Endocrinol Metab.  2022 Apr;37(2):290-302. 10.3803/EnM.2021.1343.

Developmental Hypothyroidism Influences the Development of the Entorhinal-Dentate Gyrus Pathway of Rat Offspring

• Jin T
• Wang R
• Peng S
• Liu X
• Zhang H
• He X
• Teng W
• Teng X

• KMID: 2529221
• DOI: http://doi.org/10.3803/EnM.2021.1343

Abstract

Background
Developmental hypothyroidism impairs learning and memory in offspring, which depend on extensive neuronal circuits in the entorhinal cortex, together with the hippocampus and neocortex. The entorhinal-dentate gyrus pathway is the main entrance of memory circuits. We investigated whether developmental hypothyroidism impaired the morphological development of the entorhinal-dentate gyrus pathway.
Methods
We examined the structure and function of the entorhinal-dentate gyrus pathway in response to developmental hypothyroidism induced using 2-mercapto-1-methylimidazole.
Results
1,1´-Dioctadecyl-3,3,3´,3´-tetramethylindocarbocyanine perchlorate tract tracing indicated that entorhinal axons showed delayed growth in reaching the outer molecular layer of the dentate gyrus at postnatal days 2 and 4 in hypothyroid conditions. The proportion of fibers in the outer molecular layer was significantly smaller in the hypothyroid group than in the euthyroid group at postnatal day 4. At postnatal day 10, the pathway showed a layer-specific distribution in the outer molecular layer, similar to the euthyroid group. However, the projected area of entorhinal axons was smaller in the hypothyroid group than in the euthyroid group. An electrophysiological examination showed that hypothyroidism impaired the long-term potentiation of the perforant and the cornu ammonis 3–cornu ammonis 1 pathways. Many repulsive axon guidance molecules were involved in the formation of the entorhinaldentate gyrus pathway. The hypothyroid group had higher levels of erythropoietin-producing hepatocyte ligand A3 and semaphorin 3A than the euthyroid group.
Conclusion
We demonstrated that developmental hypothyroidism might influence the development of the entorhinal-dentate gyrus pathway, contributing to impaired long-term potentiation. These findings improve our understanding of neural mechanisms for memory function.

Keyword

Hypothyroidism; Neural pathways; Neuronal tract-tracers; Synaptic potentials; Axon guidance

Figure

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  Fig. 1. Representative images of the entorhinal-hippocampal pathway. (A) Schematic drawing of the development of the entorhinalhippocampal pathway. (B) Brain slices containing the maximum cross-sectional areas of the hippocampus were chosen for the electrophysiological experiments. (C, D) Microelectrode array covering the perforant pathway (C) or CA3-CA1 pathway (D). One microelectrode was selected as the stimulation site (red circle), which can induce the best synaptic responses in the recording site (green circle). CA, cornu ammonis; DG, dentate gyrus; EC, entorhinal cortex.

• 

  Fig. 2. Tracing the entorhinal-hippocampal pathway of (A) the euthyroid group and (B) the hypothyroid group at P2. DiI-labeled entorhinal fibers branched into the alvear path and the perforant pathway, which both appear in the euthyroid group and the hypothyroid group. Sections (n=10–12) were from different pups (n=4–6 pups from 4–6 dams) at P2. Scale bar, 200 μm. ab, angular bundle; AP, alvear path; CA, cornu ammonis; DG, dentate gyrus; EC, entorhinal cortex; PP, perforant pathway; SLE, stratum lacunosum moleculare; P, postnatal day.

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  Fig. 3. Distribution of entorhinal axons in the dentate gyrus at (A, B) P2, (C, D) P4, and (E, F) P10. Photomicrographs of brain sections during the layer-specific distribution from (A, C, E) the euthyroid group and (B, D, F) the hypothyroid group. Euthyroid pup axons stared to distribute into the outer molecular layer (OML) at P2 (A, arrow), and displayed a layer-specific distribution in the OML at P10 (E, arrow). Hypothyroid pup axons reached the OML, as in euthyroid pups, but showed delayed development. Sections (n=10–12) were from different pups (n=4–6 pups from 4–6 dams) at P2, P4, and P10. Scale bar, 50 μm. DG, dentate gyrus; GL, granule layer; ML, molecular layer; SLE, stratum lacunosum moleculare; P, postnatal day.

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  Fig. 4. The proportion of DiI-labeled fibers in the outer molecular layer (OML) at (A) P2, (B) P4, and (C) the projected area in the OML at P10. Slices (n=5–7) from different pups (n=5–7 pups from 5–7 dams) in each group. aP<0.001; bP=0.027, compared with the euthyroid group.

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  Fig. 5. Long-term potentiation in the perforant pathway and CA3-CA1 pathway assessed as the percent change in the field excitatory postsynaptic potential (fEPSP) slope and fEPSP amplitude (Amp) from baseline at P10. (A, B) For the perforant pathway, there was no significant difference in the fEPSP slope; however, the fEPSP Amp of the hypothyroid pups was lower than that of the euthyroid pups. Slices (n=6–9) were from different pups (n=6–9 pups from 6–9 dams) at P10. (C, D) For the CA3-CA1 pathway, maternal hypothyroidism reduced both the fEPSP slope and the Amp in the pups. Slices (n=6–7) were from different pups (n=6–7 pups from 6–7 dams) at P10. (E, F) Inset: representative waveforms recorded in the perforant and CA3-CA1 pathways before high-frequency stimulation (HFS) and after HFS in the euthyroid and hypothyroid groups. CA, cornu ammonis. aP=0.036; bP=0.038; cP=0.008, compared with the euthyroid group.

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  Fig. 6. Expression of (A) netrin 1 (Ntn1), (B) repulsive guidance molecule BMP co-receptor A (Rgma), (C) erythropoietin-producing hepatocyte ligand A3 (Efna3), and (D) semaphorin 3A (Sema3a) mRNA in the hippocampus of the euthyroid and hypothyroid groups (n=6–8 pups from 5–6 dams in each group). aP<0.05; bP<0.01, compared with the euthyroid group.

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  Fig. 7. (A, B, C) Western blotting analysis of ephrin A3 and Sema3a proteins in the hippocampus of the euthyroid and hypothyroid groups (n=6 pups from 4–6 dams in each group). The bands (A) depicted representative findings for the euthyroid and hypothyroid groups. The bar graphs (B, C) showed the results of semiquantitative measurements of ephrin A3 and Sema3a. E, euthyroid; H, hypothyroid. aP<0.05; bP<0.01, compared with the euthyroid group.

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