Role of TRPC channels in temporal association tasks

The first study, which involved a a conditional knock out of TRPC 5 channel expression using an eGFP-Cre fusion protein introduced via virus injection, partially showed the predicted results. Both, training day and tone test day showed differences in freezing levels between CTRL and EXP, with EXP showing lower level of freezing score. That was especially visible during the tone epochs, which, together with the trace epochs during the training day, are expected to be the periods most affected by PF. It still remains unclear if post-tone interval during the second day of protocol, which are expressed as ITI in the graph (Fig. 8), did not reach significance due to the number of training trials during the first day, that could have appealed to a compensation mechanism, or because of non optimal protocol execution. However, Cohen’s d measure of effect size was elaborated to test the statistical weight of each subject within group, and it always showed reliability of the results.
The second study (cannula implant) clearly showed no-significant result.

Mice were successful conditioned. However, no trends were visible and CTRL and EXP had very similar performance. We believe that the principal cause of such a result is the one discussed above: The drugs were not properly tested, and most of their effects under certain conditions were not well-established. Thus, for the next attempt it is reasonable to conduct a pilot study in which the drugs and their effects will be further studied first in vitro and then in vivo.

A more complex view: The possibility of an intervention by a compensation mechanism in order to explain learning process that occurred in the EXP after the third trial of the training day (Fig. 9) opens to new questions about the system that underlies temporal association memory, which might be more complex and not only relying on TRPC 4 and 5 channels activity.
There is evidence in rat that only a total hippocampectomy is able to disrupt auditory fear conditioning (McEchron et al., 1998). Moreover, another group found that also ventral hippocampus and not only the dorsal part of it, was involved in trace fear conditioning impairments (Gilmartin, Kwapis & Helmstetter, 2012).

A recent study pointed worse freezing performance in TRPC1 KO mice during a trace fear conditioning task out. They explained the result basing on other mechanisms and not relying on PF (Xing et al., 2016). However, they also claimed that the deficit caused by the inactivation of TRPC1 channels could be rescued by an environmental enriched intervention (EE). It is well established that EE brings the development of more and longer dendritic connections (Holloway Jr., 1966) and it has been recently shown that TRPC 4 and TRPC 5 channels have an important role in dendritic growth process (Puram et al., 2016). All these findings seem to strengthen the theory that TRPC channels, especially TRPC 4/5, have a central role in temporal memory but at the same time, they might not act alone.

From another point of view, Riccio and colleagues (2009) demonstrated that TRPC4/5 KO mice seem to show diminished innate fear, which was tested in tasks such as elevated plus-maze, open field, social interaction, and novelty suppressed feeding tests. However, in the same study, 24 hours after a classical fear conditioning protocol, TRPC5 KO mice showed lower freezing score in response to the CS when compared to control mice, suggesting the participation of TRPC5 channels in the mechanism of conditioned fear memory, at least under certain training conditions. Moreover, this finding could rely only on amygdala TRPC4 and 5 blockage but, as it could have had impacts on our results, should be further investigated also in dorsal hippocampal area.
Controversal studies showed that TRPC5 KO mice have motor coordination problems (Riccio et al., 2014) whereas the same group in another experiment tested motor function in TRPC5 KO mice, not revealing any deficit. (Riccio at al., 2009).
A similar problem was found in the literature with two different studies regarding LTP. Based on our hypothesis, TRPC4 and TRPC5 channels ,as a base mechanism for temporal association memory encoding, should impair LTP.
One study affirms that no detectable difference between TRPC4/5 KO mice and wild type mice were found in LTP performance (Riccio et al., 2014); but again, this discovery referred to amygdala and not to hippocampus. On the other hand, Phelan and colleagues (2013) found that TRPC1 channels did not effect LTP, while TRPC5 contributed to LTP.