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First published online January 31, 2006
Journal of Experimental Biology 209, 766-779 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02049

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Classical conditioning of activities of salivary neurones in the cockroach

Hidehiro Watanabe and Makoto Mizunami^*

Graduate School of Life Sciences, Tohoku University, Katahira 2-1-1, Sendai 980-8577, Japan

View larger version (16K): [in a new window]   Fig. 1. Experimental procedure. (A) Arrangement of extracellular recording from an SDN, lateral view. The salivary duct nerve (SDN) originates from the suboesophageal ganglion (SOG) and runs along the surface of the salivary duct (SD) to innervate the salivary glands. The SD and SDN were hooked by a pair of tungsten electrodes. CC, cervical connective; COC, circumoesophageal connective; A, anterior; P, posterior; V, ventral; D, dorsal. (B,C) Stimulus schedules for forward-pairing (B) and backward-pairing (C) trials. Five sets of differential conditioning trials were carried out. For each set of P+V- and V+P- forward-pairing trials, peppermint (hatched squares) or vanilla (shaded squares) odour was presented 2 s before the presentation of sucrose solution (open squares) and then vanilla or peppermint odour was presented alone, respectively. For each set of P+V- backward-pairing trials, peppermint odour was presented 4 s after the presentation of sucrose solution and then vanilla odour was presented alone. (D) Stimulus schedule for unpaired presentation of peppermint and vanilla odours (CS alone). Peppermint and vanilla odours were alternately presented five times without pairing with sucrose solution. (E) Stimulus schedule for unpaired presentation of sucrose solution (US alone). Sucrose solution was presented five times without pairing with odour. The inter-stimulus intervals were 5 min in B-D and 10 min in E. The durations of odour and sucrose stimulations were 4 s in B-E.

View larger version (23K): [in a new window]   Fig. 2. Arrangement for the measurement of saliva secretion upon electrical stimulation of the salivary duct nerve (SDN), lateral view. The distal cut-stamp of the salivary duct (SD) was inserted into a small plastic chamber, and the tip of the plastic chamber was covered with white Vaseline (dotted area) to prevent leakage of saliva. Saliva (shaded area) secreted from the SD was collected at 1 min intervals using a plastic capillary attached to a syringe. The amount of secreted fluid was calculated from the length (L) of the fluid column. The SDN was electrically stimulated by a pair of hook electrodes, and the resulting spikes were monitored by another pair of hook electrodes. Bars, 100 µm. CC, cervical connective; COC, circumoesophageal connective. A, anterior; P, posterior; V, ventral; D, dorsal.

View larger version (89K): [in a new window]   Fig. 3. Two large salivary neurones (SN1 and SN2) in the suboesophageal ganglion (SOG), stained by metal backfilling of one salivary duct nerve (SDN), viewed dorsally (A) and laterally (B). Areas surrounded by broken lines are mandibular (MD), maxillary (MX) and labial (LB) neuromeres, respectively. CC, cervical connective; COC, circumoesophageal connective. (C) An SDN at the surface of a salivary duct (SD), filled with metal. The broken line indicates the outline of the SDN. Two large-diameter axons (black arrowheads) and one small-diameter axon (white arrowhead) are visible. SG, salivary gland; A, anterior; P, posterior; V, ventral; D, dorsal.

View larger version (83K): [in a new window]   Fig. 4. Effects of surgical ablation of one of the salivary neurones SN1 or SN2 on unit activities of a salivary duct nerve (SDN). (A,B) Spontaneous spike activity of an SDN 10 min before (left) and 10 min after (right) surgical ablation of a part of the suboesophageal ganglion (SOG). In A, a lower-frequency unit with the largest amplitude disappeared after surgery, and post-mortal backfilling of the SDN revealed elimination of the cell body and some dendrites of SN1 (C). In B, a higher-frequency unit with the second-largest amplitude disappeared after surgery, and post-mortal histological examination revealed ablation of the cell body and some dendrites of SN2 (D). Vertical bars, 2 mV; horizontal bars, 1 s (A,B); 100 µm (C,D). The SOG is viewed dorsally in C and D.

View larger version (73K): [in a new window]   Fig. 5. Responses of a salivary duct nerve (SDN) to distilled water, 10% sucrose solution and 20% sodium chloride (NaCl) solution applied to the mouth. Both of the two large units, a low spontaneous frequency unit with the largest amplitude (black circle) and a higher frequency unit with the second-largest amplitude, exhibited strong responses to water, sucrose and NaCl solution. Coincident occurrence of the two large units resulted in larger-amplitude potential (triangles). The short arrows indicate artefacts caused by movement of the mouth or the oesophagus. The broken line indicates the onset of taste stimulation. All four recordings were from the same preparation. Vertical bar, 0.2 mV; horizontal bar, 1 s.

View larger version (60K): [in a new window]   Fig. 6. Responses of a salivary duct nerve (SDN) to vanilla or peppermint odour presented to an antenna. (A) Activities of an SDN during 2 s vanilla or peppermint odour stimulations. The responses of the salivary neurones, SN1 (black circle, largest unit) and SN2 (second-largest unit), to vanilla or peppermint odour were very weak and barely detectable in these recordings. (B) Responses of the SDN to vanilla or peppermint odour 30 min after five sets of P+V- differential conditioning trials recorded in the same preparation. SN1 and SN2 exhibited prominent responses to conditioned odour (peppermint), but their responses to control odour (vanilla) were barely detectable. Broken lines indicate the onset and outset of odour stimulation. Vertical bars, 0.2 mV; horizontal bars, 1 s.

View larger version (17K): [in a new window]   Fig. 7. Effects of forward-pairing trials (A,B) and unpaired presentation of odours (C) on responses of the salivary neurones (SN1 and SN2). Summed responses of SN1 and SN2 to peppermint or vanilla odour before and at 5 min after the first, second, third and fourth sets of P+V- (A) or P-V+ (B) conditioning trials or unpaired presentations of odours (C) are shown. Relative responses, measured as the relative increase in spike frequency for the first 2 s of odour stimulation compared to that during a 2 s period before odour stimulation, are shown as means ± s.e.m.; N=20 (A,B), N=21 (C). Asterisks indicate the results of statistical comparison with responses to peppermint or vanilla odour before conditioning (NS, P>0.05; *P<0.05; **P<0.01; t-test).

View larger version (31K): [in a new window]   Fig. 8. Effects of forward and backward pairing trials and of non-associative control. (A,B) Summed responses of salivary neurones (SN1 and SN2) to peppermint (hatched bars) or vanilla (shaded bars) odour before and at 1 min and 30 min after five sets of P+V- (A) or V+P- (B) forward-pairing trials. (C) Summed responses of SN1 and SN2 to odours before and at 1 min and 30 min after five sets of P+V- backward-pairing trials. (D) Summed responses of SN1 and SN2 to odours before and at 6 min and 35 min after five presentations of sucrose solution without pairing with odour (US alone). The responses are shown as means ± s.e.m. The results of statistical comparison are shown above the bars (NS, P>0.05, *P<0.05, **P<0.01, ***P<0.001; t-test).

View larger version (24K): [in a new window]   Fig. 9. Summed responses of salivary neurones (SN1 and SN2) to peppermint (hatched bars) or vanilla (shaded bars) odour 1 day after five sets of P+V- forward-pairing or backward-pairing trials. The responses are shown as means ± s.e.m. The results of statistical comparisons are shown above the bars (NS, P>0.05, **P<0.01; t-test).

View larger version (17K): [in a new window]   Fig. 10. Responses of salivary neurones, SN1 (A) and SN2 (B), to peppermint (hatched bars) and vanilla (shaded bars) odours before and at 5 min and 30 min after five sets of P+V- forward-pairing trials. The responses are shown as means ± s.e.m. The results of statistical comparison are shown above the bars (NS, P>0.05, *P<0.05, **P<0.01, ***P<0.001; WCX-test in Fig. 8B; t-test in Fig. 8B).

View larger version (16K): [in a new window]   Fig. 11. Changes in the level of salivation upon electrical stimulations of one salivary duct nerve (SDN). The amount of saliva secreted from a salivary duct was measured every minute while brief (0.2 ms) electric pulses were delivered to the SDN at 5 Hz for 2, 5, 10, 20 and 40 s with intervals of 6 min. Averaged data from 12 preparations are shown as means ± s.e.m. The amounts of secretion (broken lines) before and after the onset of electrical stimulation were statistically compared, and asterisks indicate the level of significance (*P<0.05; **P<0.01; WCX-test).