Roles of calcium/calmodulin-dependent kinase II in long-term memory formation in crickets

Abstract

Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a key molecule in many systems of learning and memory in vertebrates, but roles of CaMKII in invertebrates have not been characterized in detail. We have suggested that serial activation of NO/cGMP signaling, cyclic nucleotide-gated channel, Ca(2+)/CaM and cAMP signaling participates in long-term memory (LTM) formation in olfactory conditioning in crickets, and here we show participation of CaMKII in LTM formation and propose its site of action in the biochemical cascades. Crickets subjected to 3-trial conditioning to associate an odor with reward exhibited memory that lasts for a few days, which is characterized as protein synthesis-dependent LTM. In contrast, animals subjected to 1-trial conditioning exhibited memory that lasts for only several hours (mid-term memory, MTM). Injection of a CaMKII inhibitor prior to 3-trial conditioning impaired 1-day memory retention but not 1-hour memory retention, suggesting that CaMKII participates in LTM formation but not in MTM formation. Animals injected with a cGMP analogue, calcium ionophore or cAMP analogue prior to 1-trial conditioning exhibited 1-day retention, and co-injection of a CaMKII inhibitor impaired induction of LTM by the cGMP analogue or that by the calcium ionophore but not that by the cAMP analogue, suggesting that CaMKII is downstream of cGMP production and Ca(2+) influx and upstream of cAMP production in biochemical cascades for LTM formation. Animals injected with an adenylyl cyclase (AC) activator prior to 1-trial conditioning exhibited 1-day retention. Interestingly, a CaMKII inhibitor impaired LTM induction by the AC activator, although AC is expected to be a downstream target of CaMKII. The results suggest that CaMKII interacts with AC to facilitate cAMP production for LTM formation. We propose that CaMKII serves as a key molecule for interplay between Ca(2+) signaling and cAMP signaling for LTM formation, a new role of CaMKII in learning and memory.

Figure 1. Effects of CaMKII inhibitors on 1-day retention.

Two control groups were each injected with 3 µl of saline or saline containing 1% DMSO (designated as saline (D)) 20 min prior to 3-trial conditioning. Another four groups were each injected with 3 µl of saline containing 2 mM KN-62, 2 mM KN-93, 500 µM KN-62 or 500 µM KN-93 (dissolved in 1% DMSO) 20 min prior to 3-trial conditioning. Relative preference between the rewarded odor and control odor was tested before and at 1 day after training. Preferences indexes (PIs) for the rewarded odor before (white bars) and after (grey bars) training are shown as box and whisker diagrams. 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. Odor preferences before and after training are compared by WCX test. Odor preferences after training of different groups were compared by the M-W test. The results of statistical comparisons are shown by asterisks (*** P<0.001, ** P<0.01, * P<0.05, NS P>0.05, adjusted by Holm's method). The number of animals tested is shown at each data point in this figure and in subsequent figures.

Figure 2. No effects of CaMKII inhibitors on 1-hour retention.

Three groups of animals were each injected with 3 µl of saline containing 1% DMSO (designated as saline (D)) or saline containing 2 mM KN-62 or 2 mM KN-93 (dissolved in 1% DMSO) 20 min prior to 3-trial conditioning. Relative preference between the rewarded odor and control odor was tested before and at 1 hour after training. PIs for the rewarded odor before (white bars) and after (grey bars) training are shown as box and whisker diagrams. Odor preferences before and after training are compared by WCX test. Odor preferences after training of different groups were compared by the M-W test. The results of statistical comparisons are shown by asterisks (*** P<0.001, NS P>0.05, adjusted by Holm's method).

Figure 3. Effective time window of injection of a CaMKII inhibitor.

Two groups of animals were each injected with 3 µl of saline containing 1% DMSO (saline (D)) or saline containing 2 mM KN-62 (dissolved in 1% DMSO) 60 min prior to 3-trial conditioning. Another two groups of animals were each injected with 3 µl of saline (D) or saline containing 2 mM KN-62 20 min after 3-trial conditioning. Relative preference between the rewarded odor and control odor was tested before and at 1 day after training. PIs for the rewarded odor before (white bars) and after (grey bars) training are shown as box and whisker diagrams. Odor preferences before and after training were compared by WCX test and odor preferences after training of different groups were compared by the M-W test. The results of statistical comparisons are shown by asterisks (** P<0.01, * P<0.05, NS P>0.05, adjusted by Holm's method).

Figure 4. Effects of co-injection of a CaMKII inhibitor and 8br-cGMP or CaMKII inhibitor and 8br-cAMP on LTM formation.

Five groups of animals were each injected with 3 µl of saline or saline containing 8br-cGMP (500 µM), 8br-cAMP (200 µM), 8br-cGMP (500 µM) and KN-62 (2 mM) or 8br-cAMP (500 µM) and KN-62 (2 mM) 20 min prior to 1-trial conditioning. KN-62 was dissolved in DMSO (1%). Relative preference between the rewarded odor and control odor was tested before and at 1 day after training. PIs for the rewarded odor before (white bars) and after (grey bars) training are shown as box and whisker diagrams. Odor preferences before and after training are compared by WCX test. Odor preferences after training of different groups were compared by the M-W test. The results of statistical comparisons are shown by asterisks (*** P<0.001, ** P<0.01, * P<0.05, NS P>0.05, adjusted by Holm's method).

Figure 5. Effects of co-injection of a CaMKII inhibitor and ionomycin on LTM formation.

Four groups of animals were each injected with 3 µl of saline or saline containing 20 µM ionomycin, 200 µM ionomycin, or 200 µM ionomycin and 2 mM KN-62 20 min prior to 1-trial conditioning. The saline contained 1% DMSO. Relative preference between the rewarded odor and control odor was tested before and at 1 day after training. PIs for the rewarded odor before (white bars) and after (grey bars) training are shown as box and whisker diagrams. Odor preferences before and after training are compared by WCX test. Odor preferences after training of different groups were compared by the M-W test. The results of statistical comparisons are shown by asterisks (** P<0.01, * P<0.05, NS P>0.05, adjusted by Holm's method).

Figure 6. Effects of co-injection of a CaMKII inhibitor and forskolin on LTM formation.

Five groups of animals were each injected with 3 µl of saline containing 200 µM 1,9-dideoxyforskolin, 200 µM forskolin, 20 µM forskolin, 200 µM forskolin and 2 mM KN-62 or 200 µM forskolin and 2 mM KN-93 20 min prior to 1-trial conditioning. All of these drugs were dissolved in DMSO (1%). Relative preference between the rewarded odor and control odor was tested before and at 1 day after training. PIs for the rewarded odor before (white bars) and after (grey bars) training are shown as box and whisker diagrams. Odor preferences before and after training are compared by WCX test. Odor preferences after training of different groups were compared by the M-W test. The results of statistical comparisons are shown by asterisks (*** P<0.001, NS P>0.05, adjusted by Holm's method).

Figure 7. A model of the biochemical pathways for LTM formation in crickets.

A model proposed for the biochemical pathways for olfactory LTM formation in crickets, in which our previous model , was modified to account for findings in this study. Single-trial conditioning induces only short-term synaptic plasticity that underlies amnesic treatment-sensitive short-term memory (STM) and amnesic treatment-resistant mid-term memory (MTM) . Multiple-trial conditioning activates NO/cGMP signaling, and this activates cyclic nucleotide-gated (CNG) channel, Ca²⁺/CaM, CaMKII and then adenylyl cyclase (AC)/cAMP/PKA signaling. This in turn activates cAMP responsive element-binding protein (CREB), which results in transcription and translation of genes that are necessary for achieving long-term plasticity of synaptic connection (a column of gray triangles) upon other neurons that underlies LTM. Thus, CaMKII intermediates between Ca²⁺ signaling and cAMP signaling.