Name: hippocampal insulin microinjection and in vivo microdialysis during spatial memory testing
Uploaded: 2013-01-11T17:00:00
Description: Watch this Scientific Journal Video about Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing at JoVE.com

This work was supported by the NIH/NIDDK (DK077106 to ECM).

1. Surgery Preparation
<ol>
 <li>Handling. Animals (most commonly, rats or mice, although the method for microdialysis and combination with behavioral testing is largely a general one with any required species-specific adaptations needed, e.g. for anesthesia) are handled for a minimum of 10 min/day for at least two days prior to surgery. Extensive handling has been shown to leave animals in an unstressed state at the time of testing, avoiding possible confound4-5,7,8. We will use rats throughout thi...

<ol>
	<li>McNay, E. C., Fries, T. M., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreases+in+rat+extracellular+hippocampal+glucose+concentration+associated+with+cognitive+demand+during+a+spatial+task.">Decreases in rat extracellular hippocampal glucose concentration associated with cognitive demand during a spatial task.</a> Proc. Natl. Acad. Sci. U.S.A. 97 (6), 2881 (2000).</li><li>McNay, E. C., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+differences+in+hippocampal+extracellular+fluid+glucose+concentration+during+behavioral+testing+and+following+systemic+glucose+administration.">Age-related differences in hippocampal extracellular fluid glucose concentration during behavioral testing and following systemic glucose administration.</a> J. Gerontol. A. Biol. Sci. Med. Sci. 56 (2), 66 (2001).</li><li>McNay, E. C., McCarty, R. C., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluctuations+in+brain+glucose+concentration+during+behavioral+testing:+dissociations+between+brain+areas+and+between+brain+and+blood.">Fluctuations in brain glucose concentration during behavioral testing: dissociations between brain areas and between brain and blood.</a> Neurobiol. Learn. Mem. 75 (3), 325 (2001).</li><li>McNay, E. C., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+glucose+concentrations+in+the+rat+hippocampus+measured+by+zero-net-flux:+effects+of+microdialysis+flow+rate.">Extracellular glucose concentrations in the rat hippocampus measured by zero-net-flux: effects of microdialysis flow rate.</a> 72 (2), 785 (1999).</li><li>McNay, E. C., Sherwin, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+recurrent+hypoglycemia+on+spatial+cognition+and+cognitive+metabolism+in+normal+and+diabetic+rats.">Effect of recurrent hypoglycemia on spatial cognition and cognitive metabolism in normal and diabetic rats.</a> Diabetes. 53 (2), 418 (2004).</li><li>McNay, E. 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=Hippocampal+memory+processes+are+modulated+by+insulin+and+high-fat-induced+insulin+resistance.">Hippocampal memory processes are modulated by insulin and high-fat-induced insulin resistance.</a> Neurobiology of Learning and Memory. 93 (4), 546 (2010).</li><li>McNay, E. C., McCarty, R. C., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluctuations+in+brain+glucose+concentration+during+behavioral+testing:+dissociations+between+brain+areas+and+between+brain+and+blood.">Fluctuations in brain glucose concentration during behavioral testing: dissociations between brain areas and between brain and blood.</a> Neurobiology of Learning &amp; Memory. 75 (3), 325 (2001).</li><li>McNay, E. C., Williamson, A., McCrimmon, R. J., Sherwin, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+and+neural+hippocampal+effects+of+long-term+moderate+recurrent+hypoglycemia.">Cognitive and neural hippocampal effects of long-term moderate recurrent hypoglycemia.</a> Diabetes. 55 (4), 1088 (2006).</li><li>McNay, E. C., Sherwin, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+artificial+cerebro-spinal+fluid+(aCSF)+to+artificial+extracellular+fluid+(aECF):+microdialysis+perfusate+composition+effects+on+in+vivo+brain+ECF+glucose+measurements.">From artificial cerebro-spinal fluid (aCSF) to artificial extracellular fluid (aECF): microdialysis perfusate composition effects on in vivo brain ECF glucose measurements.</a> Journal of Neuroscience Methods. 132 (1), 35 (2004).</li><li>McNay, E. 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=Hippocampal+memory+processes+are+modulated+by+insulin+and+high-fat-induced+insulin+resistance.">Hippocampal memory processes are modulated by insulin and high-fat-induced insulin resistance.</a> Neurobiology of Learning and Memory. 93 (4), 546 (2010).</li><li>Benveniste, H., Drejer, J., Schousboe, A., Diemer, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+cerebral+glucose+phosphorylation+and+blood+flow+after+insertion+of+a+microdialysis+fiber+through+the+dorsal+hippocampus+in+the+rat.">Regional cerebral glucose phosphorylation and blood flow after insertion of a microdialysis fiber through the dorsal hippocampus in the rat.</a> Journal of Neurochemistry. 49 (3), 729 (1987).</li><li>Benveniste, H., Diemer, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+reactions+to+implantation+of+a+microdialysis+tube+in+the+rat+hippocampus.">Cellular reactions to implantation of a microdialysis tube in the rat hippocampus.</a> Acta Neuropathologica. 74 (3), 234 (1987).</li><li>McNay, E. C., Fries, T. M., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreases+in+rat+extracellular+hippocampal+glucose+concentration+associated+with+cognitive+demand+during+a+spatial+task.">Decreases in rat extracellular hippocampal glucose concentration associated with cognitive demand during a spatial task.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (6), 2881 (2000).</li><li>McNay, E. 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=Hippocampal+memory+processes+are+modulated+by+insulin+and+high-fat-induced+insulin+resistance.">Hippocampal memory processes are modulated by insulin and high-fat-induced insulin resistance.</a> Neurobiol. Learn Mem. 93 (4), 546 (2010).</li><li>Lonnroth, P., Jansson, P. -. A., Smith, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microdialysis+method+allowing+characterization+of+intercellular+water+space+in+humans.">A microdialysis method allowing characterization of intercellular water space in humans.</a> American Journal of Physiology. 1987, E228 (1987).</li><li>McNay, E. C., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+glucose+concentrations+in+the+rat+hippocampus+measured+by+zero-net-flux:+effects+of+microdialysis+flow+rate.">Extracellular glucose concentrations in the rat hippocampus measured by zero-net-flux: effects of microdialysis flow rate.</a> 72 (2), 785 (1999).</li><li>Lalonde, R. o. b. e. 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=The+neurobiological+basis+of+spontaneous+alternation.">The neurobiological basis of spontaneous alternation.</a> Neuroscience &amp; Biobehavioral Reviews. 26 (1), 91 (2002).</li><li>Richman, C., Dember, W., Kim, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+alternation+behavior+in+animals:+A+review.">Spontaneous alternation behavior in animals: A review.</a> Current Psychology. 5 (4), 358 (1986).</li><li>McNay, E. C., Canal, C. E., Sherwin, R. S., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+memory+with+septal+injections+of+morphine+and+glucose:+effects+on+extracellular+glucose+levels+in+the+hippocampus.">Modulation of memory with septal injections of morphine and glucose: effects on extracellular glucose levels in the hippocampus.</a> Physiol. Behav. 87 (2), 298 (2006).</li><li>McNay, E. C., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+modulation+across+neural+systems:+intra-amygdala+glucose+reverses+deficits+caused+by+intraseptal+morphine+on+a+spatial+task+but+not+on+an+aversive+task.">Memory modulation across neural systems: intra-amygdala glucose reverses deficits caused by intraseptal morphine on a spatial task but not on an aversive task.</a> Journal of Neuroscience. 18 (10), 3853 (1998).</li><li>Rex, A., Bert, B., Fink, H., Voigt, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulus-dependent+changes+of+extracellular+glucose+in+the+rat+hippocampus+determined+by+in+vivo+microdialysis.">Stimulus-dependent changes of extracellular glucose in the rat hippocampus determined by in vivo microdialysis.</a> Physiol. Behav. 98 (4), 467 (2009).</li></ol>

All solutions used in microdialysis should be filtered immediately prior to use, using a .2 &mu;m filter. After insertion of the probe into the guide cannula, observe to confirm that flow is unimpeded and sample collection is occurring. If flow stops after insertion, the most likely cause is damage to the probe membrane caused by insufficient care on insertion, and a fresh probe must be substituted.
As noted above, a key advantage to these methods is the lack of confound, permitting both beha...

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 <td></td>
 <td></td>
 <td></td>
 <td>The majority of reagents are standard laboratory grade and can be obtained from a supplier of choice. Similarly, equipment such as syringe pumps and tubing can be used from any of several manufacturers. Specific items used here for which details are important include:</td>
 </tr>
 <tr>
 <td>CMA 12 microdialysis probes</td>
 <td>CMA/ Microdialysis</td>
 <td>CMA-12-XXX</td>
 <td>These are available in various membrane lengths and cutoffs, indicated by specific codes in the 'XXX.'</td>
 </tr>
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 <td>Human insulin (Humulin)</td>
 <td>Eli Lilly</td>
 <td>N/a</td>
 <td></td>
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 <td>Liquid swivel</td>
 <td>Instech</td>
 <td>375/D/22QM</td>
 <td>This specific swivel has very low torque and internal volume, as well as a nonreactive quartz lining.</td>
 </tr>
 </tbody>
 </table>

hippocampal insulin microinjection and in vivo microdialysis during spatial memory testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Rats should recover rapidly from surgery and be alert, motile and active within 30 min following cessation of anesthesia. The impact of the cannula cap should be minimal if the surgery is performed cleanly and minimal dental cement is used. If any sign of infection is noticed during the post-operative monitoring, or the rat is in any way showing signs of distress or discomfort, immediately terminate the experiment; this should be extremely rare. During the handling prior to testing, animals should be alert, freely motile...

Watch this Scientific Journal Video about Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing at JoVE.com

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

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