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First published online January 31, 2006
Journal of Experimental Biology 209, 711-721 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02047

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Perturbation of the activity of a single identified neuron affects long-term memory formation in a molluscan semi-intact preparation

M. R. Lowe and G. E. Spencer^*

Department of Biological Sciences, Brock University, Ontario, Canada, L2S 3A1

View larger version (43K): [in a new window]   Fig. 1. Illustration of pneumostome opening and the underlying circuitry in Lymnaea. (A) Schematic diagram of the three neuron CPG and motorneurons innervating the pneumostome muscles. RPeD1, right pedal dorsal 1 neuron; VD4, visceral dorsal 4; IP3, input 3 interneuron; VK, visceral K closer motorneurons (MN); VI/J, visceral I/J opener motorneurons; filled circles, inhibitory connections; open triangles, excitatory connections; grey triangles, biphasic connections (excitation followed by inhibition). (B) An example of pneumostome opening in a typical semi-intact preparation. The pneumostome is located on the bottom right of each panel and is closed, but reared upward in (i) and fully open in (ii). The two photographs were taken in quick succession (1 s apart) after lowering the saline level to expose the pneumostome to the water surface. Scale bar, 3 mm.

View larger version (26K): [in a new window]   Fig. 2. Operantly conditioned semi-intact preparations demonstrated a reduction in total breathing following conditioning for learning and LTM. Conditioned and yoked control preparations were given a 20 min pre-observation period and a 20 min post observation period to assess total breathing time before and after their operant and yoked sessions, respectively. During the pre- and post-observation periods both conditioned and yoked preparations were allowed to breathe freely. The total breathing time of conditioned and yoked preparations during the pre-observation period did not significantly differ from each other (unpaired t-test=0.4451, P>0.05). Only the conditioned preparations demonstrated a significant reduction in total breathing time during the post observation period, which followed the LTM test (paired t-test=3.090, P=0.01). **P=0.01.

View larger version (26K): [in a new window]   Fig. 3. Operantly conditioned semi-intact preparations demonstrated learning and long-term memory, but yoked and naïve preparations did not. The number of openings (and total breathing time for controls) were assessed during each session. (A) Conditioned preparations showed a significant reduction in attempted pneumostome openings when comparing session 1 to 4 (learning; t=3.583, P<0.01) and session 1 to the memory test (LTM; t=3.539, P<0.01). In addition, there was also a significant reduction in attempted pneumostome openings in the conditioned group between session 1 and 2 (t=3.280, P<0.01). **P<0.01. (B) A two-way RM-ANOVA demonstrated that the number of pneumostome openings in yoked (grey bars) and naïve preparations (white bars) remained unchanged across the training sessions and in the memory test 18 h later (P>0.05 for treatment, sessions and interaction effects). There was no significant difference in number of openings in Session 1 across all three groups (P>0.05). (C) Although the total breathing time of yoked preparations was lower than that of the naïve preparations (F(1,18)=5.879, P<0.05) there was no significant decline in the total breathing time within each treatment group when comparing session 1 to 4 (yoked: t=0.0303, P>0.05; naïve: t=1.115, P>0.05) and session 1 to the memory test (yoked: t =0.401, P>0.05; naïve: t=0.7139, P>0.05).

View larger version (21K): [in a new window]   Fig. 4. Operantly conditioned semi-intact preparations given only two training sessions demonstrated learning, but not long-term memory. Conditioned preparations demonstrated a significant reduction in attempted pneumostome openings between session 1 and 2 (t=2.711, P<0.05) indicating learning, but not between session 1 and the 18 h memory test (t=0.1196, P>0.05). *P<0.05.

View larger version (26K): [in a new window]   Fig. 5. Operantly conditioned sham controls demonstrated learning, but not LTM. Conditioned sham controls were conditioned preparations (two sessions only) in which RPeD1 was impaled by an electrode but was not hyperpolarized, in order to determine if impaling the soma of RPeD1 with sharp electrodes affected the preparation's behaviour. (A) The soma of RPeD1 was impaled for up to 20 min immediately following the completion of training session 1. (B) Sample recordings of RPeD1 impulse activity recorded in conditioned and naïve preparations after the completion of training session 1, after the saline level had been raised. (C) Impaling RPeD1 with sharp electrodes did not affect behaviour, since the conditioned sham preparations still showed learning between session 1 and 2 (t=2.818, P<0.05), but no LTM was formed (t=0.8309, P>0.05). Within C, arrowheads denote time of electrode insertion. *P<0.05.

View larger version (29K): [in a new window]   Fig. 6. Preventing RPeD1 impulse activity in operantly conditioned preparations augmented the formation of long-term memory. (A) Upon completion of session 1 in conditioned, yoked, and naïve control preparations, RPeD1 was penetrated with a microelectrode followed by the immediate injection of hyperpolarizing current to prevent impulse activity for up to 20 min. (B) Sample recordings in which RPeD1 impulse activity was prevented after the completion of training session 1 (arrowheads denote the time of hyperpolarizing current injection in B and C). (C) Conditioned preparations significantly reduced their number of attempted pneumostome openings between sessions 1 and 2 (learning; t=4.014, P<0.001) and session 1 and the memory test 18 h later (LTM; t=4.176, P<0.001). Naïve preparations also significantly reduced their pneumostome openings in session 2 (t=3.733, P<0.01), but not in the memory test (t=0.6616, P>0.05) and thus did not show LTM. **P<0.01; ***P<0.001.

View larger version (35K): [in a new window]   Fig. 7. Increasing RPeD1 impulse activity did not augment long-term memory formation in operantly conditioned preparations. (A) In conditioned and naïve preparations RPeD1 was depolarized (i.e. by injecting positive current) for up to 20 min immediately after the completion of session 1 (and after saline level had been raised). (B) Sample recordings in which RPeD1 impulse activity was increased in conditioned and naïve preparations after the completion of training session 1 (arrowheads denote the time of the depolarizing current injection in B and C). (C) Only the conditioned preparations significantly reduced their number of attempted pneumostome openings between sessions 1 and 2 (learning; t=2.477, P<0.05), but not between session 1 and the memory test 18 h later (LTM; t=1.012, P>0.05).