This work was supported by Project no. RRF-2.3.1-21-2022-00011, titled National Laboratory of Translational Neuroscience has been implemented with the support provided by the Recovery and Resilience Facility of the European Union within the framework of Programme Sz&#233;chenyi Plan Plus.

The present protocol was reviewed and approved by the Animal Welfare Committee at the Institute of Experimental Medicine (PE/EA/1490-7/2017, PE/EA/106-2/2021). The presented data is from FVB/Ant background14, 3-month-old male THAI mice (n = 3-6/group). FVB/Ant background THAI animals tend to have highly pigmented spots on their skin that may distort measurements. Hence, search for pigmented spots on the skin of the imaged area after fur removal. Animals do not require special housing conditions unless the experiment specifically requires so (e.g., a special diet).
1. Hyperthyroid treatment
NOTE: A general protocol for inducing hyperthyroidism in mice is provided here. The ATA guide19 offers detailed explanations on the background of the methods with alternatives mentioned.
<ol>
	<li>Dissolve T3 (3,5,3&#39;-triiodothyronine, see Table of Materials) in 40 mM NaOH to create a stock solution with a concentration between 5-10 mg/mL.</li>
	<li>Dilute the stock solution with saline to a final concentration of 0.1 &#181;g/&#181;L.</li>
	<li>Inject the diluted T3 solution intraperitoneally (i.p.) into awake animals at a volume of 10 &#181;L per gram of body weight (bwg). After 24 h, the animals will be considered hyperthyroid. 
	NOTE: T3 treatment can be replaced by any other kind of treatment. The treatment does not affect the protocol of in vivo imaging.</li>
</ol>
2. Hypothyroid treatment
NOTE: Here, only a general protocol for inducing hypothyroidism in mice is provided. ATA guide19 describes detailed explanations on the background of the methods with alternatives mentioned.
<ol>
	<li>Switch the diet to an iodine-free chow diet and add KClO4 and methimazole to the drinking water (0.01 % methimazole, 0.05 % KClO4) (see Table of Materials).</li>
	<li>Replace the drinking solution regularly (every 2-3 days) with a fresh drinking solution because methimazole is light-sensitive and degrades quickly.</li>
	<li>Keep up the treatment regime for at least 2 weeks, no more than 4 weeks. Animals will lose weight and will show discomfort. If animals are hardly moving around, have lost much of their hair, or are barely conscious, use a humane endpoint and terminate them by any method (following institutionally approved protocols).</li>
	<li>Continue hypothyroid treatment when combined with other treatments to prevent potential recovery from hypothyroidism.</li>
</ol>

3. In vivo imaging
<ol>
	<li>Start the software compatible with the in vivo imaging system (see Table of Materials).</li>
	<li>Log in and wait for the &#34;Imaging Wizard&#34; panel to load. It is a smaller panel on the bottom left of the window.</li>
	<li>On the &#34;Imaging Wizard&#34; initiate camera cooling by clicking on Initialize in the &#34;Image Wizard panel&#34;. This makes the instrument run a setup protocol, wait for it to complete. The &#34;Imaging Wizard panel&#34; turns blue, and a green light will turn on in the panel when the camera temperature is low enough and the instrument is ready.</li>
	<li>Set the temperature of the heating pad to 30-37 &#176;C to keep the measured animals warm. 
	NOTE: Continue with the protocol while waiting for the camera temperature to be optimal.</li>
	<li>Do not keep or treat animals near the instrument. Avoid excess amounts of hair circulating in the air around the instrument.</li>
	<li>Anesthetize 1-3 animals with ketamine-xylazine i.p. injection (ketamine 50 mg/kg body weight, xylazine 10 mg/kg body weight, see Table of Materials). Alternatively, if an isoflurane anesthetic system is installed, follow institutionally approved protocols to use isoflurane anesthesia, replacing the ketamine-xylazine mixture. 
	NOTE: Hypothyroid mice are more sensitive to ketamine-xylazine; use half dose.</li>
	<li>Use eye protection gel during anesthesia.</li>
	<li>Check pedal reflex by pinching the footpads. No pedal reflex confirms the state of surgical plane anesthesia.</li>
	<li>After the anesthesia takes effect, remove fur from the imaged body parts using the most suitable fur removal method (epilator, shaving, cream, etc.). Ensure no fur is left on the imaged body parts to prevent the scattering of luminescent light.</li>
	<li>Dissolve Na-luciferin (see Table of Materials) in 1x phosphate-buffered saline (PBS) at a concentration of 15 mg/mL. Luciferin is light-sensitive; avoid direct light exposure. Store the solution in amber tubes or wrap it in aluminum foil.</li>
	<li>Treat shaven animals with luciferin solution 10 &#181;L/bwg i.p.</li>
	<li>Place the animals into the instrument with the camera&#39;s center point marked as a &#39;+&#39; on the pad. Ensure proper placement by checking the gridlines and confirming with a single &#39;Photo&#39; capture if unsure.</li>
	<li>Wait for 15 min after substrate administration before taking the first measurement. During this time, set the imaging time in the &#34;Image Wizard&#34; panel to 3 min for luminescence and check the boxes for Photo and Luminescence. The &#39;Photo&#39; is necessary to coincide with luminescence to identify the source of the measured signal. 
	NOTE: 15 min is required for optimal substrate uptake and tissue distribution. The luminescent signal plateaus 15-20 min after luciferin administration. The signal starts to slowly decrease after the plateau.</li>
	<li>Take the first measurement by clicking on Measure in the &#34;Imaging Wizard&#34; panel.</li>
	<li>If both ventral and dorsal imaging are performed, rearrange animals for the second body part to be imaged immediately after concluding the first.</li>
	<li>After imaging is done, return the animals to their cages and continue the experiment with the next set of animals.</li>
	<li>Allow the animals to recover, which typically takes 1-2 h at most. Place a tube filled with warm water near the animals to facilitate recovery, and monitor vital signs such as breathing and perfusion.</li>
	<li>Decide the fate of measured animals. In the data presented in this article, the measured animals were euthanized following institutionally approved protocols for ex vivo measurements. However, this is not necessary. Consider whether euthanasia or follow-up experiments are ethical.</li>
</ol>

4. Data analysis
<ol>
	<li>Open the &#34;ClickInfo&#34; file in the software. A panel named &#34;Tool Palette&#34; for image analysis and editing will open on the right side of the window.</li>
	<li>Convert scale to radiance in the top left corner of the image.</li>
	<li>Click on&#160;&#34;Image Adjust&#34;.</li>
	<li>Decide on the optimal binning and color scale of the images. Make all images utilize the same settings.</li>
	<li>Click on &#34;ROI tools&#34; on the &#34;Tool palette&#34;.</li>
	<li>Select areas of interest by clicking on &#34;Place ROI&#34; in the &#34;ROI tools&#34;. Using the same sized ROIs or different sized ROIs can also be meaningful depending on the experimental design.</li>
	<li>Click on &#34;Measure ROIs&#34;. A new window will open with the data of placed ROIs. Export the data with ctr + c-ctrl + v Windows command into an organizing or statistical software of choice.</li>
	<li>Data can be exported as total flux or average radiance. Choose which variable is the most relevant in the current experimental setting.</li>
	<li>Continue data analysis in accordance with the experimental design. Individual calculation of (treated-background) values as &#34;measured effect in one animal&#34; is recommended.</li>
</ol>

Imaging of Thyroid Hormone Action Indicator Mice to Assess Endocrine Disruption

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+regulation+of+hypothalamic-pituitary-thyroid+axis+under+physiological+and+pathophysiological+conditions.">Central regulation of hypothalamic-pituitary-thyroid axis under physiological and pathophysiological conditions.</a> Endocr Rev. 35 (2), 159-194 (2014).</li><li>Bianco, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paradigms+of+Dynamic+Control+of+Thyroid+Hormone+Signaling.">Paradigms of Dynamic Control of Thyroid Hormone Signaling.</a> Endocr Rev. 40 (4), 1000-1047 (2019).</li><li>Zoeller, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocrine+disrupting+chemicals+and+thyroid+hormone+action.">Endocrine disrupting chemicals and thyroid hormone action.</a> Adv Pharmacol. 92, 401-417 (2021).</li><li>Guarnotta, V., Amodei, R., Frasca, F., Aversa, A., Giordano, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+chemical+endocrine+disruptors+and+hormone+modulators+on+the+endocrine+system.">Impact of chemical endocrine disruptors and hormone modulators on the endocrine system.</a> Int J Mol Sci. 23 (10), 5710 (2022).</li><li>La Merrill, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consensus+on+the+key+characteristics+of+endocrine-disrupting+chemicals+as+a+basis+for+hazard+identification.">Consensus on the key characteristics of endocrine-disrupting chemicals as a basis for hazard identification.</a> Nat Rev Endocrinol. 16 (1), 45-57 (2020).</li><li>Fini, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+multiwell-based+fluorescent+screen+for+monitoring+vertebrate+thyroid+hormone+disruption.">An in vivo multiwell-based fluorescent screen for monitoring vertebrate thyroid hormone disruption.</a> Environ Sci Technol. 41 (16), 5908-5914 (2007).</li><li>Mughal, B. B., Fini, J. B., Demeneix, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thyroid-disrupting+chemicals+and+brain+development:+an+update.">Thyroid-disrupting chemicals and brain development: an update.</a> Endocr Connect. 7 (4), 160-186 (2018).</li><li>Dong, M., Li, Y., Zhu, M., Li, J., Qin, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrabromobisphenol+a+disturbs+brain+development+in+both+thyroid+hormone-dependent+and+-independent+manners+in+xenopus+laevis.">Tetrabromobisphenol a disturbs brain development in both thyroid hormone-dependent and -independent manners in xenopus laevis.</a> Molecules. 27 (1), 249 (2021).</li><li>Beck, K. R., Sommer, T. J., Schuster, D., Odermatt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+tetrabromobisphenol+A+effects+on+human+glucocorticoid+and+androgen+receptors:+A+comparison+of+results+from+human-+with+yeast-based+in+vitro+assays.">Evaluation of tetrabromobisphenol A effects on human glucocorticoid and androgen receptors: A comparison of results from human- with yeast-based in vitro assays.</a> Toxicology. 370, 70-77 (2016).</li><li>Li, J., Li, Y., Zhu, M., Song, S., Qin, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multiwell-based+assay+for+screening+thyroid+hormone+signaling+disruptors+using+thibz+expression+as+a+sensitive+endpoint+in+xenopus+laevis.">A multiwell-based assay for screening thyroid hormone signaling disruptors using thibz expression as a sensitive endpoint in xenopus laevis.</a> Molecules. 27 (3), 798 (2022).</li><li>Myosho, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preself-feeding+medaka+fry+provides+a+suitable+screening+system+for+in+vivo+assessment+of+thyroid+hormone-disrupting+potential.">Preself-feeding medaka fry provides a suitable screening system for in vivo assessment of thyroid hormone-disrupting potential.</a> Environ Sci Technol. 56 (10), 6479-6490 (2022).</li><li>Mohacsik, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Transgenic+mouse+model+for+detection+of+tissue-specific+thyroid+hormone+action.">A Transgenic mouse model for detection of tissue-specific thyroid hormone action.</a> Endocrinology. 159 (2), 1159-1171 (2018).</li><li>Sinko, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrabromobisphenol+A+and+diclazuril+evoke+tissue-specific+changes+of+thyroid+hormone+signaling+in+male+thyroid+hormone+action+indicator+Mice.">Tetrabromobisphenol A and diclazuril evoke tissue-specific changes of thyroid hormone signaling in male thyroid hormone action indicator Mice.</a> Int J Mol Sci. 23 (23), 14782 (2022).</li><li>Sinko, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+hypothalamic+mechanisms+control+decreased+circulating+thyroid+hormone+levels+in+infection+and+fasting-induced+non-thyroidal+illness+syndrome+in+male+thyroid+hormone+action+indicator+mice.">Different hypothalamic mechanisms control decreased circulating thyroid hormone levels in infection and fasting-induced non-thyroidal illness syndrome in male thyroid hormone action indicator mice.</a> Thyroid. 33 (1), 109-118 (2023).</li><li>Salas-Lucia, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+T3+uptake+and+transport+can+trigger+thyroid+hormone+signaling+in+the+brain.">Axonal T3 uptake and transport can trigger thyroid hormone signaling in the brain.</a> Elife. 12, 82683 (2023).</li><li>Liu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triiodothyronine+(T3)+promotes+brown+fat+hyperplasia+via+thyroid+hormone+receptor+alpha+mediated+adipocyte+progenitor+cell+proliferation.">Triiodothyronine (T3) promotes brown fat hyperplasia via thyroid hormone receptor alpha mediated adipocyte progenitor cell proliferation.</a> Nat Commun. 13 (1), 3394 (2022).</li><li>Bianco, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=American+thyroid+association+guide+to+investigating+thyroid+hormone+economy+and+action+in+rodent+and+cell+models.">American thyroid association guide to investigating thyroid hormone economy and action in rodent and cell models.</a> Thyroid. 24 (1), 88-168 (2014).</li><li>Silva, J. E., Larsen, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenergic+activation+of+triiodothyronine+production+in+brown+adipose+tissue.">Adrenergic activation of triiodothyronine production in brown adipose tissue.</a> Nature. 305 (5936), 712-713 (1983).</li><li>Bianco, A. C., Silva, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+conversion+of+thyroxine+to+triiodothyronine+is+required+for+the+optimal+thermogenic+function+of+brown+adipose+tissue.">Intracellular conversion of thyroxine to triiodothyronine is required for the optimal thermogenic function of brown adipose tissue.</a> J Clin Invest. 79 (1), 295-300 (1987).</li><li>Caporale, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+cohorts+to+molecules:+Adverse+impacts+of+endocrine+disrupting+mixtures.">From cohorts to molecules: Adverse impacts of endocrine disrupting mixtures.</a> Science. 375 (6582), 8244 (2022).</li></ol>

The authors have nothing to disclose.

The threats posed by Endocrine-Disrupting Chemicals (EDCs) to human health are well recognized; however, research on EDCs faces formidable challenges. These challenges are partially a consequence of the complexity of the endocrine system. Many EDCs have been identified to simultaneously disrupt multiple endocrine systems22. Additionally, in the context of Thyroid Hormone (TH) economy, there exists an additional layer of complexity due to tissue-specific differences in regulating TH action. This co...

Thyroid hormone (TH) signaling is a fundamental regulator of cellular metabolism, essential for normal development and optimal tissue function in adulthood1. Within tissues, TH action is finely controlled by a complex molecular machinery, allowing for tissue-specific maintenance of local TH levels. This autonomy of different tissues from circulating TH levels is of great importance2,3,4.
Numerous chemicals have the potential to disrupt endocrine functions and are found in the environment as pollutants. It is a growing concern that these molecules can enter the food chain through wastewater and agricultural production, thereby impacting the health of livestock and humans5,6,7.
One of the significant challenges in addressing this issue is the sheer number of compounds involved, including both authorized and already banned, but still persistently present, molecules. In recent years, substantial efforts have been made to develop test systems for screening and identifying the disruptive potential of various chemicals8,9,10,11. While these methods excel in high-throughput screening of thousands of compounds and identifying potential threats, a detailed analysis of specific in vivo effects of these molecules is essential to establish the hazards of human exposure. Thus, a multifaceted approach is necessary when studying and characterizing endocrine-disrupting chemicals (EDCs).
In the context of TH regulation, understanding the tissue-specific consequences of EDC exposure requires quantifying local TH action. Although several in vivo models have been developed for this purpose, most rely on endogenous markers as their output measure. Despite being physiological, these markers are subject to numerous regulatory mechanisms, both direct and indirect, making their interpretation more challenging. Therefore, characterizing EDC effects on TH regulation at the tissue level remains a significant challenge12,13.
To address the challenges of measuring tissue-specific TH signaling, the Thyroid Hormone Action Indicator (THAI) mouse model was recently developed. This model allows for specific quantification of changes in local TH action under endogenous conditions. A luciferase transgene was introduced into the mouse genome, which is highly sensitive to regulation by TH action14. This model has demonstrated effectiveness in answering various research questions that require quantifying changes in local tissue TH signaling14,15,16,17,18.
Recognition of one potential use of the THAI model is characterizing tissue-specific effects of EDCs on TH signaling. The model has recently been employed successfully to investigate the tissue-specific effects of tetrabromobisphenol A and diclazuril on TH signaling15. Here, baseline protocols are presented for utilizing in vivo imaging techniques on the THAI model as a test system for characterizing EDCs that disrupt TH function. This method leverages the bioluminescent nature of the luciferin-luciferase reaction. Essentially, the transgenically expressed luciferase enzyme catalyzes the oxidation of administered luciferin, generating luminescent light proportional to the amount of luciferase in the tissue (Figure 1). Consequently, the biological response measured is luciferase activity, which has been validated as a suitable measure of local TH action14. While the THAI model is applicable for quantifying TH action in virtually all tissues, in vivo imaging primarily focuses on TH action in the small intestine (ventral imaging) and the interscapular brown adipose tissue (BAT, dorsal imaging)14.
A significant advantage of the in vivo imaging technique is that it eliminates the need to sacrifice animals for measurements. This allows investigators to design longitudinal and follow-up experiments as self-controlled studies, reducing between-subjects bias and the number of animals used. This aspect is particularly crucial in EDC characterization, and the method&#39;s strength and versatility for this purpose have been previously demonstrated14,15.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3,5,3&#39;-triiodothyronine (T3)</td>
			<td>Merck</td>
			<td>T2877</td>
			<td></td>
		</tr>
		<tr>
			<td>Animals, mice</td>
			<td></td>
			<td></td>
			<td>THAI mouse</td>
		</tr>
		<tr>
			<td>Eye protection gel</td>
			<td>Oculotect</td>
			<td></td>
			<td>1000 IU/g</td>
		</tr>
		<tr>
			<td>Falcon tube</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>50 mL volume</td>
		</tr>
		<tr>
			<td>Iodine-free chow diet</td>
			<td>Research Diets</td>
			<td>custom</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina II in vivo imaging system</td>
			<td>Perkin Elmer</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Vetcentre</td>
			<td>E1857</td>
			<td></td>
		</tr>
		<tr>
			<td>Living Image software 4.5</td>
			<td>Perkin Elmer</td>
			<td>-</td>
			<td>provided with the instrument</td>
		</tr>
		<tr>
			<td>Measuring cylinder</td>
			<td></td>
			<td></td>
			<td>250 mL</td>
		</tr>
		<tr>
			<td>methimazole</td>
			<td>Merck</td>
			<td>M8506</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfuge tubes</td>
			<td>Eppendorf</td>
			<td></td>
			<td>For diluting treatment materials</td>
		</tr>
		<tr>
			<td>NaClO4</td>
			<td>Merck</td>
			<td>71852</td>
			<td></td>
		</tr>
		<tr>
			<td>Na-luciferin, substrate</td>
			<td>Goldbio</td>
			<td>103404-75-7</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Merck</td>
			<td>101052833</td>
			<td></td>
		</tr>
		<tr>
			<td>Phoshphate buffer saline</td>
			<td>Chem Cruz</td>
			<td>sc-362302</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Gilson</td>
			<td></td>
			<td>For diluting treatment materials</td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>Axygen</td>
			<td></td>
			<td>For diluting treatment materials</td>
		</tr>
		<tr>
			<td>Shaving cream/epilator/shaver</td>
			<td></td>
			<td></td>
			<td>Personal preference</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>B Braun</td>
			<td></td>
			<td>1 mL volume</td>
		</tr>
		<tr>
			<td>Syringe needle</td>
			<td>B Braun</td>
			<td></td>
			<td>0.3 x 12 mm</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Vetcentre</td>
			<td>E1852</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo characterization of endocrine disrupting chemical effects via thyroid hormone action indicator mouse

Thyroid hormones (TH) play a critical role in cell metabolism and tissue function. TH economy is susceptible to endocrine disrupting chemicals (EDCs) that can disturb hormone production or action. Many environmental pollutants are EDCs, representing an emerging threat to both human health and agricultural production. This has led to an increased demand for proper test systems to examine the effects of potential EDCs. However, current methodologies face challenges. Most test systems use endogenous markers regulated by multiple, often complex regulatory processes, making it difficult to distinguish direct and indirect effects. Moreover, in vitro test systems lack the physiological complexity of EDC metabolism and pharmacokinetics in mammals. Additionally, exposure to environmental EDCs usually involves a mixture of multiple compounds, including in vivo generated metabolites, so the possibility of interactions cannot be ignored. This complexity makes EDC characterization difficult. The Thyroid Hormone Action Indicator (THAI) mouse is a transgenic model that carries a TH-responsive luciferase reporter system, enabling the assessment of tissue-specific TH action. One can evaluate the tissue-specific effects of chemicals on local TH action by quantifying luciferase reporter expression in tissue samples. Furthermore, with in vivo imaging, the THAI mouse model allows for longitudinal studies on the effects of potential EDCs in live animals. This approach provides a powerful tool for testing long-term exposure, complex treatment structures, or withdrawal, as it enables the assessment of changes in local TH action over time in the same animal. This report describes the process of in vivo imaging measurements on THAI mice. The protocol discussed here focuses on developing and imaging hyper- and hypothyroid mice, which can serve as controls. Researchers can adapt or expand the treatments presented to meet their specific needs, offering a foundational approach for further investigation.

Generally, the measured radiance ranges from magnitudes of 105 to 1010 p/s/cm2/sr. However, exact values can vary among animals within the same image and across different images. Therefore, comparing raw data might be misleading. It&#39;s crucial to establish control and background signals in all experiments, making self-controlled designs highly recommended.
Figure 2</strong...

Watch this Scientific Journal Video about In vivo Characterization of Endocrine Disrupting Chemical Effects via Thyroid Hormone Action Indicator Mouse at JoVE.com

Author Spotlight: In Vivo Assessment of Thyroid Hormone Disruption Using the THAI Mouse Model

The Thyroid Hormone Action Indicator mouse model was developed to enable tissue-specific quantification of local thyroid hormone action using its endogenous regulatory machinery. Recently, it has been shown that the model is suitable for characterizing endocrine-disrupting chemicals interacting with thyroid hormone economy, both by ex vivo and in vivo methodologies.

In vivo Characterization of Endocrine Disrupting Chemical Effects via Thyroid Hormone Action Indicator Mouse

Thyroid hormones (TH) play a critical role in cell metabolism and tissue function. TH economy is susceptible to endocrine disrupting chemicals (EDCs) that ...

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Research

JoVE Journal

Neuroscience

736 Views.  Institute of Experimental Medicine. The Thyroid Hormone Action Indicator mouse model was developed to enable tissue-specific quantification of local thyroid hormone action using its endogenous regulatory machinery. Recently, it has been shown that the model is suitable for characterizing endocrine-disrupting chemicals interacting with thyroid hormone economy, both by ex vivo and in vivo methodologies.

Video: Author Spotlight: In Vivo Assessment of Thyroid Hormone Disruption Using the THAI Mouse Model

MPI CyberMotion Simulator: Implementation of a Novel Motion Simulator to Investigate Multisensory Path Integration in Three Dimensions

Path integration is a process in which self-motion is integrated over time to obtain an estimate of one's current position relative to a starting point 1. Humans can do path integration based exclusively on visual 2-3, auditory 4, or inertial cues 5. However, with multiple cues present, inertial cues - particularly kinaesthetic - seem to dominate 6-7. In the absence of vision, humans tend to overestimate short distances (&lt;5 m) and turning angles (&lt;30&deg;), but underestimate longer ones 5. Movement through physical space therefore does not seem to be accurately represented by the brain.
Extensive work has been done on evaluating path integration in the horizontal plane, but little is known about vertical movement (see 3 for virtual movement from vision alone). One reason for this is that traditional motion simulators have a small range of motion restricted mainly to the horizontal plane. Here we take advantage of a motion simulator 8-9 with a large range of motion to assess whether path integration is similar between horizontal and vertical planes. The relative contributions of inertial and visual cues for path navigation were also assessed.
16 observers sat upright in a seat mounted to the flange of a modified KUKA anthropomorphic robot arm. Sensory information was manipulated by providing visual (optic flow, limited lifetime star field), vestibular-kinaesthetic (passive self motion with eyes closed), or visual and vestibular-kinaesthetic motion cues. Movement trajectories in the horizontal, sagittal and frontal planes consisted of two segment lengths (1st: 0.4 m, 2nd: 1 m; &plusmn;0.24 m/s2 peak acceleration). The angle of the two segments was either 45&deg; or 90&deg;. Observers pointed back to their origin by moving an arrow that was superimposed on an avatar presented on the screen.
Observers were more likely to underestimate angle size for movement in the horizontal plane compared to the vertical planes. In the frontal plane observers were more likely to overestimate angle size while there was no such bias in the sagittal plane. Finally, observers responded slower when answering based on vestibular-kinaesthetic information alone. Human path integration based on vestibular-kinaesthetic information alone thus takes longer than when visual information is present. That pointing is consistent with underestimating and overestimating the angle one has moved through in the horizontal and vertical planes respectively, suggests that the neural representation of self-motion through space is non-symmetrical which may relate to the fact that humans experience movement mostly within the horizontal plane.

An efficient way to gain insight into how humans navigate themselves in three dimensions is described. The method takes advantage of a motion simulator capable of moving observers in ways unattainable by traditional simulators. Results confirm that movement in the horizontal plane is underestimated, while vertical movement is overestimated.

Path integration is a process in which self-motion is integrated over time to obtain an estimate of one's current position relative to a starting point 1. ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors&#8212;an essential step toward a model of odor coding in the mammalian olfactory system.

Olfactory receptor activation patterns encode odor identity, but the lack of published data identifying odorant ligands for mammalian olfactory receptors hinders the development of a comprehensive model of odor coding. This protocol describes a method to facilitate high-throughput identification of olfactory receptor ligands and quantification of receptor activation.

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

Preparation of Single-cohort Colonies and Hormone Treatment of Worker Honeybees to Analyze Physiology Associated with Role and/or Endocrine System

Honeybee workers are engaged in various tasks related to maintaining colony activity. The tasks of the workers change according to their age (age-related division of labor). Young workers are engaged in nursing the brood (nurse bees), while older workers are engaged in foraging for nectar and pollen (foragers). The physiology of the workers changes in association with this role shift. For example, the main function of the hypopharyngeal glands (HPGs) changes from the secretion of major royal jelly proteins (MRJPs) to the secretion of carbohydrate-metabolizing enzymes. Because worker tasks change as the workers age in typical colonies, it is difficult to discriminate the physiological changes that occur with aging from those that occur with the role shift. To study the physiological changes in worker tissues, including the HPGs, in association with the role shift, it would be useful to manipulate the honeybee colony population by preparing single-cohort colonies in which workers of almost the same age perform different tasks. Here we describe a detailed protocol for preparing single-cohort colonies for this analysis. Six to eight days after single-cohort colony preparation, precocious foragers that perform foraging tasks earlier than usual appear in the colony. Representative results indicated role-associated changes in HPG gene expression, suggesting role-associated HPG function. In addition to manipulating the colony population, analysis of the endocrine system is important for investigating role-associated physiology. Here, we also describe a detailed protocol for treating workers with 20-hydroxyecdysone (20E), an active form of ecdysone, and methoprene, a juvenile hormone analogue. The survival rate of treated bees was sufficient to examine gene expression in the HPGs. Gene expression changes were observed in response to 20E- and/or methoprene-treatment, suggesting that hormone treatments induce physiological changes of the HPGs. The protocol for hormone treatment described here is appropriate for examining hormonal effects on worker physiology.

Here we describe our detailed protocol for the preparation of single-cohort honeybee colonies&#160;&#8211; a useful tool for analyzing the role-associated worker physiology. We also describe detailed protocols for treating workers with juvenile hormone and ecdysone to evaluate the involvement of these hormones in the regulation of worker behavior and/or physiology.

Honeybee workers are engaged in various tasks related to maintaining colony activity. The tasks of the workers change according to their age (age-related ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...