**3. Signaling pathway involved in epicatechin effect**

EpiC effects on LTM formation may be due to its ability to drive an increase in intracellular kinase activity [51] in neurons such as RPeD1. It has also been shown that activation of CREB is necessary for LTM formation in *Lymnaea* [52] and EpiC increases CREB-regulated gene expression in neurons [53]. Further, there is increasing evidence implying that EpiC can drive rapid signaling intracellular as it increases phosphorylation of protein kinase B (Akt)/PI3K, PKC and Erk MAPK and induces cellular survival/proliferation in human hepatoma cells [54]. This is important since LTM following operant conditioning in *Lymnaea* requires activations of PKC and MAPK [55]. In addition, EpiC appears to be able to directly alter DNA methylation activity [56], which has been shown in *Lymnaea* to alter LTM formation [57]. EpiC

has been shown in the mammalian brain to cross the blood brain barrier and directly affect CNS function possibly by enhancing 5HT function [58]. EpiC may also activate NOS and stimulate NO production [53]. It is known in the *Lymnaea* model system that 5HT and NO are involved in LTM formation [50, 59]. It remains to be elucidated whether EpiC brings about its enhancing effects on LTM formation via these molecules. EpiC effects on cognitive enhancement in mammalian preparations has been shown, but it is unclear whether the enhanced cognitive benefit is directly due to altering neuronal activity or through effects on blood flow to the brain as a result of increased angiogenesis [8].

It is reported that exposure to crayfish effluent (CE), which also enhances LTM formation and significantly decreases RPeD1 excitability [60], works a serotonergic pathway that can be blocked by mianserin, a serotonin receptor antagonist [50]. As previously shown, however, mianserin does not affect the LTM formation enhancement induced by EpiC [4]. In addition, once the osphradial nerve that connects the osphradium (a sensory organ) to the CNS is severed, CE no longer enhances LTM formation [50]. Thus, the osphradial nerve must be intact in order to cause enhancement of LTM formation by exposing CE. Whereas, EpiC enhanced LTM formation after severing the osphradial nerve [4]. Thus, it appears that EpiC acts via a different mechanism and a different pathway from those caused by the perception of CE.

McComb and collaborators demonstrated the memory formation by using *in vitro* semi-intact preparations [26]. After operant conditioning of intact snails, semi-intact preparations were dissected so that changes in the respiratory behavior (pneumostome openings) and underlying activity of the identified CPG neuron, RPeD1, could be monitored simultaneously.

Our group can perform "*in vitro*" operant conditioning in semi-intact preparations from naïve snails. In the training, we applied a gentle tactile stimulus to the pneumostome area whenever the snail began to open it. Following the training, the respiratory behavior decreased. After the training, naïve snails exposed to EpiC (15 mg/L) prior to recording exhibited significantly increased RPeD1 excitability compared with non-exposed snails. This experiment can help to understand how EpiC alters RPeD1 excitability to drive aerial respiratory behavior and leads to enhanced LTM formation.

These results provide the basis of future studies in *Lymnaea* to elucidate how EpiC enhances LTM formation of respiratory conditioning.

## **4. Effects of intaking of catechin-rich foods**

Does exposure to food products containing EpiC during the training elicit similar effects seen for exposure to pure EpiC? Lukowiak et al. demonstrated interesting experiments whether foods containing substantial amounts of EpiC, such as green tea, cocoa, apple peel and black tea, can enhance memory formation in *Lymnaea* [7]. Exposed to pond water containing green tea or pure cocoa powder in concentration comparable to human consumption level (approximately 1 g/day) during training, the memory enhancement was comparable to that elicited by pure EpiC experiments [7].

Interestingly, black tea does not only enhance LTM formation but suppresses LTM formation in *Lymnaea* [61]. Black tea is made from the same plant as green-tea through an oxidation process called "fermentation" and becomes stronger in flavor than green tea. However, the content of EpiC in black tea (0.49 mg/100 g) reduces compared with green tea (6.16 mg/100 g) [3]. Black tea substantially contains more other flava-3-nols, thearubigins and theaflavins, than green tea [3, 62]. These flava-3-nols are formed during the fermentation reaction in black tea. As far as we

#### *Green Tea-Derived Catechins Have Beneficial Effects on Cognition in the Pond Snail DOI: http://dx.doi.org/10.5772/intechopen.99789*

know, no studies have investigated direct effects of these flava-3-nols on memory formation. However, it is reported that intake of theaflavins is associated with longterm language and verbal memory in human [63]. In another study, theaflavins are reported to improve memory impairment [64, 65]. Thus, thearubigins and theaflavins may be not candidate substances for blocking memory formation in black tea.

Another component of black tea, caffeine, can inhibit cognitive function. In drosophila, caffeine reduces the performance for light aversive conditioning [66]. However, both green tea and black tea contain a same amount of caffeine. It is possible that L-theanine included in green tea in comparatively large amounts is thought to balance the effects of caffeine. The combination of these two substances may be synergistic, as one study found that people who ingested L-theanine and caffeine together had better attention than when either was used alone [67, 68]. Therefore, investigating catechin-rich foods is difficult to permit a full understanding of the specific effect of these phytochemicals.

#### **5. Rescue effect of epicatechin on stress-impaired memory**

Green tea-derived catechins do not only enhance memory formation, but also rescue impaired cognitive functions due to environmental stressors. Catechin-rich foods have been considered to improve various aspects of cognitive functions in rodents and humans, and some reports suggest that it has positive effects on mild cognitive impairment [69–71]. EpiC administration improves spatial memory in mice via an increase in cerebral angiogenesis or a direct effect on neural elements [8]. In *Lymnaea*, there are some reports for the recovery effect of EpiC on impaired function by environmental stressor [5, 38].

Most of freshwater mollusks including *Lymnaea* are dependent on calcium intake directly from the environment through their skin [72] and exhibit reduced shell growth in the environment containing less than 20 mg/L calcium [73, 74]. It is considered that this level of calcium acts as a stressor on the snail. Following 1 h exposure to a low calcium environment, *Lymnaea* was not able to form LTM, although it still had an ability of ITM [75]. Following a 2 h-TS in EpiCsupplemented low-calcium pond water, snails persist a decrease of respiratory behavior both 24 hours and 72 hours after the training [5]. In addition, memory formation of the training in EpiC-suppelmented pondwater was not diminished by the combination of a low- calcium pond water environment and 1 hour of crowding immediately prior to operant conditioning training, which blocks all forms of memory (short-term, intermediate-term and long-term memory) in *Lymnaea* [38]. These results suggest that EpiC reverses an imposed memory deficit by exposure to memory 'unfriendly' stress.

### **6. Enhancing effect of epicatechin on memory formation by classical conditioning of feeding behavior**

*Lymnaea* can be classically, as well as operantly, conditioned [28, 29]. Conditioned taste aversion (CTA) is a classical conditioning, which is based on pairing sucrose as a conditioned stimulus (CS) with an aversive chemical unconditioned stimulus (UCS) such as KCl, which inhibits feeding and evokes a withdrawal response. After this procedure, trained snails show a significantly weaker feeding response to sucrose than controls. We here introduced the enhancing effect of EpiC on LTM formed by CTA. In the previous study, we showed that EpiC increases the persistence of LTM as mentioned below [76].

#### *Update on Malacology*

CTA training procedure we performed is briefly as follows. Adult snails randomly chosen were food deprived for 24 hours before being subjected to CTA training. Snails were then immersed in an appetitive solution (10 mM sucrose) for 15 s. Then, the sucrose solution was quickly replaced with distilled water, and the feeding response (i.e. number of bites) was measured in distilled water for 5 minutes (pre-test). Ten minutes after the pre-test, CTA training was performed. In CTA training, snails were immersed for 15 s in 10 mM sucrose, which were immediately immersed for 15 s in 10 mM KCl solution (i.e. the UCS). The UCS

#### **Figure 4.**

*Change in feeding response after conditioned taste aversion (CTA) training. (A) Histogram showing the decreasing rate of the feeding response at the 24 h post-test n = 48). The cumulant (the ratio of the cumulative number of snails at each rate, from low to high, out of the total number, n = 48; black circles) is also shown for reference. Snails that showed at least a 40% decrease in the number of bites were defined as good learners, while poor learners were defined as snails whose post-test scores decreased by less than 40%. (B) Feeding responses in the pre- and post-tests for good learners trained without Epi (control, blue circles, n = 14) and with Epi (green circles, n = 12). \*\*\*\*P < 0.0001, \*P < 0.05. These figures are reproduced from [76] with permission.*

#### *Green Tea-Derived Catechins Have Beneficial Effects on Cognition in the Pond Snail DOI: http://dx.doi.org/10.5772/intechopen.99789*

.inhibits the feeding response. After the UCS was presented, snails were immersed either in distilled water (control) or EpiC solution (15 mg/L) for 9.5 minutes. This procedure was repeated 5 times. After CTA training, snails were kept in distilled water for 24, 48 or 72 hours and then the post-test was performed, which was exactly the same as the pre-test. By comparing the number of bites in the pretest with that in the 24 h post-test, we determined whether the snail was a 'good' learner or a 'poor' learner.

**Figure 4A** shows a histogram of the decreasing rate of the feeding response in the 24 h post-test (i.e. 24 hours after training). The decreasing rate was measured for 26 snails trained without EpiC and 22 snails trained with EpiC, and the data from all snails (n = 48) are combined in the histogram. As shown in **Figure 4A**, from their responses, the snails were roughly divided into two groups: 'good' and 'poor' learners. Good learners were defined as snails that showed at least a 40% decrease in the number of bites in the 24 h post-test compared with that in the pre-test. Thus, poor learners were defined as snails whose post-test scores decrease by less than 40%. In this data, 32 of the 48 snails (i.e. 67%) were classified as good learners.

For the good learners, we statistically analyzed on the data presented in **Figure 4B** (control group, n = 14; EpiC group, n = 12). In both groups, snails showed a significant decrease in the number of bites in the 24 h post-test (control group, 37.3 ± 3.5 to 8.8 ± 1.6 bites per 5 min, *P* < 0.0001; EpiC group: 39.9 ± 2.6 to 12.3 ± 2.4 bites per 5 min, *P* < 0.0001). Thus, placing snails in the EpiC solution in CTA training did not alter their 24 h memory performance.

We next compared the post-test scores between two groups (control group versus EpiC group) at 24, 48 and 72 hours after CTA training. A statistical analyze showed that there was a significant difference in the memory scores at 72 hours between the two groups (control group versus EpiC group, *P* < 0.05) while there was no significant difference in the 24 h and 48 h post-tests. Thus, we concluded that exposing snails to EpiC solution resulted in significantly longer memory persistence.

An identified spontaneously active pair of neurons, the cerebral giant cells (CGCs), has been shown to both modulate the neuronal network underling the feeding behavior and be necessary for LTM and its retrieval following CTA training (**Figure 4**) [31, 32]. Therefore, a possible mechanism underlying the significant effect of EpiC on LTM persistence is an alteration in CGC activity. Our data supported this possibility [76]. Additionally, our data suggested that a GABAergic neuron may play a significant role in mediating CTA-LTM and the EpiC effect on the CGC may involve a GABAergic neuron. For example, the GABA sensitivity of a neuron (maybe the CGC itself) might be enhanced in good learners or in snails exposed to EpiC.

### **7. Conclusion**

Studies described in this chapter have provided valuable information on a possibility of EpiC-rich foods contributing to cognition ability in *Lymnaea*. In addition, animal models in *Lymnaea* contribute to new evidences for the generality of mechanisms for the effects of EpiC on learning and memory formation, across learning paradigms (e.g. classical or operant conditioning).

These studies suggest that EpiC has not only antioxidant properties but also targets molecules (e.g. specific receptors) directly to affect the signaling pathway. Then, the results may yield the basis of future studies to elucidate how EpiC enhances LTM formation of classical and operant conditioning in *Lymnaea*.

*Update on Malacology*
