First published online March 12, 2009
Journal of Experimental Biology 212, 922-933 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.023069
Novel neural correlates of operant conditioning in normal and differentially reared Lymnaea
Abdullah M. Khan and
Gaynor E. Spencer*
Department of Biological Sciences, Brock University, 500 Glenridge
Avenue, St Catharines, Ontario, Canada L2S 3A1
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Fig. 2. Only the normally reared conditioned animals demonstrated a reduction in
behaviour across the training sessions and memory test. Whereas normally
reared conditioned animals (Cond.) showed a significant reduction
(**P<0.01) in the number of attempted openings from
session 1 (TS1: 20.4±0.6) to session 4 (TS4: 6.7±0.8) and the
memory test (MT: 6.9±0.6), differentially reared conditioned (DR cond.)
animals did not show a reduction in aerial respiratory behaviour as a result
of the operant conditioning (TS1: 6.2±1.1, TS4: 5.1±0.8, MT:
4.6±1.0).
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Fig. 3. Pneumostome opening behaviour coincides closely with the number of IP3
events recorded in the VI cell in semi-intact preparations and reflects the
behaviour of the intact animal. Both the number of pneumostome openings (A,C)
and the number of IP3 events in the VI cell (B,D) were recorded. (A) The
differentially reared naïve preparations showed significantly fewer
pneumostome openings (DR naïve: 4.7±0.7;
*P<0.05) than the normally reared naïve
preparations (8.6±0.7). (B) Accordingly, the number of IP3 events was
significantly lower (**P<0.01) in the differentially
reared naïve preparations (5.0±0.8) compared with the normally
reared naïve preparations (8.8±0.7). (C) Normally reared
conditioned (Cond.) preparations performed aerial respiration significantly
less often (4.1±0.9) than normally reared yoked controls
(7.7±0.8; **P<0.01). The differentially reared
conditioned (DR cond.) preparations, however, did not demonstrate a
significant reduction in aerial respiration compared with their corresponding
yoked controls (DR yoked: 5.1±0.7, DR cond.: 5.4±0.6;
P>0.05). (D) The number of IP3 events was also significantly
reduced in the normally reared conditioned animals (4.7±0.8) compared
with normally reared yoked preparations (7.7±1.0;
*P<0.05), but not in the differentially reared
conditioned preparations compared with their yoked controls (DR yoked:
5.2±0.6, DR cond.: 6.0±0.5). (E) Representative
electrophysiological recordings in a normally reared yoked control preparation
showing an IP3 event in the VI and RPeD1 cells and corresponding pneumostome
opening. (F) Representative electrophysiological recordings in a normally
reared conditioned preparation showing the absence of an IP3 event or
pneumostome opening.
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Fig. 4. Conditioning induces novel changes in the latency from IP3 events to
pneumostome opening in normal and differentially reared preparations. (A)
Intra-burst spike frequency of IP3 events was significantly reduced
(**P<0.01) in normally reared conditioned (Cond.)
preparations (5.2±0.3 Hz) compared with the normally reared yoked
controls (7.3±0.5 Hz), but was not altered in the differentially reared
preparations (DR yoked: 7.6±0.4 Hz, DR cond.: 7.4±0.5). (B) The
latency from IP3 events in the VI motorneuron to the pneumostome opening was
determined for all preparations. A significant increase in the latency was
observed in both normally reared conditioned preparations (Cond.:
1.1±0.5 s) and differentially reared conditioned preparations (DR
cond.: 1.1±0.3 s) compared with their corresponding yoked controls
(Yoked: 0.0±0.3 s, DR yoked: 0.3±0.2 s;
*P<0.05). (C–E) Representative recordings showing
IP3 events in the VI cell and pneumostome openings in (C) normally reared
yoked control, (D) normally reared conditioned, and (E) differentially reared
conditioned preparations, demonstrate the reduced IP3 event frequency and
increased latency to openings in conditioned preparations.
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Fig. 5. Coincident IP3 events and pneumostome activity were significantly reduced
in both normal and differentially reared conditioned preparations. Coincident
activity was monitored by determining the percentage of IP3 events that
produced a pneumostome opening. (A) In both normally reared and differentially
reared conditioned (Cond.) preparations, significantly fewer IP3 events
resulted in pneumostome openings compared with the yoked controls (Yoked:
98±1%, Cond.: 71±6%, DR yoked: 97±2%, DR cond.:
84±6%; **P<0.01;
*P<0.05). (B) Representative electrophysiological
recordings in a normally reared yoked control preparation showing a coincident
IP3 event with pneumostome opening. (C,D) Representative recordings from a
normally reared (C) and differentially reared (D) conditioned preparation
illustrate IP3 events that did not result in pneumostome openings.
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Fig. 6. Following the contingent application of the punishing stimulus to the
semi-intact preparations, only conditioned preparations showed a reduction in
behaviour and neural activity. (A) A two-way ANOVA revealed a significant
effect of training on the change in pneumostome openings after presentation of
the punishing stimulus (F(1,47)=4.36; P<0.05).
In both the normally reared and differentially reared conditioned
preparations, there was a reduction in the number of pneumostome openings
after application of a punishing stimulus to an open pneumostome in the
semi-intact preparation. Yoked preparations did not demonstrate a reduction in
respiratory activity (Yoked: 0.00±0.77, Cond.: –1.01±0.91,
DR yoked: 0.14±0.65, DR cond.: –1.91±0.56). (B) A two-way
ANOVA revealed a significant effect of training on the change in number of IP3
events after the punishing stimulus (F(1,47)=6.42;
P<0.05). There was a corresponding reduction in the number of IP3
events in the normally reared and differentially reared conditioned groups
compared with their yoked controls after the punishing stimulus (Yoked:
0.15±0.71, Cond.: –0.92±0.66, DR yoked: 0.14±0.67,
DR cond.: –2.18±0.62).
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Fig. 7. Immediate behavioural and neural responses occur following the punishing
stimulus in the semi-intact preparation. (A) The latency from the stimulus to
the next pneumostome opening was used as an indication of the preparation's
behavioural response to the aversive stimulus. A two-way ANOVA revealed a
significant effect of training on the latency following the punishing stimulus
(P<0.005). There was a significant increase in this latency in
normally reared conditioned preparations (Cond.: 116±19 s) compared
with yoked preparations (Yoked: 44±12 s;
*P<0.05). Although differentially reared preparations
also showed the same trend, this difference was not significant (DR yoked:
50±8 s, DR cond.: 87±24 s). (B) RPeD1 responds to a mechanical
stimulus to the pneumostome with an inhibition of firing. The latency from the
stimulus to the next action potential in RPeD1 was thus used as an indication
of the neural response to the aversive stimulus. A two-way ANOVA revealed a
significant effect of training on the latency following the punishing stimulus
(P<0.005). Operant conditioning resulted in a significantly
increased latency to the next action potential in both normally reared
conditioned (32±7 s) and differentially reared conditioned preparations
(36±8 s) compared with their corresponding yoked controls (Yoked:
13±4 s, DR yoked: 19±4 s; *P<0.05).
(C–E) Representative traces showing RPeD1 activity and pneumostome
openings from (C) a normally reared yoked control, (D) a normally reared
conditioned, and (E) a differentially reared conditioned preparation,
illustrating increased latencies in RPeD1 firing and pneumostome openings in
the conditioned preparations following the punishing stimulus.
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© The Company of Biologists Ltd 2009
P<0.01) in the pre-observation period than
normally reared naïve animals (6.0±0.6). (B) Differentially reared
animals also showed a reduced total breathing time (37±7 s;