We would like to acknowledge VPC, CJR, and MCP for the development of this protocol. EMM received financial support from the American University College of Arts and Sciences Graduate Student Support to carry out this research.&#160;This work was also supported by an American University Faculty Mellon Award and funding through the American University College of Arts and Sciences (both to VPC).

All procedures were approved by the Institutional Animal Care and Use Committee at American University.
1. Preparing the Solution Tanks
<ol>
	<li>Obtain six tanks, two for each experimental group (glucose, mannitol, and water). Label one of the two tanks &#8216;housing tank&#8217; (it will house the fish) and label the other &#8216;solution tank&#8217; (it will hold the solution). 
	NOTE: The mannitol treatment group is the osmotic control, and the water treatment group is a handling/stress control. It is important to keep the tanks, airlines/airstones, lids, and cleaning supplies separate for each treatment group</li>
	<li>Use a 2 L tank if the total number of fish used is less than 20. Use a 4 L tank if the total number of fish used is more than 20. 
	NOTE: Use an N of 5-10 per treatment group per sampling time point.</li>
	<li>Keep the tanks in a water bath at 28-29 &#176;C to maintain water temperature.</li>
	<li>On Day 1, place the fish into their respective treatment solutions (glucose, mannitol, water) for 24-hours (&#8216;Water to Treatment&#8217;). On Day 2, transfer the fish from their treatment solutions to water for 24-hours (&#8216;Treatment to Water&#8217;). On Day 3, transfer the fish from water to treatment solutions (&#8216;Water to Treatment&#8217;). This alternating exposure continues for the remainder of the experiment (Figure 1). Transfer water-treated control fish from water to water daily.</li>
	<li>Ensure that the fish are fed and transferred within the same 2-hour window each day throughout the duration of the experiment.</li>
</ol>
2. Preparing the fish
<ol>
	<li>Use adult zebrafish (4 months &#8211; 1 year)5.</li>
	<li>Feed the fish ground Tetramin flakes daily upon arrival to the lab.</li>
	<li>Record the pH and the temperature of all the tanks and record the general condition of the fish.</li>
</ol>
3. Transferring fish
<ol>
	<li>Transfer fish in each treatment group from the housing tank to the corresponding solution tank using a standard fish net.</li>
	<li>Place the tank containing the fish back in the water bath, replace the airstone and tank lid. This tank is now the &#8216;housing tank&#8217; and the tank that previously held the fish is now the &#8216;solution tank&#8217;.</li>
	<li>Discard the old solution and clean the tank, along with the tank lids, airlines, airstones, and nets to prevent buildup of glucose and mannitol. 
	NOTE: Do not wash items with soap. Use water and a dedicated scrub brush/sponge for each treatment condition to properly clean the tanks.</li>
	<li>Dry the newly cleaned &#8216;solution tanks&#8217; with a paper towel. Prepare the solutions for the following day using this tank. Ensure the other items are dried and separated by appropriate treatment groups. 
	NOTE: Keep a log of what solutions the fish are being transferred out of and into each day, as well as the solutions that are prepared for the following day. For example: Fish transferred from glucose to H2O, new 1% glucose solution prepared for tomorrow.</li>
</ol>
4. Post-transfer solution preparation
<ol>
	<li>Preparing sugar solutions
	<ol>
		<li>Fill each solution tank with 2 L (or 4 L) of System Water (system water is defined as water that has been treated with the correct ratio of salt solution and is at the same temperature as stock and treatment tanks).</li>
		<li>Measure the correct amount of glucose and mannitol (see step 5 below) using a top loading scale and separate weigh boats for each chemical.</li>
		<li>Add the weighed glucose or mannitol aliquot to the appropriate, cleaned solution tank, which contains only system water.</li>
		<li>Stir the glucose and mannitol solutions with separate glass stir rods until the sugars are completely dissolved.</li>
		<li>Return solution tanks to the water bath and cover with their corresponding lids.</li>
	</ol>
	</li>
	<li>Preparing water solutions
	<ol>
		<li>Fill experimental tanks (2 L or 4 L) with System Water.</li>
		<li>Return these &#8216;solution tanks&#8217; to the water bath and cover with their corresponding lids.</li>
	</ol>
	</li>
</ol>
5. Changing percentages
<ol>
	<li>Maintain the fish in a 1% solution during the first 2-weeks of treatment: 40 g of glucose or mannitol in a 4 L tank.</li>
	<li>Maintain the fish in a 2% solution during weeks 3 and 4 of treatment: 80 g of glucose or mannitol in a 4 L tank.</li>
	<li>Maintain the fish in a 3% solution for the final 4 weeks of treatment: 120 g of glucose or mannitol in a 4 L tank.</li>
</ol>
6. Measuring blood glucose levels and collecting tissue
<ol>
	<li>Anesthetize fish 2 at a time in a 0.02% Tricaine solution.</li>
	<li>Decapitate the fish directly behind the gills using a razorblade.</li>
	<li>Measure blood sugar value. 
	NOTE: We use a blood glucose meter (e.g., Freestyle Lite) to measure blood glucose and place the test strip directly on the exposed heart (cardiac blood sample).</li>
	<li>Dissect the wanted tissue from the fish (brain, muscle, etc.).</li>
	<li>Store collected tissue by flash freezing on dry ice and storing in a -80 &#176;C freezer, fixing in 4% paraformaldehyde, or placing in a buffer solution for immediate use.</li>
</ol>

<ol>
	<li>Rubinstein, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+from+disease+modeling+to+drug+discovery.">Zebrafish: from disease modeling to drug discovery.</a> Current Opinion in Drug Discovery and Development. 6 (2), 218-223 (2003).</li><li>Gerlai, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associative+learning+in+zebrafish+(Danio+rerio).">Associative learning in zebrafish (Danio rerio).</a> Methods in Cell Biology. 101, 249-270 (2011).</li><li>Goldsmith, J. R., Jobin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Think+small:+zebrafish+as+a+model+system+of+human+pathology.">Think small: zebrafish as a model system of human pathology.</a> BioMed Research International. , (2012).</li><li>Moss, 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=Regeneration+of+the+pancreas+in+adult+zebrafish.">Regeneration of the pancreas in adult zebrafish.</a> Diabetes. 58 (8), 1844-1851 (2009).</li><li>Connaughton, V. P., Baker, C., Fonde, L., Gerardi, E., Slack, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternate+immersion+in+an+external+glucose+solution+differentially+affects+blood+sugar+values+in+older+versus+younger+zebrafish+adults.">Alternate immersion in an external glucose solution differentially affects blood sugar values in older versus younger zebrafish adults.</a> Zebrafish. 13 (2), 87-94 (2016).</li><li>Etuk, E. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+studying+diabetes+mellitus.">Animal models for studying diabetes mellitus.</a> Agriculture and Biology Journal of North America. 1 (2), 130-134 (2010).</li><li>Agardh, E., Bruun, A., Agardh, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+glial+cell+immunoreactivity+and+neuronal+cell+changes+in+rats+with+STZ-induced+diabetes.">Retinal glial cell immunoreactivity and neuronal cell changes in rats with STZ-induced diabetes.</a> Current Eye Research. 23 (4), 276-284 (2001).</li><li>Carmo, A., Cunha-Vaz, J. G., Carvalho, A. P., Lopes, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+synthase+activity+in+retinas+from+non-insulin-dependent+diabetic+Goto-Kakizaki+rats:+correlation+with+blood-retinal+barrier+permeability.">Nitric oxide synthase activity in retinas from non-insulin-dependent diabetic Goto-Kakizaki rats: correlation with blood-retinal barrier permeability.</a> Nitric Oxide. 4 (6), 590-596 (2000).</li><li>Ramsey, D. J., Ripps, H., Qian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+electrophysiological+study+of+retinal+function+in+the+diabetic+female+rat.">An electrophysiological study of retinal function in the diabetic female rat.</a> Investigative Ophthalmology &amp; Visual Science. 47 (11), 5116-5124 (2006).</li><li>Biessels, G. J., Gispen, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+diabetes+on+cognition:+what+can+be+learned+from+rodent+models.">The impact of diabetes on cognition: what can be learned from rodent models.</a> Neurobiology of Aging. 26 (1), 36-41 (2005).</li><li>Intine, R. V., Olsen, A. S., Sarras, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+zebrafish+model+of+diabetes+mellitus+and+metabolic+memory.">A zebrafish model of diabetes mellitus and metabolic memory.</a> Journal of Visualized Experiments. (72), e50232 (2013).</li><li>Sarras, M. P., Intine, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+zebrafish+as+a+disease+model+provides+a+unique+window+for+understanding+the+molecular+basis+of+diabetic+metabolic+memory.">Use of zebrafish as a disease model provides a unique window for understanding the molecular basis of diabetic metabolic memory.</a> Research on Diabetes. , (2013).</li><li>Gleeson, M., Connaughton, V., Arneson, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+hyperglycaemia+in+zebrafish+(Danio+rerio)+leads+to+morphological+changes+in+the+retina.">Induction of hyperglycaemia in zebrafish (Danio rerio) leads to morphological changes in the retina.</a> Acta Diabetologica. 44 (3), 157-163 (2007).</li><li>Capiotti, K. M., 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=Hyperglycemia+induces+memory+impairment+linked+to+increased+acetylcholinesterase+activity+in+zebrafish+(Danio+rerio).">Hyperglycemia induces memory impairment linked to increased acetylcholinesterase activity in zebrafish (Danio rerio).</a> Behavioural Brain Research. 274, 319-325 (2014).</li><li>Capiotti, K. M., 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=Persistent+impaired+glucose+metabolism+in+a+zebrafish+hyperglycemia+model.">Persistent impaired glucose metabolism in a zebrafish hyperglycemia model.</a> Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. 171, 58-65 (2014).</li><li>Capiotti, K. M., 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=Hyperglycemia+alters+E-NTPDases,+ecto-5'-nucleotidase,+and+ectosolic+and+cytosolic+adenosine+deaminase+activities+and+expression+from+encephala+of+adult+zebrafish+(Danio+rerio).">Hyperglycemia alters E-NTPDases, ecto-5'-nucleotidase, and ectosolic and cytosolic adenosine deaminase activities and expression from encephala of adult zebrafish (Danio rerio).</a> Purinergic Signaling. 12 (2), 211-220 (2016).</li><li>Ali, Z., 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=Photoreceptor+Degeneration+Accompanies+Vascular+Changes+in+a+Zebrafish+Model+of+Diabetic+Retinopathy.">Photoreceptor Degeneration Accompanies Vascular Changes in a Zebrafish Model of Diabetic Retinopathy.</a> Investigative Ophthalmology &amp; Visual Science. 61 (2), 43 (2020).</li><li>Wiggenhauser, L. M., 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=Activation+of+Retinal+Angiogenesis+in+Hyperglycemic+pdx1-/-+Mutants.">Activation of Retinal Angiogenesis in Hyperglycemic pdx1-/- Mutants.</a> Diabetes. 69 (5), 1020-1031 (2020).</li><li>Chen, X. L., 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=Involvement+of+HMGB1+mediated+signalling+pathway+in+diabetic+retinopathy:+evidence+from+type+2+diabetic+rats+and+ARPE-19+cells+under+diabetic+condition.">Involvement of HMGB1 mediated signalling pathway in diabetic retinopathy: evidence from type 2 diabetic rats and ARPE-19 cells under diabetic condition.</a> Journal of Ophthalmology. 97, 1598-1603 (2013).</li><li>Costa, E., 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=Effects+of+light+exposure,+pH,+osmolarity,+and+solvent+on+the+retinal+pigment+epithelial+toxicity+of+vital+dyes.">Effects of light exposure, pH, osmolarity, and solvent on the retinal pigment epithelial toxicity of vital dyes.</a> American Journal of Ophthalmology. 155, 705-712 (2013).</li><li>Alvarez, Y., 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=Predominant+cone+photoreceptor+dysfunction+in+a+hyperglycemic+model+of+non-proliferative+diabetic+retinopathy.">Predominant cone photoreceptor dysfunction in a hyperglycemic model of non-proliferative diabetic retinopathy.</a> Disease Models and Mechanisms. 3, 236-245 (2010).</li><li>Fletcher, E. L., Phipps, J. A., Wilkinson-Berka, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysfunction+of+retinal+neurons+and+glia+during+diabetes.">Dysfunction of retinal neurons and glia during diabetes.</a> Clinical and Experimental Optometry. 88, 132-145 (2005).</li><li>Fletcher, E. L., Phipps, J. A., Ward, M. M., Puthussery, T., Wilkinson-Berka, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+and+glial+abnormality+as+predictors+of+progression+of+diabetic+retinopathy.">Neuronal and glial abnormality as predictors of progression of diabetic retinopathy.</a> Current Pharmaceutical Design. 13, 2699-2712 (2007).</li><li>Agardh, E., Bruun, A., Agardh, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+glial+cell+immunoreactivity+and+neuronal+cell+changes+in+rats+with+STZ-+induced+diabetes.">Retinal glial cell immunoreactivity and neuronal cell changes in rats with STZ- induced diabetes.</a> Current Eye Research. 23, 276-284 (2001).</li><li>Barber, A. J., Antonetti, D. A., Gardner, T. W., Group, T. P. S. R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+expression+of+retinal+occludin+and+glial+fibrillary+acidic+protein+in+experimental+diabetes.">Altered expression of retinal occludin and glial fibrillary acidic protein in experimental diabetes.</a> Investigative Ophthalmology &amp; Visual Science. 41, 3561-3568 (2000).</li><li>Lieth, E., 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=Glial+reactivity+and+impaired+glutamate+metabolism+in+short-term+experimental+diabetic+retinopathy.">Glial reactivity and impaired glutamate metabolism in short-term experimental diabetic retinopathy.</a> Diabetes. 47, 815-820 (1998).</li><li>Rungger-Brandle, E., Dosso, A. A., Leuenberger, P. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+and+microglial+response+in+the+retina+of+streptozotocin-induced+diabetic+rats.">Neuronal and microglial response in the retina of streptozotocin-induced diabetic rats.</a> Visual Neuroscience. 17, 463-471 (2000).</li><li>Mizutani, M., Gerhardinger, C., Lorenzi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muller+cell+changes+in+human+diabetic+retinopathy.">Muller cell changes in human diabetic retinopathy.</a> Diabetes. 47, 445-449 (1998).</li><li>Tanvir, Z., Nelson, R., DeCicco-Skinner, K., Connaughton, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+month+of+hyperglycemia+alters+spectral+responses+of+the+zebrafish+photopic+electroretinogram.">One month of hyperglycemia alters spectral responses of the zebrafish photopic electroretinogram.</a> Disease Models and Mechanisms. 11, (2018).</li><li>Hancock, H. A., Kraft, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillatory+potential+analysis+and+ERGs+of+normal+and+diabetic+rats.">Oscillatory potential analysis and ERGs of normal and diabetic rats.</a> Investigative Ophthalmology &amp; Visual Science. 45, 1002-1008 (2004).</li><li>Layton, C. J., Safa, R., Osborne, N. 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The authors declare no conflicts of interest.

Diabetes is a nationwide problem. Studies show that by 2030, an estimated 400 million people will have some form of diabetes. In rodent models, Type 2 DM is studied using genetic manipulation. In rats, the Zucker diabetic fatty rats (ZDF), and the Otsuka Long-Evans Tokushima fatty rats (OLETF), are providing more information on the effects of Type 2 DM10. In addition, high fat diets have been used in rodent to induce hyperglycemia. This mirrors the noninvasive procedure proposed in this paper. Usi...

Zebrafish (Danio rerio) are quickly becoming a widely used animal model to study both disease and cognition1. The ease of genetic manipulation and embryonic transparency through the early developmental stages, make them a prime candidate to study human diseases with a known genetic basis. For example, zebrafish have been used to study Holt-Oram syndrome, cardiomyopathies, glomerulocystic kidney disease, muscular dystrophy, and diabetes mellitus (DM) among other diseases1. In addition, the zebrafish model is ideal because of the species&#8217; small size, ease of maintenance, and high fecundity2,3.
The zebrafish pancreas is both anatomically and functionally similar to the mammalian pancreas4. Thus, the unique characteristics of size, high fecundity, and similar endocrine structures make zebrafish a suitable candidate for studying DM-related complications. In zebrafish, there are two experimental methods used to induce the prolonged hyperglycemia that is characteristic of DM: an influx of glucose (modeling Type 2) and cessation of insulin secretion (modeling Type 1)5,6. Experimentally, to stop insulin secretion, pancreatic &#946;-cells can be chemically destroyed using either Streptozotocin (STZ) or Alloxan injections. STZ has been used successfully in rodents and zebrafish, resulting in complications associated with retinopathy7,8,9, cognitive impairments10, and limb regeneration11. However, in zebrafish, &#946;-cells regenerate after treatment, causing &#8220;booster injections&#8221; of STZ to be necessary to maintain diabetic conditions12. Alternatively, the pancreas of the zebrafish can be removed6. These are both highly invasive procedures, due to the multiple injections, and extensive recovery time.
Conversely, hyperglycemia can be induced noninvasively through exposure to exogenous glucose. In this protocol, fish are submerged in a highly concentrated glucose solution for 24-hours5,13 or continually for 2-weeks14,15,16. Exogenous glucose is taken up transdermally, by ingestion, and/or across the gills resulting in elevated blood sugar levels. Since this non-invasive technique does not directly manipulate insulin levels, it cannot claim to induce Type 2 DM. However, it can be used to examine complications induced by hyperglycemia, which is one of the main symptoms of Type 2 DM.
Recently, the zebrafish mutant pdx1-/- was developed by manipulating the pancreatic and duodenal homeobox 1 gene, a gene linked to the genetic cause of Type 2 DM in humans. Using this mutant, researchers have been able to replicate pancreatic development disruption, high blood sugar, and study hyperglycemia-induced diabetic retinopathy17,18.
In this paper, we describe a noninvasive hyperglycemia induction method that uses an alternating immersion protocol. This protocol maintains hyperglycemic conditions for up to 8 weeks with subsequent complications observed. In brief, adult zebrafish are placed in a sugar solution for 24 hours and then a water solution for 24 hours. As opposed to continuous immersion in external glucose solutions, alternating days between sugar and water mimics the rise and fall of blood sugar in diabetes. An alternating glucose protocol additionally allows hyperglycemia to be induced for longer periods of time, as the zebrafish are not as able to compensate for the high external glucose conditions. As proof of principle, we provide data showing that hyperglycemia induced using this protocol alters retinal chemistry and physiology.

<table>
	<tbody>
		<tr>
			<td>Airline Tubing</td>
			<td>petsmart</td>
			<td>5291863</td>
			<td>This can be used in the tank to circulate air</td>
		</tr>
		<tr>
			<td>Airpump</td>
			<td>petsmart</td>
			<td>5094984</td>
			<td>This can be used in the tank to circulate air</td>
		</tr>
		<tr>
			<td>Airstones</td>
			<td>petsmart</td>
			<td>5149683</td>
			<td>This can be used in the tank to circulate air</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma</td>
			<td>G8270-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>D-mannitol</td>
			<td>Acros Organics</td>
			<td>AC125340050</td>
			<td></td>
		</tr>
		<tr>
			<td>Freestyle Lite Meter</td>
			<td>Amazon</td>
			<td>B01LMOMLTU</td>
			<td></td>
		</tr>
		<tr>
			<td>Freestyle Lite Strips</td>
			<td>Amazon</td>
			<td>B074ZN3H2Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Net</td>
			<td>petsmart</td>
			<td>5175115</td>
			<td></td>
		</tr>
		<tr>
			<td>Tanks</td>
			<td>Amazon</td>
			<td>B0002APZO4</td>
			<td></td>
		</tr>
	</tbody>
</table>

alternate immersion in glucose to produce prolonged hyperglycemia in zebrafish

Zebrafish (Danio rerio) are an excellent model to investigate the effects of chronic hyperglycemia, a hallmark of Type II Diabetes Mellitus (T2DM). This alternate immersion protocol is a noninvasive, step-wise method of inducing hyperglycemia for up to eight weeks. Adult zebrafish are alternately exposed to sugar (glucose) and water for 24 hours each. The zebrafish begin treatment in a 1% glucose solution for 2 weeks, then a 2% solution for 2 weeks, and finally a 3% solution for the remaining 4 weeks. Compared to water-treated (stress) and mannitol-treated (osmotic) controls, glucose-treated zebrafish have significantly higher blood sugar levels. The glucose-treated zebrafish show blood sugar levels of 3-times that of controls, suggesting that after both four and eight weeks hyperglycemia can be achieved. Sustained hyperglycemia was associated with increased Glial Fibrillary Acidic Protein (GFAP) and increased nuclear factor Kappa B (NF-kB)&#160;levels in retina and decreased physiological responses, as well as cognitive deficits suggesting this protocol can be used to model disease complications.

Using this protocol (Figure 1), blood sugar values are significantly elevated after both 4-weeks and 8-weeks of treatment (Figure 2A), with hyperglycemia defined as 3x the control averages from both water-treated and mannitol-treated groups. Water-treated controls are transferred in and out of water daily, providing a stress/handling control. Mannitol serves as an osmotic control in in vitro glucose studies19,...

Watch this Scientific Journal Video about Alternate Immersion in Glucose to Produce Prolonged Hyperglycemia in Zebrafish at JoVE.com

Alternate Immersion in Glucose to Produce Prolonged Hyperglycemia in Zebrafish

This protocol noninvasively induces hyperglycemia in zebrafish for up to 8 weeks. Using this protocol, an in-depth study of the adverse effects of hyperglycemia can be made.

Zebrafish (Danio rerio) are an excellent model to investigate the effects of chronic hyperglycemia, a hallmark of Type II Diabetes Mellitus (T2DM). This ...

alternate-immersion-glucose-to-produce-prolonged-hyperglycemia

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4.7K Views.  American University. This protocol noninvasively induces hyperglycemia in zebrafish for up to 8 weeks. Using this protocol, an in-depth study of the adverse effects of hyperglycemia can be made.

Video: Alternate Immersion in Glucose to Produce Prolonged Hyperglycemia in Zebrafish

Human Fear Conditioning Conducted in Full Immersion 3-Dimensional Virtual Reality 

Fear conditioning is a widely used paradigm in non-human animal research to investigate the neural mechanisms underlying fear and anxiety. A major challenge in conducting conditioning studies in humans is the ability to strongly manipulate or simulate the environmental contexts that are associated with conditioned emotional behaviors. In this regard, virtual reality (VR) technology is a promising tool. Yet, adapting this technology to meet experimental constraints requires special accommodations. Here we address the methodological issues involved when conducting fear conditioning in a fully immersive 6-sided VR environment and present fear conditioning data.
In the real world, traumatic events occur in complex environments that are made up of many cues, engaging all of our sensory modalities. For example, cues that form the environmental configuration include not only visual elements, but aural, olfactory, and even tactile. In rodent studies of fear conditioning animals are fully immersed in a context that is rich with novel visual, tactile and olfactory cues. However, standard laboratory tests of fear conditioning in humans are typically conducted in a nondescript room in front of a flat or 2D computer screen and do not replicate the complexity of real world experiences. On the other hand, a major limitation of clinical studies aimed at reducing (extinguishing) fear and preventing relapse in anxiety disorders is that treatment occurs after participants have acquired a fear in an uncontrolled and largely unknown context. Thus the experimenters are left without information about the duration of exposure, the true nature of the stimulus, and associated background cues in the environment1. In the absence of this information it can be difficult to truly extinguish a fear that is both cue and context-dependent. Virtual reality environments address these issues by providing the complexity of the real world, and at the same time allowing experimenters to constrain fear conditioning and extinction parameters to yield empirical data that can suggest better treatment options and/or analyze mechanistic hypotheses.
In order to test the hypothesis that fear conditioning may be richly encoded and context specific when conducted in a fully immersive environment, we developed distinct virtual reality 3-D contexts in which participants experienced fear conditioning to virtual snakes or spiders. Auditory cues co-occurred with the CS in order to further evoke orienting responses and a feeling of "presence" in subjects 2 . Skin conductance response served as the dependent measure of fear acquisition, memory retention and extinction.

Human Fear Conditioning Conducted in Full Immersion 3-Dimensional Virtual Reality

Classical fear conditioning paradigm was adapted for human participants in a fully immersive virtual reality setting. Using a discrimination paradigm, conditioned fear, cue and context memory retention, and extinction was measured with skin conductance response to dynamic virtual snakes and spiders (the conditioned stimuli) in two distinct virtual contexts.

Fear conditioning is a widely used paradigm in non-human animal research to investigate the neural mechanisms underlying fear and anxiety. A major ...

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

A Revised Surgical Approach to Induce Endolymphatic Hydrops in the Guinea Pig

Endolymphatic hydrops is an enlargement of scala media that is most often associated with Meniere&#39;s disease, though the pathophysiologic mechanism(s) remain unclear. In order to adequately study the attributes of endolymphatic hydrops, such as the origins of low-frequency hearing loss, a reliable model is needed. The guinea pig is a&#160;good model because it hears in the low-frequency regions that are putatively affected by endolymphatic hydrops. Previous research has demonstrated that endolymphatic hydrops can be induced surgically via intradural or extradural approaches that involve drilling on the endolymphatic duct and sac. However, whether it was possible to create an endolymphatic hydrops model using an extradural approach that avoided dangerous drilling on the endolymphatic duct and sac was unknown. The objective of this study was to demonstrate a revised extradural approach to induce experimental endolymphatic hydrops at 30 days post-operatively by obliterating the endolymphatic sac and injuring the endolymphatic duct with a fine pick. The sample size consisted of seven guinea pigs. Functional measurements of hearing were made and temporal bones were subsequently harvested for histologic analysis. The approach had a success rate of 86% in achieving endolymphatic hydrops. The risk of cerebrospinal fluid leak was minimal. No perioperative deaths or injuries to the posterior semicircular canal occurred in the sample. The presented method demonstrates a safe and reliable way to induce endolymphatic hydrops at a relatively quick time point of 30 days. The clinical implications are that the presented method provides a reliable model to further explore the origins of low-frequency hearing loss that can be associated endolymphatic hydrops.

This article demonstrates an extradural approach to obliterate the guinea pig endolymphatic sac and injure the endolymphatic duct with a fine pick in order to induce experimental endolymphatic hydrops.

Endolymphatic hydrops is an enlargement of scala media that is most often associated with Meniere&#39;s disease, though the pathophysiologic mechanism(s) ...

Combining Behavior and EEG to Study the Effects of Mindfulness Meditation on Episodic Memory

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has examined the behavioral and neural effects of mindfulness meditation on episodic memory. Here we present a protocol that combines mindfulness meditation training, an episodic memory task, and EEG to examine how mindfulness meditation changes behavioral performance and the neural correlates of episodic memory. Subjects in a mindfulness meditation experimental group were compared to a waitlist control group. Subjects in the mindfulness meditation experimental group spent four weeks training and practicing mindfulness meditation. Mindfulness was measured before and after training using the Five Facet Mindfulness Questionnaire (FFMQ). Episodic memory was measured before and after training using a source recognition task. During the retrieval phase of the source recognition task, EEG was recorded. The results showed that mindfulness, source recognition behavioral performance, and EEG theta power in right frontal and left parietal channels increased following mindfulness meditation training. In addition, increases in mindfulness correlated with increases in theta power in right frontal channels. Therefore, results obtained from combining mindfulness meditation training, an episodic memory task, and EEG reveal the behavioral and neural effects of mindfulness meditation on episodic memory.

Here we present a protocol for combining mindfulness meditation training, an episodic memory task, and EEG to understand the behavioral and neural effects of mindfulness meditation on episodic memory.

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has ...

Alternate Immersion in Glucose to Produce Prolonged Hyperglycemia in Zebrafish

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Chapters in this video

Human Fear Conditioning Conducted in Full Immersion 3-Dimensional Virtual Reality

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

A Revised Surgical Approach to Induce Endolymphatic Hydrops in the Guinea Pig

Combining Behavior and EEG to Study the Effects of Mindfulness Meditation on Episodic Memory