Inventory of Supplemental Materials
Supplemental Methods
Supplemental Figure 1
RT-PCR analysis of 5-ht3ar from the total RNA of mouse brains.
Supplemental Table 1
Behavior in the elevated plus maze test.
Supplemental References
Supplemental Methods
Mice
The generation of 5-ht3ar-/- mice has been described previously (Zeitz et al. 2002) and these mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). We confirmed no expression of 5-HT3A receptor in the brains of 5-ht3ar-/- mice by RT-PCR analysis (Supplemental Fig. 1), consistent with previous reports (Zeitz et al. 2002). The mice had been backcrossed to C57BL/6J mice for six to ten generations. For behavioral experiments, we used 10- to 12-week-old male wild-type (C57BL/6J) and 5-ht3ar-/- mice. Two or three naive mice were housed per cage and all behavioral experiments were performed between 9:00 and 17:00.
RT-PCR analysis
RT-PCR analysis was performed as previously described (Kondo et al.2012). Total RNA was isolated from mouse whole brain (beyond doubt, including the hippocampus, amygdala, and frontal cortex) using ISOGEN (Nippon gene), and cDNA was synthesized using reverse transcriptase (Toyobo) according to the manufacturers’ instructions. Then, cDNA samples were subjected to amplification with AmpliTaq DNA Polymerase (Applied Biosystems) in a programmable thermal cycler with denaturation at 94°C for 30 sec, annealing at 57°C for 30sec, and extension at 72°C for 30 sec for 35 cycles. The PCR primers were as follows: for the 5-HT3A receptor, 5’-CCC AGT TCA AGG AGT TCA GC-3’ and 5’-CCC AGA AGG AGT GTG ATC TTG-3’; for actin (as a control), 5’-CTG TAT TCC CCT CCA TCG TG-3’and 5’-GGT GTT GAA GGT CTC AAA CAT G-3’.
Fear conditioning test
The fear conditioning test, which assesses a form of associative learning between an aversive footshock (unconditioned stimulus; US) and a conditioned stimulus (CS), was performed as previously described (Kondo et al. 2012) with slight modifications. On the conditioning session day (defined as ‘Day 0’), mice were placed in the conditioning chamber (Context A) for 120 s, and then presented with a tone (CS: 85 dB, 3000 Hz) for 30 s. At the end of the tone presentation, the mice were given a footshock (US: 2 sec, 0.6 mA). One more tone-shock pairing was presented with a 120 s inter-stimulus interval. Thirty seconds after the final footshock, the mice were returned to their home cages. After the conditioning day, the procedures for the contextual and tone-cued fear tests were different. For the contextual fear test, the mice were placed in the conditioning chamber (Context A), whereas for the tone-cued fear test, the mice were placed in a novel chamber (Context B) with contexts different from those of the conditioning chamber, and then exposed to tone presentation. Freezing was defined as no movement other than respiration, and was recorded using a 5-second sampling method (Wehner and Radcliffe 2004; Joseph et al. 2013).
Fear extinction protocol
The fear conditioning session was performed in the conditioning chamber (Context A) on Day 0 as described above.Subsequently, the mice were subjected to a daily extinction trial.Freezing responses were recorded on 6 consecutive days (Days 1–6) (Sananbenesi et al. 2007). In the first extinction paradigm, which assessed the extinction process of contextual fear, each trial consisted of a 5 min re-exposure to the conditioned context (context A) without the footshock. Freezing responses on each extinction day were recorded. In the second extinction paradigm, which assessed the extinction process of tone-cued fear, each trial consisted of a 3 min exposure to the tone presentation in a novel chamber (context B) without the footshock. Tone-cued freezing responses on each extinction day were recorded.
Supplemental Figure 1
RT-PCR analysis of 5-ht3ar from the total RNA of mouse brains.
Supplemental Table 1
Behavior in the elevated plus maze test.
WT (n = 12) / KO (n = 12) / Two-tailed t-testTotal distance traveled (cm) / 106.9 ± 9.9 / 92.0 ± 5.5 / ns / p = 0.2024
Time in open arms (%) / 18.71 ± 1.89 / 29.74 ± 4.31 / * / p = 0.0333
Entries into open arms (%) / 40.49 ± 1.56 / 47.77 ± 2.81 / * / p = 0.0337
Time in closed arms (%) / 64.66 ± 2.44 / 54.72 ± 3.90 / * / p = 0.0417
Entries into closed arms (%) / 59.51 ± 1.56 / 52.23 ± 2.81 / * / p = 0.0337
*p < 0.05; ns, not significant (two-tailed t-test). Values are means ± SEM.
Supplemental References
Kondo M, Takei Y, Hirokawa N. 2012. Motor protein KIF1A is essential for hippocampal synaptogenesis and learning enhancement in an enriched environment. Neuron73: 743–757.
Joseph A, Tang M, Mamiya T, Chen Q, Yang LL, Jiao J, Yu N, Tang YP. 2013. Temporal association of elevated cholecystokininergic tone and adolescent trauma is critical for posttraumatic stress disorder-like behavior in adult mice. Proc Natl Acad Sci USA110: 6589–6594.
Sananbenesi F, Fischer A, Wang X, Schrick C, Neve R, Radulovic J, Tsai LH. 2007. A hippocampal Cdk5 pathway regulates extinction of contextual fear. Nat Neurosci10: 1012–1019.
Wehner JM, Radcliffe RA. 2004. Cued and contextual fear conditioning in mice. Curr Protoc Neurosci8.5C.1–8.5C.14
Zeitz KP, Guy N, Malmberg AB, Dirajlal S, Martin WJ, Sun L, Bonhaus DW, Stucky CL, Julius D, Basbaum AI. 2002. The 5-HT3 subtype of serotonin receptor contributes to nociceptive processing via a novel subset of myelinated and unmyelinated nociceptors. J Neurosci22: 1010–1019.
1