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Roles of NO signaling in long-term memory formation in visual learning in an insect.

Matsumoto Y, Hirashima D, Terao K, Mizunami M - PLoS ONE (2013)

Bottom Line: In this study, we examined the possible contribution of NO-cGMP signaling and cAMP signaling to LTM formation in visual pattern learning in crickets.The results indicate that cAMP signaling is downstream of NO signaling for visual LTM formation.We conclude that visual learning and olfactory learning share common biochemical cascades for LTM formation.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Life Science, Hokkaido University, Sapporo, Japan.

ABSTRACT
Many insects exhibit excellent capability of visual learning, but the molecular and neural mechanisms are poorly understood. This is in contrast to accumulation of information on molecular and neural mechanisms of olfactory learning in insects. In olfactory learning in insects, it has been shown that cyclic AMP (cAMP) signaling critically participates in the formation of protein synthesis-dependent long-term memory (LTM) and, in some insects, nitric oxide (NO)-cyclic GMP (cGMP) signaling also plays roles in LTM formation. In this study, we examined the possible contribution of NO-cGMP signaling and cAMP signaling to LTM formation in visual pattern learning in crickets. Crickets that had been subjected to 8-trial conditioning to associate a visual pattern with water reward exhibited memory retention 1 day after conditioning, whereas those subjected to 4-trial conditioning exhibited 30-min memory retention but not 1-day retention. Injection of cycloheximide, a protein synthesis inhibitor, into the hemolymph prior to 8-trial conditioning blocked formation of 1-day memory, whereas it had no effect on 30-min memory formation, indicating that 1-day memory can be characterized as protein synthesis-dependent long-term memory (LTM). Injection of an inhibitor of the enzyme producing an NO or cAMP prior to 8-trial visual conditioning blocked LTM formation, whereas it had no effect on 30-min memory formation. Moreover, injection of an NO donor, cGMP analogue or cAMP analogue prior to 4-trial conditioning induced LTM. Induction of LTM by an NO donor was blocked by DDA, an inhibitor of adenylyl cyclase, an enzyme producing cAMP, but LTM induction by a cAMP analogue was not impaired by L-NAME, an inhibitor of NO synthase. The results indicate that cAMP signaling is downstream of NO signaling for visual LTM formation. We conclude that visual learning and olfactory learning share common biochemical cascades for LTM formation.

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Effects of cycloheximide on formation of 1-day memory after visual pattern conditioning.(A) Three groups of animals were each subjected to injection of 3 μl of saline or saline containing 1 mM or 10 mM cycloheximide (CHX) at 20 min prior to 8-trial conditioning in which a visual pattern was paired with water reward. Relative preference between the rewarded pattern and control pattern was tested before and at 1 day after training. (B) Another two groups were each injected with 3 μl of saline or saline containing 10 mM CHX at 20 min prior to 8-trial visual conditioning. Relative preference between the rewarded pattern and control pattern was tested before and at 30 min after training. Preferences indexes (PIs) for the rewarded patterns before (white bars) and after (grey bars) training are shown as box and whisker diagrams and are statistically compared. The line in the box is the median and the box represents the 25-75 percentiles. Whiskers extend to extreme values as long as they are within a range of 1.5× box length from the upper or lower quartiles. Any data not included between the whiskers are plotted as outliers with dots. The results of statistical comparisons of visual pattern preferences before and after training are shown by asterisks (*** P<0.001, ** P<0.01, NS P>0.05, WCX test). The number of animals tested is shown at each data point in this figure and in subsequent figures.
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pone-0068538-g002: Effects of cycloheximide on formation of 1-day memory after visual pattern conditioning.(A) Three groups of animals were each subjected to injection of 3 μl of saline or saline containing 1 mM or 10 mM cycloheximide (CHX) at 20 min prior to 8-trial conditioning in which a visual pattern was paired with water reward. Relative preference between the rewarded pattern and control pattern was tested before and at 1 day after training. (B) Another two groups were each injected with 3 μl of saline or saline containing 10 mM CHX at 20 min prior to 8-trial visual conditioning. Relative preference between the rewarded pattern and control pattern was tested before and at 30 min after training. Preferences indexes (PIs) for the rewarded patterns before (white bars) and after (grey bars) training are shown as box and whisker diagrams and are statistically compared. The line in the box is the median and the box represents the 25-75 percentiles. Whiskers extend to extreme values as long as they are within a range of 1.5× box length from the upper or lower quartiles. Any data not included between the whiskers are plotted as outliers with dots. The results of statistical comparisons of visual pattern preferences before and after training are shown by asterisks (*** P<0.001, ** P<0.01, NS P>0.05, WCX test). The number of animals tested is shown at each data point in this figure and in subsequent figures.

Mentions: First, we studied whether the formation of 1-day memory after visual conditioning can be characterized as protein synthesis-dependent LTM. We used 8-trial conditioning to associate a visual pattern with water reward, with an inter-trial interval of 5 min, because we observed that it leads to formation of 1-day memory, whereas 4-trial conditioning leads to formation of 30-min memory but not 1-day memory [11]. Three groups of animals were each injected with 3 µl of cricket saline or saline containing 1 mM or 10 mM cycloheximide, a protein synthesis inhibitor, into the hemolymph of the head at 20 min prior to 8-trial conditioning. The relative preference between the conditioned pattern and control pattern was tested before and 1 day after conditioning. The group injected with saline exhibited significantly increased preference for the conditioned pattern at 1 day after conditioning compared to that before conditioning (Figure 2A; W=72, p=0.0039, WCX test, the sample number shown in legends). On the other hand, the group injected with 10 mM cycloheximide exhibited no significant level of 1-day memory retention (W=117, p=0.77, WCX test). The group injected with 1 mM cycloheximide exhibited a significant level of 1-day retention (W=9, p=0.0010, WCX test), indicating that the effect of cycloheximide is dose-dependent. We conclude that memory at 1 day after 8-trial visual conditioning can be characterized as protein synthesis-dependent LTM.


Roles of NO signaling in long-term memory formation in visual learning in an insect.

Matsumoto Y, Hirashima D, Terao K, Mizunami M - PLoS ONE (2013)

Effects of cycloheximide on formation of 1-day memory after visual pattern conditioning.(A) Three groups of animals were each subjected to injection of 3 μl of saline or saline containing 1 mM or 10 mM cycloheximide (CHX) at 20 min prior to 8-trial conditioning in which a visual pattern was paired with water reward. Relative preference between the rewarded pattern and control pattern was tested before and at 1 day after training. (B) Another two groups were each injected with 3 μl of saline or saline containing 10 mM CHX at 20 min prior to 8-trial visual conditioning. Relative preference between the rewarded pattern and control pattern was tested before and at 30 min after training. Preferences indexes (PIs) for the rewarded patterns before (white bars) and after (grey bars) training are shown as box and whisker diagrams and are statistically compared. The line in the box is the median and the box represents the 25-75 percentiles. Whiskers extend to extreme values as long as they are within a range of 1.5× box length from the upper or lower quartiles. Any data not included between the whiskers are plotted as outliers with dots. The results of statistical comparisons of visual pattern preferences before and after training are shown by asterisks (*** P<0.001, ** P<0.01, NS P>0.05, WCX test). The number of animals tested is shown at each data point in this figure and in subsequent figures.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3722230&req=5

pone-0068538-g002: Effects of cycloheximide on formation of 1-day memory after visual pattern conditioning.(A) Three groups of animals were each subjected to injection of 3 μl of saline or saline containing 1 mM or 10 mM cycloheximide (CHX) at 20 min prior to 8-trial conditioning in which a visual pattern was paired with water reward. Relative preference between the rewarded pattern and control pattern was tested before and at 1 day after training. (B) Another two groups were each injected with 3 μl of saline or saline containing 10 mM CHX at 20 min prior to 8-trial visual conditioning. Relative preference between the rewarded pattern and control pattern was tested before and at 30 min after training. Preferences indexes (PIs) for the rewarded patterns before (white bars) and after (grey bars) training are shown as box and whisker diagrams and are statistically compared. The line in the box is the median and the box represents the 25-75 percentiles. Whiskers extend to extreme values as long as they are within a range of 1.5× box length from the upper or lower quartiles. Any data not included between the whiskers are plotted as outliers with dots. The results of statistical comparisons of visual pattern preferences before and after training are shown by asterisks (*** P<0.001, ** P<0.01, NS P>0.05, WCX test). The number of animals tested is shown at each data point in this figure and in subsequent figures.
Mentions: First, we studied whether the formation of 1-day memory after visual conditioning can be characterized as protein synthesis-dependent LTM. We used 8-trial conditioning to associate a visual pattern with water reward, with an inter-trial interval of 5 min, because we observed that it leads to formation of 1-day memory, whereas 4-trial conditioning leads to formation of 30-min memory but not 1-day memory [11]. Three groups of animals were each injected with 3 µl of cricket saline or saline containing 1 mM or 10 mM cycloheximide, a protein synthesis inhibitor, into the hemolymph of the head at 20 min prior to 8-trial conditioning. The relative preference between the conditioned pattern and control pattern was tested before and 1 day after conditioning. The group injected with saline exhibited significantly increased preference for the conditioned pattern at 1 day after conditioning compared to that before conditioning (Figure 2A; W=72, p=0.0039, WCX test, the sample number shown in legends). On the other hand, the group injected with 10 mM cycloheximide exhibited no significant level of 1-day memory retention (W=117, p=0.77, WCX test). The group injected with 1 mM cycloheximide exhibited a significant level of 1-day retention (W=9, p=0.0010, WCX test), indicating that the effect of cycloheximide is dose-dependent. We conclude that memory at 1 day after 8-trial visual conditioning can be characterized as protein synthesis-dependent LTM.

Bottom Line: In this study, we examined the possible contribution of NO-cGMP signaling and cAMP signaling to LTM formation in visual pattern learning in crickets.The results indicate that cAMP signaling is downstream of NO signaling for visual LTM formation.We conclude that visual learning and olfactory learning share common biochemical cascades for LTM formation.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Life Science, Hokkaido University, Sapporo, Japan.

ABSTRACT
Many insects exhibit excellent capability of visual learning, but the molecular and neural mechanisms are poorly understood. This is in contrast to accumulation of information on molecular and neural mechanisms of olfactory learning in insects. In olfactory learning in insects, it has been shown that cyclic AMP (cAMP) signaling critically participates in the formation of protein synthesis-dependent long-term memory (LTM) and, in some insects, nitric oxide (NO)-cyclic GMP (cGMP) signaling also plays roles in LTM formation. In this study, we examined the possible contribution of NO-cGMP signaling and cAMP signaling to LTM formation in visual pattern learning in crickets. Crickets that had been subjected to 8-trial conditioning to associate a visual pattern with water reward exhibited memory retention 1 day after conditioning, whereas those subjected to 4-trial conditioning exhibited 30-min memory retention but not 1-day retention. Injection of cycloheximide, a protein synthesis inhibitor, into the hemolymph prior to 8-trial conditioning blocked formation of 1-day memory, whereas it had no effect on 30-min memory formation, indicating that 1-day memory can be characterized as protein synthesis-dependent long-term memory (LTM). Injection of an inhibitor of the enzyme producing an NO or cAMP prior to 8-trial visual conditioning blocked LTM formation, whereas it had no effect on 30-min memory formation. Moreover, injection of an NO donor, cGMP analogue or cAMP analogue prior to 4-trial conditioning induced LTM. Induction of LTM by an NO donor was blocked by DDA, an inhibitor of adenylyl cyclase, an enzyme producing cAMP, but LTM induction by a cAMP analogue was not impaired by L-NAME, an inhibitor of NO synthase. The results indicate that cAMP signaling is downstream of NO signaling for visual LTM formation. We conclude that visual learning and olfactory learning share common biochemical cascades for LTM formation.

Show MeSH
Related in: MedlinePlus