Dio2 is upregulated in brain capillaries following imprinting

We attempted to identify the genes differentially expressed at an earlier phase of imprinting than reported previously20. One-day-old chicks were trained for imprinting for 1 h and whole brains were collected and used for complementary DNA microarray and quantitative reverse transcription–polymerase chain reaction. We identified 18 genes, including Dio2, Type 2 iodothyronine deiodinase, that upregulated accompanying imprinting (Fig. 1a; Supplementary Tables S1 and S2). We focused on Dio2, the enzyme that catalyses the intracellular deiodination of thyroxine (T 4 ) prohormone to the active T 3 , because the concentrations of thyroid hormones in plasma were reported to peak around hatching in precocial birds22. Thyroid hormone is synthesized as T 4 in thyroid, circulates in blood vessels and reaches the endothelial cells of brain capillaries23. Using in situ hybridization, mRNA for Dio2 was found to be ubiquitously accumulated in the imprinted chicks' brains, including the IMM, and its expression was enriched in brain capillaries (Fig. 1b–h). Immunohistochemistry showed that Dio2 co-localized with P-glycoprotein, a marker of brain capillaries (Fig. 1i–k), western blotting showed that Dio2 was fractionated in the capillary fraction (Fig. 1l) and Dio2 was recognized as a single band using anti-Dio2 antibody in the whole brain lysate (Supplementary Fig. S1). Dio2 was also enriched in brain capillaries of the MNM, which has been identified as the auditory-imprinting-relevant region17,18, suggesting that visual imprinting may have some influence on the auditory-imprinting-relevant region (Fig. 1m–p). These results imply that Dio2 converts T 4 to T 3 in endothelial cells of brain capillaries and provides T 3 to brain cells for imprinting.

Figure 1: Dio2 is upregulated in brain capillaries following imprinting. (a) Quantitative reverse transcription–polymerase chain reaction showed upregulation of Dio2 associated with imprinting. Mean±s.e.m. (U-test, *P<0.05). The numbers of animals used are indicated in parentheses. (b–d) In situ hybridization showed Dio2 was upregulated ubiquitously in brains, including the IMM. (b,c) Antisense probe for Dio2 was used (b, imprinted; c, dark-reared). (d) Sense probe as a negative control was used. (e) Diagram of coronal section of left brain hemisphere. (f–h) High magnification of IMM (boxes). Dio2 was upregulated in brain capillaries associated with imprinting. (i–k) Dio2 protein was co-localized with P-glycoprotein. (i,j) Labelling of Dio2 protein (i) and P-glycoprotein (j), a marker for brain capillaries in the same section. (k) Images of i and j have been combined. (l) Immunoblotting showed that Dio2 was enriched in the capillary fraction. (m–p) In situ hybridization showed that Dio2 was upregulated in brain capillaries associated with imprinting in the MNM. (m) Diagram of coronal section of the left brain hemisphere. (n–p) High magnification of MNM (box). (n,o) Antisense probes for Dio2 were used. (p) Sense probe as a negative control was used. (b–e,m) Scale bars, 2 mm. (f–k, n–p) Scale bar, 200 μm. GFAP, glial fibrillary acidic protein ; MAP, Microtubule-associated protein. Full size image

Thyroid hormone signalling is involved in imprinting

Indeed, intravenous injection of Dio2 inhibitors, iopanoic acid (IOP) and phloretin, impaired visual imprinting (Fig. 2a–c). To confirm the conversion from T 4 to T 3 by Dio2, we injected 125I-labelled T 4 intravenously to detect 125I-labelled T 3 converted from 125I-labelled T 4 in brains. As a result, 125I-labelled T 3 was detected mostly in the brain (Fig. 2d). Furthermore, intravenous injection of IOP reduced the amount of 125I-labelled T 3 in brain (Fig. 2e), indicating that Dio2 did convert T 4 to T 3 in chick brains. Thus, it may be concluded that Dio2 converts T 4 to T 3 in endothelial cells of brain capillaries, providing T 3 to brain cells for imprinting. The IMM in chicks has a critical role in visual imprinting2,13,14. As shown in Fig. 2f,g, bilateral ablation of the IMM prevented imprinting, and abolished the acquisition of filial preferences as reported previously13. The converted T 3 in endothelial cells is assumed to be transported by a monocarboxylate transporter towards brain cells, incorporated in the cytoplasm and binds to thyroid hormone receptors (TRs) whose gene expressions were detected in the IMM (Supplementary Fig. S2). In fact, imprinting was impaired by IMM injection with inhibitors of thyroid hormone signalling molecules (IOP; a monocarboxylate transporter 8 inhibitor, BSP; a thyroid hormone receptor antagonist, NH-3 (ref. 24)), suggesting that accumulation of T 3 in the IMM of chick brain by thyroid hormone signalling in the blood–brain barrier is important for imprinting (Fig. 2h). As the TRs are reported to distribute in neuronal and glial cells in mammals25, T 3 probably affects neuron and/or glia through its receptor in chick. As Dio2 is upregulated in broad areas of chick brain including the IMM and MNM, thyroid hormone may be involved in a variety of brain functions around the time of hatching. Northern blotting showed Dio2 expression to be stronger in the brain and lung than in the liver, testis, thymus and skin (Supplementary Fig. S3). In brains, Dio2 mRNA gradually accumulated from 3 days before hatching, peaked around hatching then leveled off and remained in steady state until 5 days after hatching (Supplementary Fig. S4). As Dio2 is known to be induced by infectious stress in the brain26, Dio2 in other parts of the brain may be involved in the immune response against microorganisms under new circumstances after hatching. However, Dio2 upregulation by imprinting training is learning-dependent, but not infection-dependent, because the chicks reared as the control group in light-exposed condition without imprinting training did not show Dio2 upregulation. Another possibility is that the function of Dio2 in the neuronal network of other areas may be linked with the molecular events in the IMM and MNM, which would be necessary for the acquisition of imprinting behaviour.

Figure 2: Thyroid hormone signalling is involved in imprinting. (a) The schemes to test the effect of inhibitors for imprinting. Imprinting training and simultaneous choice test were carried out by the method of Izawa et al.44 with modification (see detailed information in the Methods section). After hatching, chicks were kept in dark enclosures to prevent exposure to light until day 1. Then chicks were trained with a yellow LEGO object, and the preference for the yellow LEGO object was evaluated 1 h later. Imprinting training was initiated 30 min after the inhibitor injection. (b) Preference score was measured by the difference in approach time during 120 s; the time chicks spent near the control object (red) was subtracted from that chicks spent near the training object (yellow). Chicks stayed in each approach area or in the intermediate areas between the two areas during simultaneous choice test. Side walls on the alley and the arms are not drawn in this figure. (c) Imprinting was impaired by intravenous injection of Dio2 inhibitors (IOP and phloretin). (d,e) Converted T 3 flowed in brain. (d) Intravenously injected 125I-labelled T 4 was detected mostly as T 3 in brains, but as T 4 in serum (n=4). (e) Amount of 125I-labelled T 3 detected in brains was reduced by IOP (n=4). (f,g) IMM is required for imprinting on day 1. Ibotenic acid lesions were performed on day 0 and chicks were trained and tested on day 1. Training and testing for the preference followed the experimental scheme shown in a,b. (f) Histological reconstruction of ibotenic acid lesions in IMM. The lesioned areas are superimposed over coronal sections of telencephalon. (g) Effects of bilateral lesions of IMM on the acquisition of imprinting on day 1. (h) Imprinting was impaired by the IMM injection of inhibitors (Dio2 inhibitor, IOP; a monocarboxylate transporter 8 inhibitor, BSP; a thyroid hormone receptor antagonist, NH-3). Training and testing for preference followed the experimental scheme shown in a,b. (c,h) Mean±s.e.m. (Kruskal–Wallis test and subsequent multiple comparisons, *P<0.05). (d,e,g) Mean±s.e.m. (U-test, *P<0.05). The numbers of animals used are indicated in each figure (c,g,h). Full size image

Thyroid hormones are critical factors for imprinting

The concentration of thyroid hormones in brain and serum during development was determined using embryos and chicks. In brains, T 3 gradually accumulated from 6 days before hatching, peaked around hatching then decreased to background level 5 days after hatching, while T 4 became almost undetectable (Fig. 3a). In serum, concentrations of both T 3 and T 4 peaked around hatching (Fig. 3b). Also, the amount of T 3 in imprinted chicks was 1.7-fold higher than in dark-reared chicks, IOP-injected chicks or light-exposed control chicks kept in a training chamber where light was turned on and off but with no training object (Fig. 3c). No significant difference was found in serum thyroid hormones in these animals (Fig. 3d). The preference score in the test of imprinting was positively correlated with T 3 in brain, the amount of T 3 in good learners being higher than in poor learners (Fig. 3e). In this experiment, the time of the training session was shortened so a weak training protocol was used to yield good or poor learners. Because IOP injection impaired imprinting, as shown in Fig. 2c,h, a further increase in T 3 converted by Dio2 in the process of training was likely to be required for imprinting. We then examined whether thyroid hormones are determinants in imprinting. When T 3 was intravenously injected to 1-day-old chicks, chicks' preference was facilitated and not affected by IOP. Such facilitation was also found for T 4 , but the chicks' preference greatly decreased in the presence of IOP, suggesting that brain T 3 converted from T 4 by Dio2 is the determinant for imprinting (Fig. 3f).

Figure 3: Thyroid hormones are critical factors for imprinting. (a,b) Amount of thyroid hormones in brains (n=4, a) and in serum (n=4, b) during development. (c) Amount of thyroid hormones increased in brains by imprinting, but not in serum (d). (e) The amount of T 3 in good learners (n=12) was higher than in poor learners (n=13). Preference for the training object (yellow) was evaluated in the same way as shown in Fig. 2b. In this experiment, the time of the training session was shortened so a weak training protocol was used to yield good or poor learners. The preference scores in the simultaneous choice test (during 120 s) of the good learners were over 80 s in the test for imprinting, and those of the poor learners were under 40 s. (f) Intravenous injection of thyroid hormones facilitated imprinting on day 1. (g) Effect of wortmannin on imprinting of 1-day-old chicks. (c–g) Mean±s.e.m. (Kruskal–Wallis test and subsequent multiple comparisons, *P<0.05). (c,d,f,g) The training, testing and injection were performed following the experimental scheme shown in Fig. 2a. (e,f,g) The preference for the training object (yellow) was evaluated in the same way as shown in Fig. 2b. The numbers of animals used are indicated in each figure (e–g). NS, nonsignificant. Full size image

We next examined the mode of action of T 3 in the imprinting process. T 3 deficiency during brain development is associated with neurological disorder27 through genomic action23. The non-genomic action of T 3 has also been described28. For example, the PI 3 Kinase/Akt pathway was reported to mediate the rapid non-genomic cardiovascular effects of TR29. Because injected T 3 affects imprinting rapidly within 30 min, it probably functions via a non-genomic action. We injected wortmannin, a PI 3 Kinase inhibitor, into the IMM to examine its effect on imprinting. Wortmannin injection 30 min before intravenous injection of T 3 hampered the effect of T 3 . However, wortmannin injected 30 min after T 3 treatment did not, indicating that the rapid non-genomic action of T 3 before the training contributed to imprinting (Fig. 3g).

T 3 acts as a determinant for the sensitive period

As increase in T 3 facilitated chicks' preference on day 1, we investigated whether increase in T 3 could affect the sensitive period of imprinting. We found that the sensitive period for imprinting was restricted to the first 3 days after hatching and that the period closed at day 4 when dark-reared chicks could no longer be imprinted (Fig. 4a,b). However, the dark-reared chicks injected with T 3 intravenously 30 min before the imprinting training showed strong preference on day 4 or day 6, showing that thyroid hormone acts as the determinant of the opening of the sensitive period (Fig. 4b; Supplementary Movie 1). The effect of T 3 on the acquisition of imprinting on day 4 was also shown using a running wheel apparatus30, which is different from the apparatus equipped with a computer-controlled rubber belt and the infrared sensor used in other experiments (Supplementary Fig. S5). The injection of thyroid hormones and various inhibitors affected the acquisition of memory itself but not the locomotor activity of chicks (Supplementary Fig. S6). However, on day 8 or day 10, thyroid hormones did not effectively enhance the preference of chicks, suggesting the presence of unidentified factors, which may contribute to the closing of the sensitive period (Fig. 4b). The effect of thyroid hormones on establishing chicks' preference on day 4 was dose-dependent (Fig. 4c,d), and the effective concentrations were consistent with those detected in 1-day-old chicks. These results indicate that a simple hormone, T 3 , may regulate the beginning of the sensitive period in imprinting.

Figure 4: T 3 acts as a determinant for the sensitive period. (a) The scheme for testing the effect of thyroid hormones for imprinting. (b) Dark-reared chicks were trained for imprinting with a yellow LEGO object and subsequently the preference for the yellow LEGO object was evaluated on a single day after hatching (from day 1 to day 10). The sensitive period closed on day 4 and chicks were not imprinted, whereas chicks injected with T 3 30 min before the training became imprintable from day 4 to day 6. (c) The scheme for testing the effect of thyroid hormones on 4-day-old chicks in d–f. Dark-reared chicks were trained for imprinting with a yellow LEGO object and the preference for the yellow LEGO object was evaluated on day 4 after hatching in the same way as shown in Fig. 2b. (d) Dose-dependent effects of thyroid hormones on 4-day-old chicks. (e) IMM injection of inhibitors (a thyroid hormone receptor antagonist, NH-3; a monocarboxylate transporter 8 inhibitor, BSP; Dio2 inhibitor, IOP) hampered the effect of thyroid hormones on 4-day-old chicks. (f) Effect of wortmannin on imprinting of 4-day-old chicks. (b,d,e) Mean±s.e.m. (Kruskal–Wallis test and subsequent multiple comparisons, *P<0.05). (f) Mean±s.e.m. (U-test, *P<0.05). The number of animals used are indicated in each figure (b,d,e,f). DMSO, dimethylsulphoxide. Full size image

Next we examined whether the mode of action of thyroid hormones in opening the sensitive period is a TR-mediated non-genomic action. IMM injection of NH-3 (ref. 24) or BSP hampered the effect of T 3 on 4-day-old animals, and IOP injection into the IMM eliminated the effect of T 4 (Fig. 4e), suggesting that T 3 converted from T 4 reopened a once closed sensitive period. Furthermore, wortmannin injection into the IMM 30 min before intravenous injection of T 3 hampered the effect of T 3 in 4-day-old animals (Fig. 4f), indicating that the action of thyroid hormone was short term, that is, a TR-mediated non-genomic action.

The effects of exogenous T 3 in 4-day-old chicks

Control experiments were performed. Chicks administered with norepinephrine, testosterone, caffeine, serotonin, or dopamine in the IMM did not demonstrate strong preference on day 4 after the sensitive period closed (Fig. 5a). In particular, drugs inducing arousal, activity, for example, caffeine, did not have the effect on chicks' preference that is seen with T 3 , indicating that T 3 has a specific role in imprinting and is not just producing a general effect of enhancing arousal, activity and so on. These results indicate that T 3 exhibits specific activity determining the start of the sensitive period. Exogenous T 3 injection 30 min before the imprinting training on day 4 after the sensitive period closed, light-exposed chicks showed the same strong preference for the yellow object as dark-reared chicks (Fig. 5b). These data indicate that T 3 as a sensitive period determinant enabled imprinting in chicks, regardless of whether they were reared in darkness or light. T 3 has an effect on the acquisition of memory during training, but does not directly affect preference during the test (Fig. 5c). The effect of exogenous T 3 on day 4 after the sensitive period had closed was mediated by the IMM region (Fig. 5d,e).

Figure 5: The effects of exogenous T 3 on 4-day-old chicks. (a) Chicks administered with norepinephrine, caffeine, serotonin, testosterone or dopamine in the IMM as control reagents of thyroid hormones did not show strong preference on day 4. These reagents, including some neurotransmitters, a steroid hormone and a stimulant drug, did not induce strong preference after the sensitive period closed, suggesting that thyroid hormone exhibits specific activity determining the sensitive period. (b) In light-exposed chicks individually reared in separate cages, T 3 injection resulted in strong preference on day 4. The chicks were not able to see each other and kept in separate cages under light condition to prevent exposure to stimuli from moving objects. With exogenous T 3 injection 30 min before the imprinting training on day 4 after the sensitive period closed, light-exposed chicks showed the same strong preference for the yellow object as dark-reared chicks. (c) T 3 has an effect on the acquisition of memory during training but does not directly affect preference during the test. T 3 injected before the training allowed chicks to be imprinted on day 4, but T 3 injected after the training did not, suggesting that T 3 is effective on the acquisition of memory itself but not on the preference test score. (d,e) Effect of T 3 on imprinting on day 4 after the sensitive period closed was mediated by the IMM. (d) Histological reconstruction of ibotenic acid lesions in IMM. The lesioned areas are superimposed over coronal sections of telencephalon. (e) Bilateral lesion of IMM hampered the effects of injected T 3 on the acquisition of imprinting on day 4, whereas chicks without lesions could be imprinted with an injection of T 3 on day 4 after the sensitive period closed. (a,c,e) Mean±s.e.m. (Kruskal–Wallis test and subsequent multiple comparisons, *P<0.05). (b) Mean±s.e.m. (U-test, *P<0.05). (a,b,c,e) The training, testing and injection were performed following the experimental scheme shown in Fig. 4c. The preference for the training object (yellow) was evaluated in the same way as shown in Fig. 2b. The number of animals used are indicated in each figure (a,b,c,e). DMSO, dimethylsulphoxide; NS, nonsignificant. Full size image

First imprinting primed the chicks for second imprinting

Moreover, we found that chicks once imprinted on day 1 showed a higher capability to learn in subsequent training on day 4, indicating that the first imprinting primed the chicks for the second imprinting (Fig. 6a,b). The priming occurred irrespective of memory content (colour: Fig. 6a,b or shape: Fig. 6c,d). The chicks did not forget the first object after learning the second object (Fig. 6e). Surprisingly, the potential to show preference was not blocked by IMM injection of NH-3 or intravenous injection of IOP just before training on day 4 (Fig. 6b). These findings suggest that the first increase in T 3 by imprinting training on day 1 was sufficient for chicks to acquire the potential to be imprinted at a later stage. In fact, T 3 concentration remained low when the imprinting training was done on day 4 (Fig. 6f).

Figure 6: Initial imprinting primed the chicks for second phase of imprinting. (a) The schemes to test the effect of initial imprinting for second phase of imprinting using objects of different colours. Chicks were trained with a yellow LEGO object on day 1. Then chicks were kept in darkness to prevent exposure to light. On day 4, the chicks were trained with a red LEGO object and the preference for the red LEGO object was evaluated 1 h later. (b) Chicks trained on day 1 became imprintable for the second training object (red LEGO object) on day 4, whereas dark-reared chicks could not be imprinted (n=10). The preference for the second training object was measured on day 4. The grey LEGO block was used as the control object in the simultaneous choice test. (c) The schemes to test the effect of first imprinting for second imprinting using the objects whose colour and shape were different. Chicks were trained with a red LEGO object on day 1. On day 4, the chicks were trained with a chick toy and the preference for the chick toy that was a different colour (yellow) and shape from the red LEGO object (first training object) was evaluated. (d) Chicks showed strong preference for the chick toy (second training object), but not for the control object (yellow) whose colour was the same as that of the chick toy (n=10). (e) The chicks were imprinted by a yellow LEGO object (first training object) on day 1 and remembered it well even after being imprinted by the red LEGO object (second training object) on day 4. The training and testing were performed following the experimental scheme shown in a. The preference for the first training object (yellow LEGO object) was measured before and after the second training. The grey LEGO block was used as the control object in the simultaneous choice test (n=10). (f) Chicks trained on day 4 (purple bars) show less brain T 3 than those trained on day 1 (yellow bar, left n=6). (b,f) Mean±s.e.m. (Kruskal–Wallis test and subsequent multiple comparisons, *P<0.05). (d,e) Mean±s.e.m. (U-test, *P<0.05). Full size image

T 3 acts as a memory priming factor for learning

As expected, the chicks treated with T 3 on day 1 were able to be imprinted on day 4 (Fig. 7a,b). This potential to show preference was not blocked by the IMM injection of NH-3 just before training on day 4 (Fig. 7b), indicating that the first increase in T 3 without visual experience on day 1 was sufficient to render chicks imprintable at a later stage. Furthermore, T 3 injection on day 1 enabled imprinting on day 8 (Fig. 7c,d), suggesting that injection of T 3 extends the sensitive period, and secondary learning can take place because the sensitive period remains open and closes later than without T 3 . We call this potential given by the wave of T 3 'memory priming' (MP). Even after the sensitive period closed, T 3 injection on day 4 enabled imprinting on day 8, while injection of T 3 before the imprinting training on day 8 did not (Fig. 7e,f), indicating that once MP is acquired, it continues to prime later learning, independent of visual experience. This led us to examine whether the first increase in T 3 could confer MP to learning other than imprinting. Chicks either injected with T 3 or trained for imprinting on day 1 showed a higher correct choice ratio in reinforcement learning of a colour discrimination pecking task on day 4 compared with those neither injected with T 3 nor trained for imprinting (Fig. 7g,h). This supports our idea that thyroid hormone confers MP and that MP which originates from imprinting may be followed by cascading layers of later learning.