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Dual antennular chemosensory pathways mediate odor-associative learning and odor discrimination in the Caribbean spiny lobster Panulirus argus

Pascal Steullet^*, Dana R. Krützfeldt, Gemma Hamidani, Tanya Flavus, Vivian Ngo and Charles D. Derby

Department of Biology and Center for Behavioral Neuroscience, Georgia State University, Atlanta, GA 30303, USA

View larger version (113K): [in a new window]   Fig. 1. Intact and ablated antennular medial and lateral flagella of the spiny lobster. (A) Drawing of a lobster showing the antennules (first antennae) and antennae II (second antennae). The higher-magnification drawing of an antennule shows the medial and lateral flagella. Aesthetascs are located exclusively on the distal half of the lateral flagellum, in the aesthetasc tuft region. Non-aesthetasc chemosensilla are located along the entire length of both the lateral and medial flagella. Letters B–G indicate the position on the antennule where respective micrographs were taken. (B) Scanning electron micrograph of the aesthetasc region of an intact lateral flagellum with rows of aesthetascs (a) and accompanying setae: companion setae (c), guard setae (g) and asymmetric setae (as). (C) Scanning electron micrograph of the aesthetasc tuft region after shaving aesthetascs (a) and asymmetric setae (arrows), but not other setae including guard setae (g). The asterisk marks an aesthetasc whose base was not completely removed by shaving (in these aesthetasc bases, dendrites were also completely disrupted, as shown by histological techniques; data not shown). (D) Light micrograph of the aesthetasc tuft region covered by cyanoacrylate glue (arrow) after shaving all setae except aesthetascs (a) and asymmetric setae (not visible on this micrograph). The asterisk indicates the original location of a guard seta prior to shaving. (E) Light micrograph of a region of an intact medial flagellum. Arrows indicate setae. (F) Scanning electron micrograph of a region of an intact medial flagellum showing two types of non-aesthetasc sensilla: hooded sensillum (hs) and plumose seta (ps). Hooded sensilla house both mechano- and chemosensory neurons [modified from (Cate and Derby, 2001)]. (G) Light micrograph of a region of a medial flagellum covered by cyanoacrylate glue (arrow) after shaving all setae. Scale bars: B, 100 µm; C, 150 µm; F, 50 µm; D,E,G, 400 µm.

View larger version (26K): [in a new window]   Fig. 2. Effect of ablations of aesthetascs and non-aesthetasc setae on odor-evoked responses recorded from nerves of antennular lateral and medial flagella. Six conditions are shown: (i) intact lateral (N=6), (ii) aesthetasc ablation (N=4); (iii) non-aesthetasc ablation of lateral (N=4), (iv) total ablation of lateral (N=5), (v) intact medial (N=4) and (vi) total ablation of medial (N=3). For more details, see Materials and methods. Values are means ± S.E.M. *Odor-evoked responses are significantly different from each other (planned-comparisons one-way ANOVA, P<0.05). For the planned comparisons, critical values for a 5 % experiment-wise error rate were determined by the sequential Bonferroni test using the Dunn–idák method (Sokal and Rohlf, 1998).

View larger version (14K): [in a new window]   Fig. 3. Effect of distilled-water treatment of antennular flagellar chemosensory neurons on search responses to a concentration series of artificial crab odor. The ordinate represents the ratio between the post-ablation and pre-ablation responses. A value of one indicates no effect of ablation treatment, and a value of zero indicates complete elimination of responses after ablation. Each column shows the upper and lower quartiles with the median (solid line) and the mean (dotted line). N=7 lobsters in each group. Distilled-water treatment of all antennular flagella significantly reduced search responses (shaded columns) [one-way within-subjects ANOVA with multiple dependent measures (MANOVA), P=0.02]. Sham control lobsters (open columns) were not affected (MANOVA, P=0.62). For further details, see Materials and methods.

View larger version (17K): [in a new window]   Fig. 4. The ability of intact (sham control) lobsters (A), aesthetasc-ablated lobsters (B) and non-aesthetasc-ablated lobsters (C) to learn an aversive associative task and to discriminate between an aversively conditioned odor (CS+=crab odor, CO) and three other complex odor mixtures (inverse crab odor, ICO; shrimp odor, SO; mullet odor, MO) following aversive conditioning with generalization testing (see Table 2 for protocol). Values are means + S.E.M. *Search responses significantly different in unconditioned and conditioned lobsters (planned-comparisons one-way ANOVA, P<0.05). Search responses significantly larger than those elicited by crab odor in conditioned lobsters (planned-comparisons one-way ANOVA, P<0.05). () Search responses close to being significantly different from those elicited by crab odor in conditioned lobsters (planned-comparisons one-way ANOVA, 0.05<P<0.10). For the planned comparisons, critical values for a 5 % experiment-wise error rate were determined by the sequential Bonferroni test using the Dunn–idák method (Sokal and Rohlf, 1998). For a description of search responses and calculation of standardized search responses relative to the responses to oyster extract in the preconditioning phase, see Materials and methods.

View larger version (28K): [in a new window]   Fig. 5. Percentage change in search responses to the aversively conditioned odor CS+ (crab odor, CO) and the novel non-conditioned odors (inverse crab odor, ICO; shrimp odor, SO; mullet odor, MO) following an aversive conditioning with generalization testing (protocol of Table 2) in intact lobsters, aesthetasc-ablated lobsters and non-aesthetasc-ablated lobsters. For each group of lobsters, the percentage change in search responses to an odorant following conditioning is the percentage difference between responses of conditioned lobsters and unconditioned lobsters relative to responses of unconditioned lobsters. Data are from Fig. 4.

View larger version (22K): [in a new window]   Fig. 6. The ability of intact (sham control) lobsters (A) and aesthetasc-ablated lobsters (B) to learn a discrimination conditioning task and to discriminate between binary mixtures of AMP+taurine with the same total concentration (1 mmol l–1) but at different blend ratios. The discrimination conditioning paradigm is described in Table 3. The aversively conditioned odorant (CS+) was the 99.9:0.1 blend ratio, and the conditioned ‘safe’ odorants (CS–) were the blend ratios 99:1, 90:10 and 50:50. Values are means + S.E.M. *Search responses significantly different in unconditioned and conditioned lobsters (planned comparisons one-way ANOVA, P<0.05); Search responses significantly larger than those elicited by the aversively conditioned blend ratio 99.9:0.1 (CS+) in conditioned lobsters (planned-comparisons one-way ANOVA, P<0.05). For the planned comparisons, critical values for a 5 % experiment-wise error rate were determined by the sequential Bonferroni test using the Dunn–idák method (Sokal and Rohlf, 1998). Search responses of conditioned lobsters to oyster extract (OE) after the post-conditioning phase are also shown. In both intact and aesthetasc-ablated lobsters, search responses to oyster extract before and after conditioning were not significantly different (P<0.05, t-test for dependent samples). For a description of search responses and calculation of the standardized search responses relative to the responses to oyster extract in the preconditioning phase, see Materials and methods.

View larger version (24K): [in a new window]   Fig. 7. Percentage change in search responses to the aversively conditioned (CS+) blend ratio of AMP:taurine mixture (99.9:0.1 blend ratio) and the conditioned safe (CS–) blend ratios 99:1, 90:10 and 50:50 following a discrimination conditioning paradigm in intact and aesthetasc-ablated lobsters. For each group of lobsters, the percentage change in search responses to an odorant following conditioning is the percentage difference between responses of conditioned lobsters and unconditioned lobsters relative to responses of unconditioned lobsters. Data are from Fig. 6.

View larger version (15K): [in a new window]   Fig. 8. Results of two cluster analyses showing the relative similarity of the chemical composition of the complex odor mixtures crab odor (CO), inverse crab odor (ICO), shrimp odor (SO) and mullet odor (MO). (A) Cluster analysis that uses ‘percentage of disagreement’ as the dissimilarity measure, which emphasizes the components unique to the odors (i.e. the presence or absence of components). (B) Cluster analysis that uses ‘1 minus the Pearson r correlation’ as the dissimilarity measure, which emphasizes differences in the relative concentrations of each component.