistance of the ORN. (B) The voltage levels were color-coded and projected into a plane (C). Before the injection amplitude and time course of the APs remained relatively constant, but 8bcGMP increased the peak-to-peak amplitude and prolonged the negative phase of the APs. (D) Average AP waveforms at the times indicated in C, normalized to the same positive peak amplitude to visualize changes in the time course. When compared to the waveform before the injection (t0, dotted line), the negative phase was prolonged and slightly reduced in amplitude (arrow) 140 min after the injection (t1, solid line). Later, the negative phase was shortened again, and 300 min after the injection the time course of the averaged and normalized waveform (t2, solid line) was identical to the pre-injection waveform (t0, dotted line). (E) Changes in the waveform of the small APs also showed increases of the peak-to-peak amplitude and a prolongation of the negative phase, but over a different time course. The prolongation of the negative phase reversed after 50 min, while with the large APs the reversal occurred after 150 min. The peak-to-peak amplitude of the small APs was transiently reduced back to the pre-injection level between 70 and 120 min after the injection, while the large APs reached their highest peak-to-peak amplitude at the same time. During the gap in the data (gray areas in C and E), small and large APs could not be distinguished.