DOI: 10.5061/dryad.rjdfn2zd1
title: Unravelling the role of thyroid hormones in seasonal neuroplasticity in European Starlings (Sturnus vulgaris)

description: Thyroid hormones clearly play a role in the seasonal regulation of
 reproduction, but any role they might play in song behavior and the
 associated seasonal neuroplasticity in songbirds remains to be elucidated.
 To pursue this question, we first established seasonal patterns in the
 expression of thyroid hormone regulating genes in male European starlings
 employing in situ hybridization methods. Thyroid hormone transporter LAT1
 expression in the song nucleus HVC was elevated during the photosensitive
 phase, pointing towards an active role of thyroid hormones during this
 window of possible neuroplasticity. In contrast, DIO3 expression was high
 in HVC during the photostimulated phase, limiting the possible effect of
 thyroid hormones to maintain song stability during the breeding season.
 Next, we studied the effect of hypothyroidism on song behavior and
 neuroplasticity using in vivo MRI. Both under natural conditions as with
 methimazole treatment, circulating thyroid hormone levels decreased during
 the photosensitive period, which coincided with the onset of
 neuroplasticity. This inverse relationship between thyroid hormones and
 neuroplasticity was further demonstrated by the negative correlation
 between plasma T3 and the microstructural changes in several song control
 nuclei and cerebellum. Furthermore, maintaining hypothyroidism during the
 photostimulated period inhibited the increase in testosterone, confirming
 the role of thyroid hormones in activating the
 hypothalamic–pituitary–gonadal (HPG) axis. The lack of high testosterone
 levels influenced the song behavior of hypothyroid starlings, while the
 lack of high plasma T4 during photostimulation affected the myelination of
 several tracts. Potentially, a global reduction of circulating thyroid
 hormones during the photosensitive period is necessary to lift the brake
 on neuroplasticity imposed by the photorefractory period, whereas local
 fine-tuning of thyroid hormone concentrations through LAT1 could activate
 underlying neuroplasticity mechanisms. Whereas an increase in circulating
 T4 during the photostimulated period potentially influences the
 myelination of several white matter tracts, which stabilizes the
 neuroplastic changes. Given the complexity of thyroid hormone effects,
 this study is a steppingstone to disentangle the influence of thyroid
 hormones on seasonal neuroplasticity.
description: 1. Subjects and experimental design Thirty male starlings (<em>Sturnus
 vulgaris</em>) were wild caught as adults in Normandy (France) in
 November 2014. All animals were housed in two large indoor aviaries (L x W
 x H: 2.2 x 1.4 x 2.1 m) at the University of Antwerp with food and water
 ad libitum with artificial light dark cycle. Starting from January 2013,
 all birds were kept in a long day photoperiod (16L/8D) in order to remain
 photorefractory. The housing and experimental procedures were performed in
 agreement with the Belgian and Flemish laws and were approved by the
 Committee on Animal Care and Use of the University of Antwerp, Belgium
 (2014-52). Starlings were
 divided into two groups: a hypothyroid group (N=16) and a control group
 (N=14). The study was started when all birds were photorefractory. Then
 they were switched from long to short (8L:16D) days to induce the return
 to photosensitivity. Methimazole (MMI) treatment was started in one group
 to induce hypothyroidism. By supplementing the drinking water with 0.05%
 MMI, the endogenous stock of THs gradually decreased until it was fully
 depleted after 2-3 weeks. MMI treatment was continued for the remainder of
 the experiment. After 12 weeks of short days, the photosensitive starlings
 were switched back to long days (16L:8D) so that they became
 photostimulated. In parallel the control group was exposed to the same
 photoperiodic regime without receiving any hormone manipulation.
   We monitored the
 neuroplasticity repeatedly at 6 different time points. The first time
 point was at the end of the photorefractory state (PR). After switching to
 short days we measured every 4 weeks to follow up the song control system
 plasticity during the photosensitive period (SD4, SD8, SD12).
 Additionally, we measured after 1 week of long days, as it is known that
 exposure to 1 long day can already affect TH. Finally, we measured after 4
 weeks on long days (LD4), when starlings were fully photostimulated. At
 each time point, songs were recorded, blood samples were taken, and MRI
 (DTI and 3D) was acquired. In addition, body weight and beak color were
 registered. 2. MRI data acquisition
   The birds were
 initially anesthetized using 2% Isoflurane (Isoflo ®, Abbot Laboratories
 Ltd.) in a mixture of 30% O<sub>2</sub> and 70%
 N<sub>2</sub> at a flow rate of 600 ml/min. Throughout the
 entire imaging procedure, respiration rate was monitored with a small
 pneumatic sensor (SA Instruments, NY, USA) positioned under the bird.
 Depending on the breathing rate, the anesthetic dose was lowered, ranging
 between 1% - 2% isoflurane. Body temperature was monitored with a cloacal
 temperature probe and kept within narrow physiological ranges (41.0 ± 0.2
 °C) using a warm air system with a feedback unit (SA Instruments, NY,
 USA). All MRI measurements
 were performed on a 7T horizontal MR system (Pharmascan 70/16 US, Bruker
 Biospin, Germany). Each imaging session started with a T2-weighted 3D
 anatomical RARE scan (TR: 2000 ms; TE: 11 ms; RARE factor: 8; zero-filled
 to a matrix of (256x92x64) with voxel resolution (0.089x0.25x0.25) mm³).
 Subsequently, a 4 shot SE-EPI DTI scan (TR: 7000 ms; TE: 23 ms; d 4ms, D
 12ms; b-value 670 s/mm²; 60 diffusion gradient directions; spatial
 resolution: (0.179x0.179x0.35) mm³; 28 coronal slices) was acquired. After
 the imaging procedure, birds were left to recover in a warmed recovery box
 before returning to the aviary. 3. MRI data
 processing  
 Diffusion data were analyzed with
 MRtrix3 version 3.0 (Tournier et al., 2012) following the same processing
 steps as described in (Orije et al., 2021b). Preprocessing of the
 individual DW-images included following steps: denoising (Veraart et al.,
 2016), correction for Gibbs ringing (Kellner et al., 2016), motion and
 distortion correction using FSL (Jenkinson et al., 2012), bias field
 correction using ANTS (Advanced Normalization Tool; Avants et al. (2010)),
 whole brain extraction and upsampling to isotropic voxels of 1.75 mm.
 These preprocessed diffusion weighted images were used to calculate
 individual diffusion maps (fractional anisotropy (FA), mean, axial and
 radial diffusivity) and fiber orientation distribution (FOD) images. The
 neuroanatomical contrast of the individual FA maps allowed the delineation
 of different ROI’s (Area X and RA) to determine their volume changes using
 AMIRA software (De Groof et al., 2006). The calculation of FOD images
 requires global intensity normalization, fiber response function
 estimation using the unsupervised Dhollander algorithm (Dhollander et al.,
 2019) and spherical deconvolution (Jeurissen et al., 2014). These FOD
 images were normalized to create an unbiased study-based FOD template,
 which involves linear and non-linear registration (Raffelt et al., 2011).
 Fiber density (FD) and fiber-bundle cross-section (FC) were estimated from
 the normalized FOD images. The transformation parameters derived from
 building the FOD template were also applied to the diffusion maps to warp
 them into the template space to perform voxel-based analysis. Next, these
 images were smoothed to double voxel size (3.5 x 3.5 x 3.5 mm³). Finally,
 all normalized diffusion maps were averaged to create a FA template that
 is used as background to display the statistical results.
description: Subject 5 and subject 16 died after
 the second and fourth time point respectively, so only MRI data later time
 points could not be acquired. Scans of subject 7 at time point 4 failed
 due to excessive movement.