Male Sprague-Dawley rats (Animal House Center, Kunming Medical University, China), weighing 250–300 g, 11–12 weeks old, were used. Rats were individually housed in home cages with ad libitum access to water and food (complying with the Chinese rat food standard GB 14924.3-2001) and subjected to a 12-h light/12-h dark cycle (lights on at 7 am) in a temperature-regulated room. Rats were allowed 1 week to acclimate our research facility before manipulations. All experiments were performed between 09:00 and 12:00. This study was carried out in accordance with the recommendations of the National Institutes of Health on experimental animal care and use. All experimental protocols were approved by the Animal Ethics Committee of the Kunming Institute of Zoology, Chinese Academy of Sciences.

The protocol of cannula implantation was similar to our previous study (Zhou et al., 2016). Surgery was performed under the anesthesia of pentobarbital sodium salt (Sigma, USA, dissolved in saline, 60 mg/kg body weight, I.P.). Rats were ventilated with 95% O₂/5% CO₂ through a mask and mounted in a stereotaxic apparatus (RWD Life Science Co., Shenzhen, China). Small holes were bilaterally drilled in the skull for the placement of two stainless-steel guide cannulae (7 mm length, 26 gauges) in the dorsal hippocampus (DH). The cannula tips were targeted to 1 mm above the DH with the coordinate of anteroposterior = −3.8 mm from the bregma, mediolateral = ±2.8 mm, dorsoventral = −3.0 mm. Cannula were affixed to the skull using dental cement. A stylet was introduced into the guide cannula to prevent possible obstruction. The rats were allowed to recover for 7 days following surgery and handled for 3 days before behavioral experiments. To test the role of hippocampal THs in our behavioral paradigm, LT₄ solution (Sigma, 0.4 μg/μl, saline as vehicle, 1 μl/side) was bilaterally injected into the hippocampus (I.H.) using a microsyringe pump at the speed of 0.2 μl/min with an injection pipe via cannula. After all behavioral tests, an injection pipe used in the experiments was inserted into the cannula for infusing 1 μl trypan blue as a marker to test the accuracy of cannula placement in each rat.

The rats were anesthetized using ether at all scheduled time points after intraperitoneal drug administration (I.P.), and their heart blood samples were obtained to test serum levels of free tetraiodothyroxine (FT₄) and free triiodothyronine (FT₃). For collecting brain tissues, rats were perfused from left ventricle with saline 500 ml for 30 min after anesthesia of pentobarbital sodium salt (Sigma, 60 mg/kg body weight). Immediately at the end of saline perfusion, dissection of their hippocampus and prefrontal cortices (PFC) were followed (Chiu et al., 2007). Sample of bilateral hippocampus or PFC was individually homogenized in artificial cerebral spinal fluid (ACSF; Mahmoud and Amer, 2014) by the ratio of 1 μg/4 μl (brain tissues/ACSF) in an ice surrounded homogenizer and centrifuged at 14,000 rpm for 15 min at 4°C. The resulting supernatant was collected and stored at −80°C until the determination of FT₄ and FT₃.

FT₄ and FT₃ were tested using time-resolved fluoroimmunoassays (TRFIA). Diagnostic kits for FT₄ and FT₃ (SYM Bio Lifescience Co., LTD, Suzhou, China) were used in conjunction with a multilabel counter (Wallac Vicotor 2 TM 1420 multilabel counter, Turku, Finland) to detect THs levels according to the manufacturer’s instructions. The detection ranges for FT₄ and FT₃ were 0–74 pmol/L and 0–54 pmol/L, respectively. Whenever the concentration of a particular hormone exceeded the corresponding upper limit, the serum was further diluted with saline to achieve a measurable concentration. All tests were done in triplicate.

Locomotor activity and thigmotaxis of rats were tested in an open field at 24 H following LT₄ or saline administration (I.P. or I.H., saline as vehicle). The rats were individually placed in the center of an open field (43 cm × 43 cm × 30 cm with virtual central zone in it, ENV-017M-027, Med Associates, Inc., St. Albans, VT, USA) and allowed to explore freely for 20 min. Rat movement was tracked using three 16-beam infrared arrays around the rat arena. The open field was cleaned with 5% ethanol after each test. The running distance reflected the locomotor activity and was automatically analyzed using the Med Associates software after test. Thigmotaxis was presented as the percentage (%) of the total distance moved (TDM) in the central zone of the test apparatus. Specifically, thigmotaxis was calculated as the ratio between TDM in the central zone and TDM over the whole test arena (including central and outer zones).

The protocol was similar to our previous study (Yu et al., 2015). The rats were placed onto a treadmill apparatus (Panlab Technology, Spain) following the same time window of LT₄ or saline administration as that in the open field tests. The running intensity was set at 25 m/min with no incline. During a 5 min test, the running distance, which reflected fatigue resistance ability, was automatically recorded.

At completion of behavioral testing, the rats were administered (I.P.) an overdose of pentobarbital sodium (Sigma), infused 1 μl trypan blue into bilateral hippocampus through the guide cannula with the same injection needle tip depth as LT₄ or saline infusion, and perfused transcardially with saline followed by 4% formaldehyde solution (in 0.1 M phosphate buffer). After extraction from the skull, the brains were removed and cryoprotected in 20% glycerol/10% formaldehyde solution for 3 days. Coronal sections (50 μm thickness) were cut through hippocampus with a freezing microtome (VT1000 S, Leica Biosystems, Germany), and wet-mounted on glass microscope slides with 70% ethanol. The nature and extent of the hippocampal lesion was verified by visual inspection of the stained brain sections. All rats had complete, bilateral lesions of the DH with sparing of axonal fibers of passage. All hippocampal lesions in this experiment were virtually identical.

The protocol was similar to that in our previous studies (Zhou et al., 2016). During the training process, rats were placed in a chamber (32 × 25 × 25 cm; Med Associates Inc., St. Albans, VT, USA), allowed to freely explore the box for 2 min, and then received 5-trial foot shocks (0.8 mA, 2 s) with 2-min intervals. The freezing time, which reflected the expression of the fear response to the context or aversive stimulus, was automatically recorded by software (Video Freeze V2.5.5.0, Med Associates, Inc., St. Albans, VT, USA) before the first conditioning trial (Pre), and the freezing times were again measured during the 2-min interval after each conditioning trial to reflect the level of acquisition. To test contextual memory, the rats were placed back in the box for 5 min without foot shocks on day 1 (test 1), day 7 (test 2), and day 14 (test 3) post training. The freezing time was recorded to represent the level of memory. All experimenters were blinded to the group assignment of the animals.

To test the systemic effects of THs on fear memory, rats were injected (I.P.) with LT₄ (Sigma, 15 μg/ml × 1 ml/kg body weight) immediately after fear conditioning. To test the contributions of THs in hippocampus to fear memory, rats were directly injected with LT₄ into DH 1 day before, immediately, 2 h or 23.5 h after fear conditioning. All control rats were injected saline (1 ml/kg body weight during systemic LT₄ administration or 1 μl/side hippocampus during hippocampal LT₄ administration) with the same injection sites as experimental rats.

The percent time freezing (total freezing time/120 s × 100%) every 120 s in six consecutive observations during fear conditioning or (total freezing time/300 s × 100%) during the fear memory test for 300 s for each rat was calculated. All data were represented as mean ± SEM. Independent Student’s t-test, repeated measures ANOVA, and one-way ANOVA followed by post hoc Bonferroni’s test were used for statistical analyses according to experimental designs (SPSS 16.0 version). P < 0.05 was considered to be statistically significant.

To examine the effects of systemic injection of LT₄ on the level of free THs in serum and hippocampus, rats were received a single LT₄ administration (15 μg/kg, I.P.). The data with one-way ANOVA analysis showed that the serum FT₄ and FT₃ was significantly increased from 2 h to 3 days and returned to baseline 7 days after administration (Figure 1A, FT₄: F_(4,15) = 163.26, P < 0.001; post hoc: 2 h, P < 0.001; 1 day, P < 0.001; 3 days, P < 0.001; 7 days, P > 0.99, respectively, compared with control group. FT₃: F_(4,15) = 39.81, P < 0.001; post hoc: 2 h, P < 0.001; 1 day, P < 0.001; 3 days, P < 0.05; 7 days, P > 0.99, respectively, compared with control group; n = 4 in each time point). Meanwhile, the hippocampal FT₄ and FT₃ was also increased from 2 h to 1 day after LT₄ administration in one-way ANOVA analysis (Figure 1B, FT₄: F_(4,15) = 7.47, P = 0.005; post hoc: 2 h, P < 0.01; 1 day, P = 0.04; 3 days, P = 0.36; 7 days, P > 0.99, respectively, compared with control group. FT₃: F_(4,15) = 6.21, P = 0.009; post hoc: 2 h, P > 0.99; 1 day, P = 0.019; 3 days, P > 0.99; 7 days, P > 0.99, respectively, compared with control group).

To examine the effects of systemic injection of LT₄ on fear memory formation, rats were injected with LT₄ (15 μg/kg, I.P.) immediately after fear conditioning (Figure 1C, left). There was no significant difference in the fear memory acquisition between control group and LT₄-injection group represented by the learning curve (Figure 1C, middle: repeated measures ANOVA, F_(1,14) = 0.18, P = 0.67), which indicated that the acquisition of fear conditioning between two groups was similar to each other. However, during the following dependent fear memory tests, the rats injected with LT₄ showed the decreased freezing level compared to control group (Figure 1C, right: repeated measures ANOVA, F_(1,14) = 14.09, P = 0.002; post hoc: 1 day, P = 0.009; 7 days, P < 0.001; 14 days, P < 0.001, respectively, compared with control group), which indicated that LT₄ administration (I.P.) immediately after fear conditioning prevented the maintenance of long-term memory.

Thus, systemic administration (I.P.) of LT₄ increased the FT₄ and FT₃ levels in serum and hippocampus, and impaired long-term fear memory maintenance.

As systemic injection of LT₄ induced dramatical changes of THs in hippocampus, we hypothesized that the hippocampal THs have critical roles on some processes. To address this question, rats were directly injected with LT₄ into DH. All of our experimental rats for drugs administration in hippocampus were histologically verified the identical loci of injection needle tips in DH and the patency of all tracts for drugs infusion, which was represented by a trypan blue-stained coronal brain from a rat having ever been injected LT₄ into the DH (Figure 2A; Bradley et al., 1992). The results showed that only FT₄ but not FT₃ was increased in hippocampus after LT₄ injection (Figure 2B, FT₄: one-way ANOVA, F_(4,15) = 113.79, P < 0.001; post hoc: 4 h, P < 0.001; 1 day, P = 0.007; 3 days, P < 0.05; 7 days, P > 0.99, respectively, compared with control group. FT₃: one-way ANOVA, F_(4,15) = 1.41, P = 0.28), in which the contribution of the local conversion of FT₄ to FT₃ was similar to the previous study (van Doorn et al., 1983). The increased concentration of FT₄ in hippocampus post LT₄ injection lasted at least 3 days and recovered to normal level 7 days later. Meanwhile, a single intrahippocampal LT₄ injection did not change the level of THs in PFC (Figure 2C, FT₄: control group vs. LT₄ group, t = 0.03, P = 0.97; FT₃: control group vs. LT₄ group, t = 0.33, P = 0.75), which was similar to the previous document (van Doorn et al., 1985). After the above LT₄ injection, FT₄ and FT₃ in serum also did not increase (Figure 2D, FT₄: control group vs. LT₄ group, t = 0.86, P = 0.42; FT₃: control group vs. LT₄ group, t = 0.09, P = 0.92).

In addition, intra-hippocampal injection of LT₄ did not affect the locomotor activity represented by the total distance in open field test (Figure 2E, control group vs. LT₄ group, t = 0.42, P = 0.67), the fatigue resistance ability represented by the total distance in treadmill test (Figure 2F, control group vs. LT₄ group, t = 0.86, P = 0.40), and the thigmotaxis represented by the percentage of the TDM in the central zone of the test apparatus (Figure 2G, control group vs. LT₄ group, t = −0.806, P = 0.44) which was similar to the previous document (Wilcoxon et al., 2007).

Taken together, LT₄ infusion in hippocampus selectively increased hippocampal FT₄ but not FT₃ concentration in rats. The high level of hippocampal FT₄ did not change the locomotor activity, the fatigue resistance ability and thigmotaxis.

To test the effects of high level of hippocampal THs on contextual fear memory acquisition, rats were directly injected with LT₄ into DH through the cannula 1 day before fear conditioning and then carried out dependent memory tests on the day 1, day 7 and day 14 post fear conditioning (Figure 3A, left). The results indicated that the high level of hippocampal THs did not impair the fear memory acquisition represented by the learning curve during the conditioning (Figure 3A, middle. Repeated measures ANOVA, F_(1,19) = 0.47, P = 0.49). However, a single LT₄ injection impaired the contextual fear memory maintenance because the rats injected with LT₄ in bilateral hippocampus showed the decreased tendency of freezing level during dependent tests (Figure 3A, right. Repeated measures ANOVA, F_(1,19) = 3.40, P = 0.08; post hoc: 1 day, P = 0.34; 7 days, P = 0.19; 14 days, P = 0.012, respectively, compared with control group). This data suggested that the abnormal high level of hippocampal THs might impair contextual fear memory consolidation.

To confirm this hypothesis, rats were injected with LT₄ into hippocampus immediately (Figure 3B, left) or 2 h (Figure 3C, left) after conditioning. The data showed that rats injected with LT₄ in hippocampus indeed revealed being decreased freezing time during the dependent contextual fear memory tests using repeated measures ANOVA (Figure 3B, right; F_(1,26) = 17.46, P < 0.001; post hoc: 1 day, P < 0.001; 7 days, P = 0.026; 14 days, P < 0.001, respectively, compared with control group; Figure 3C, right; F_(1,16) = 3.49, P = 0.049; post hoc: 1 day, P = 0.014; 7 days, P = 0.047; 14 days, P = 0.016, respectively, compared with control group), although all of these rats had similar fear learning ability represented by the learning curve using repeated measures ANOVA (Figure 3B, middle; F_(1,26) = 0.87, P = 0.35; Figure 3C, middle; F_(1,16) < 0.001, P = 0.98).

Finally, to further exclude that this result was due to that the abnormal high level of hippocampal THs impaired fear memory retrieval, we extended the intrahippocampal injection time of LT₄ to 23.5 h after fear conditioning, so that the changes of 1-D memory test should be used to reflect the roles of LT₄ injection on memory retrieval (Figure 3D, left). The data suggested that rats injected with LT₄ in hippocampus showed similar level of learning curve in repeated measures ANOVA (Figure 3D, middle; F_(1,18) = 0.38, P = 0.54) and dependent memory tests whatever the time post conditioning (Figure 3D, right; F_{(1, 18)} = 2.26, P = 0.15; post hoc: 1 day, P = 0.94; 7 days, P = 0.56; 14 days, P = 0.07, respectively, compared with control group). Thus, the high level of hippocampal THs did not impair the contextual fear memory retrieval.

Taken together, the hippocampal LT₄ injection selectively impaired contextual fear memory consolidation, but not acquisition and retrieval.