Name: Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing (Video) | JoVE Journal
Uploaded: 2013-01-11T17:00:00.000+00:00
Duration: 10 min 32 s
Description: Learn hippocampal insulin microinjection and in vivo microdialysis for measuring brain metabolism during spatial memory testing in behaving animals. This method enables real-time neurochemical analysis.

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5 minutes

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 this protocol as an example species. Handling must be done without stress caused by pulling on fur from e.g. latex or nitrile gloves: this can be best achieved using bare hands, in consultation with the facility veterinarian and employee health officials. If this is not possible, soft cotton gloves should be worn over barrier gloves to avoid pulling on the rats' fur.</li>
 <li>Sterile Field Preparation. Prior to beginning surgery, the surgical area is prepared, a sterile field prepared around the stereotaxic apparatus and a sterile drape placed across the apparatus, covering a heating pad that is used to maintain rat body temperature. A circulating warm water pad with accurate thermostat is used to prevent overheating of the animal.</li>
 <li>Anesthesia induction. The isoflurane vaporizer is checked to contain optimal levels of isoflurane and connected to the induction chamber. Connections are checked to confirm a closed loop. The air-handling vacuum system is checked to be operational. Isoflurane vaporization is switched on and the animal placed into the induction chamber using a 5% isoflurane in oxygen mix: that is, 5% isoflurane added into a stream of 100% oxygen and delivered to the induction chamber.</li>
 <li>Placement into apparatus. Animals are secured in the stereotaxic apparatus using earbars and a tooth bar. Correct earbar placement results in an immobile head and ears that lie flat along the earbars. Animals are rapidly secured and the nose inserted into a purpose-designed anesthetic nosecone; delivery of vaporized isoflurane is switched from the induction chamber to the nosecone and the anesthetic mix adjusted to deliver 2-3% isoflurane into the oxygen stream going to the nosecone.</li>
 <li>Confirmation of anesthesia. A surgical anesthetic plane is confirmed by delivering a hard pinch to the foot and a puff of air to the eye; neither should cause any response. These tests are repeated at roughly 15 min intervals throughout surgery to confirm maintenance of a surgical anesthetic plane. Hair is removed from the incision site prior to entering the sterile field.</li>
</ol>
2. Surgery
<ol>
 <li>Animal initial treatment. Ophthalmic ointment is applied to each eyeball to prevent drying. 1 ml sterile saline is given s.c. to prevent any dehydration during surgery, and the body is covered with a sterile drape. Betadine is applied to the scalp, and swabbed from the center out with a cotton swab; 70% ethanol is swabbed similarly, and the two swabbing steps are repeated twice more to ensure an appropriate incision site. Skin contact time for betadine and alcohol should be at least 3 min prior to incision. Anesthesia is maintained and checked regularly throughout surgery; a single s.c. injection of carprofen 5 g/kg) is given to initiate analgesia, and aseptic technique is used. Analgesia is given following induction of anesthesia to minimize any stress from injection.</li>
 <li>Incision and skull preparation. A 3-4 cm cut is made sagitally in the center of the skull. A 1:1 mix of bupivicaine:epinephrine is applied topically to provide further analgesia and minimize bleeding. The scalp is held away from the incision using surgical clips and sterile swabs are used to remove overlying membranes from the skull. Coordinates for drilling are measured using bregma as a reference point, marked using a sterile drill bit (or, alternatively, a hand-held cautery device), and re-confirmed for accuracy before drilling starts. Coordinates for specific brain regions of interest (e.g. here the hippocampus) are determined using a brain atlas. For hippocampal microdialysis, we use a drill site at 5.6 mm posterior to bregma, +5.0 lateral, and 3.0 ventral from dura.</li>
 <li>Drilling. Three holes are drilled through the skull, with care being taken to apply only minimal force such that trauma to the dura and underlying membranes is minimal or absent. One hole is at the measured site of cannula insertion; the other two are positioned as convenient for insertion of skull screws. Purpose-sized screws (e.g. 1.17 mm self-tapping screws; Fine Science Tools) are inserted into these holes without impacting the brain beneath, and are used as anchor points for subsequent dental cement application.</li>
 <li>Cannula placement and closure. The cannula is positioned at insertion coordinates, which are re-confirmed to be the site of the hole drilled, and is then slowly lowered to the target depth. Once correctly positioned, the cannula is secured in place with dental cement. If needed, a single sterile surgical suture is used to close the wound. A stylet is inserted into the cannula to maintain patency. In this protocol we use a CMA12 probe and guide cannula (CMA/Microdialysis).</li>
 <li>Acute post-surgical care. 3 ml sterile saline is given s.c. to continue hydration; a single s.c. injection of carprofen 5 g/kg) is also given to initiate analgesia. Animals are removed from the apparatus and placed in a warmed recovery room, in a clean cage, and monitored until they are fully recovered from anesthesia. Full recovery is assessed by restoration of righting reflex and normal locomotion. Animals are then returned to their home cage and regular holding room.</li>
 <li>Short-term follow-up care. Cages of animals that have undergone surgery are marked with the date of surgery. Animals are monitored at least once each day for at least three days following surgery, and given one carprofen chewable tablet (2 mg) on the day after surgery and each of the two following days. If animals do not consume the carprofen, injectable carprofen may be given to ensure adequate analgesia. Monitor both the overall health of the animal and the state of the wound site (for infection, redness, etc.) following institutional animal care guidelines and seeking assistance or advice from animal care staff or the veterinarian if needed. Importantly, note that appropriate handling and acclimation to the experimenter is essential: animals should be handled extensively, including manipulation of the cannula, until no sign of nervousness or stress remains when handled by the experimenter.</li>
 <li>Subsequent animal care and treatment. Animals will follow appropriate testing and euthanasia procedures according to the approved protocol and their specific experimental group.</li>
</ol>
3. Microdialysis (mD)
<ol>
 <li>Perfusate preparation. Artificial extracellular fluid (aECF) is made with 153.5 mM Na, 4.3 mM K, 0.41 mM Mg, 0.71 mM Ca, 139.4 mM Cl, 1.25 mM glucose, buffered at pH 7.49. NOTE that accurate fluid composition is essential: inaccuracies or use of other, less physiological fluids such as Ringer's or PBS for microdialysis will result in markedly erroneous results9. Specifically, note that the ionic composition of the extracellular fluid (ECF) is NOT the same as that of the CSF, as we showed in a 2004 detailed study of the hippocampal ECF9. On the day of testing, bovine serum albumin (BSA) should be added at 2% weight/weight and fully dissolved; this reduces loss of peptides such as insulin from adherence to the tubing, and also reduces the risk of fluid loss (ultrafiltration) at the probe membrane. After preparation, the perfusate should be filtered through a .2 &mu;m filter.</li>
 <li>If a treatment such as insulin is to be delivered to the brain region of interest (here the hippocampus), prepare this treatment using an aliquot of the prepared aECF with the appropriate drug concentration. For insulin, a concentration of 400 nM (66.7 &mu;U/&mu;l) for delivery via reverse microdialysis has been shown to affect hippocampal metabolic and cognitive function10. Note that the resulting tissue concentration of insulin is not measured here and remains unknown.</li>
 <li>Setting up mD probe and lines. Prepare a fresh microdialysis probe and line the day before testing. Create two separate lines for "inflow" and "outflow". Use PE50 tubing to connect two 1 meter long pieces of FEP tubing and ensure that there is minimal dead space between the lines. Connect the inflow tubing to a 1 ml Hamilton syringe filled with sterile-filtered, deionized H20 (dH20), and then attach to the probe.</li>
 <li>Microdialysis swivel. To allow for free animal movement while taking measurements, connect a liquid swivel to the inflow and outflow lines, close to the pump, using additional FEP tubing. Remember to take the internal volume of this swivel and tubing into account in considering timing of sample collection (below).</li>
 <li>Setting up mD pump. Turn on the mD pump and run at 1.5 &mu;l/min until you see dH20 exiting the outflow tube on the probe. Then, while the pump is turned off, connect the other line between the outflow tube and a sample collection tube. Run 5 ml through the tube and place the probe in a vial containing sterile dH20 overnight; ensure that the probe tip always remains wet.</li>
 <li>Pre-probing test subject. 24 hr before testing, remove the dummy stylet from the rat's head and insert an mD probe (used only for this purpose, not for sampling) for 10 min. Then replace the dummy stylet and put the rat back into its cage. This procedure is designed to minimize any effect of reactive gliosis on the day of testing11-12 and in our hands, this gives good results that match data from other techniques and appear to reflect hippocampal activation4-5,8,10,13; others have used a similar approach that leaves the probe in place for 24 hr prior to measurement, which is also a good approach if damage to the probe overnight can be avoided.</li>
 <li>Probe equilibration. On the day of microdialysis, fill the Hamilton syringe and scintillation vial with filtered aECF and pump through to equilibrate for 1 hr. If needed, fill a second Hamilton syringe with prepared treatment (for example, insulin-aECF) and place into the syringe pump.</li>
 <li>Probe insertion. When equilibration is complete, remove the dummy stylet and gently insert the equilibrated mD probe into the rat's brain via the cannula. Place the rat into a clear plastic box that contains some of the rat's home bedding. Make sure to counterbalance the tubing: attach a 1.6 ml microcentrifuge tube filled with water to the tubing outside the cage in such a way that gravity keeps the microdialysis lines unkinked but not taut. Let probe equilibrate in rat for 2 hr. At the start of this period, confirm that flow of perfusate is unblocked: this is most easily done by collecting perfusate outflow over a defined period and weighing the sample to confirm that the expected volume is exiting the system. Any probe that yields &lt;90% of expected volume, and does not self-correct after removal and re-insertion, should be replaced (otherwise edema at the probe tip will result). If flow is persistently low or absent, check all connections for leakage; failing that, disconnect tubing in stages to see whether the blockage can be isolated. If the problem is not identified, replace the probe; if the problem does not resolve, it may be necessary to start afresh with new probe and lines from Section 3.3. The two-hour equilibration period permits the blood-brain barrier to reseal around the probe and avoid any acute effect of probe insertion.</li>
 <li>Collecting samples. Ensure that the correlation between sample dialysis (from brain) and collection (into tube) is accurately calculated. For instance, using two meters of FEP tubing between swivel and probe, there is a total volume between probe and collection of 30 &mu;l and hence it takes 20 min at 1.5 &mu;l/min flow rate for sample to pass through the probe, tubing, connectors and swivel to reach the end of tubing for collection. When collecting samples ensure that you collect enough volume to have concentration necessary for your assays. Here, we will use 5 min sample bins, and thus collect 7.5 &mu;l of dialysate in each collection tube.</li>
 <li>Baseline samples. Once equilibration is complete, begin sample collection. Collect at least three samples while the rat is at rest in the home chamber to establish a stable baseline of measurements.</li>
 <li>Treatment timing. Take care to calculate required timing for initiation of treatment prior to the experiment: remember that just as there is a time lag between dialysis and sample collection, there is a lag (usually identical) between perfusate leaving the syringe and arriving at the animal's hippocampus. Hence, in our setup with a 30 &mu;l volume between syringe and probe, change the syringe to that containing treatment-perfusate 20 min before you wish the treatment to begin arriving at the hippocampus.</li>
 <li>Changing syringes. A liquid switch can be used if desired but is not necessary: at the appropriate time, simply disconnect the inflow line from the control-perfusate syringe and rapidly connect to the treatment-perfusate syringe. This should take no more than 5 sec to avoid significant interruption to the perfusate flow. NOTE that this process should be reversed after the desired dosage has been delivered, if a time-limited delivery of treatment is desired. Alternatively, treatment may continue for the duration of sampling (see Figure 2). Air bubbles should not be introduced into the line during switching of syringes, as they may accumulate at the dialysis membrane and reduce probe efficiency.</li>
 <li>Behavioral testing. If performing a behavioral task, follow that procedure (see section 4 below). NOTE that coordination with delivery of treatment to the hippocampus is important. For instance, delivery of insulin should be timed to occur 10 min prior to beginning testing 14. Hence, the switch to an insulin-containing perfusate should occur 30 min prior to behavioral testing (20 min for perfusate to pass through the lines plus 10 min desired delivery time ahead of testing).</li>
 <li>Completion of sample collection. After collecting the desired samples, gently remove the probe from the animal's head, again remembering to take into account time lag between dialysis and sample collection. Return the animal to its home cage and observe closely for any post-experimental change in health or behavior until the animal is killed and the brain removed to confirm correct probe placement. If sacrifice will not occur immediately, return the dummy stylet to the cannula to avoid any introduction of foreign material.</li>
 <li>Analysis. The analytical methodology will vary depending on the analyte(s) of interest. Because dialysis sampling does not allow for complete analyte equilibration at the probe membrane, sample concentrations should be corrected to give ECF concentrations using the zero-net-flux method15,16.</li>
</ol>
4. Behavioral Testing
<ol>
 <li>Placement on maze. After baseline samples (i.e., after at least three samples have completed dialysis, but may still be in the outflow tubing being collected), gently move the rat into the behavioral testing apparatus. Any appropriate task may be used; the data in Figure 1 were collected using a four-arm plus-shaped maze and measuring spontaneous alternation, which is a measure of spatial working memory: the rat is initially placed in the center of the maze and allowed to explore freely13,17-18. Because any behavioral test may be used, the focus of this protocol is not on the specific task used as an example here (which is detailed elsewhere13,19,20) but in brief, animals are allowed to explore the maze (the period of the grey box in Figure 1) and use hippocampally-dependent processes to retain memory of which arms have recently been visited.</li>
 <li>Dialysis tubing movement during testing. Hold the tubing such that it moves freely, neither hindering the rat's movement nor being permitted to move in front of the animal and distract it. Stay in place and minimize your own movement so that you do not affect the rat's behavior.</li>
 <li>Continued sample collection. As during baseline, move the outflow tubing to a new collection tube every 5 min.</li>
 <li>Alternation testing. Allow the animal to freely explore the maze for 20 min. Record the sequence and timing of arm entries either using a video recording or by hand for later task performance analysis 13,20.</li>
</ol>
5. Post-testing
<ol>
 <li>Final sample collection. Following testing, remove rat gently from maze and return to control chamber. Continue microdialysis sample collection for at least four samples to cover the period of recovery from task performance.</li>
 <li>Probe removal. After all samples have been collected, gently remove the probe from the animal's head and place into a storage vial; return the animal to its home cage.</li>
 <li>Probe storage. After returning animal to the home cage and completing collection of any remaining dialysate, rinse the probe thoroughly with dH20 and store in a scintillation vial filled with dH20 and covered with parafilm. Flush the tubing by switching to a syringe containing a solution of 1:10,000 Kathon in dH20 to prevent microbe growth. Probes can be re-used as long as they maintain good flow and have no damage to the membrane; with care this should routinely be in excess of ten uses.</li>
 <li>Histology. Kill the animal and remove the brain. Slice on a cryostat and use standard histological techniques (e.g. cresyl violet staining) to confirm correct probe placement.</li>
</ol>

<ol><li>McNay, E. C., Fries, T. M., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decreases+in+rat+extracellular+hippocampal+glucose+concentration+associated+with+cognitive+demand+during+a+spatial+task%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">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/pubmed?term=((Age-related+differences+in+hippocampal+extracellular+fluid+glucose+concentration+during+behavioral+testing+and+following+systemic+glucose+administration%5BTitle%5D)+AND+%22J.+Gerontol.+A.+Biol.+Sci.+Med.+Sci%22%5BJournal%5D)">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/pubmed?term=((Fluctuations+in+brain+glucose+concentration+during+behavioral+testing%3A+dissociations+between+brain+areas+and+between+brain+and+blood%5BTitle%5D)+AND+%22Neurobiol.+Learn.+Mem%22%5BJournal%5D)">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/pubmed?term=((Extracellular+glucose+concentrations+in+the+rat+hippocampus+measured+by+zero-net-flux%3A+effects+of+microdialysis+flow+rate%5BTitle%5D)+AND+%22%22%5BJournal%5D)">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/pubmed?term=((Effect+of+recurrent+hypoglycemia+on+spatial+cognition+and+cognitive+metabolism+in+normal+and+diabetic+rats%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">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/pubmed?term=((Hippocampal+memory+processes+are+modulated+by+insulin+and+high-fat-induced+insulin+resistance%5BTitle%5D)+AND+%22Neurobiology+of+Learning+and+Memory%22%5BJournal%5D)">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/pubmed?term=((Fluctuations+in+brain+glucose+concentration+during+behavioral+testing%3A+dissociations+between+brain+areas+and+between+brain+and+blood%5BTitle%5D)+AND+%22Neurobiology+of+Learning+%26+Memory%22%5BJournal%5D)">Fluctuations in brain glucose concentration during behavioral testing: dissociations between brain areas and between brain and blood.</a> Neurobiology of Learning & 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/pubmed?term=((Cognitive+and+neural+hippocampal+effects+of+long-term+moderate+recurrent+hypoglycemia%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">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/pubmed?term=((From+artificial+cerebro-spinal+fluid+%28aCSF%29+to+artificial+extracellular+fluid+%28aECF%29%3A+microdialysis+perfusate+composition+effects+on+in+vivo+brain+ECF+glucose+measurements%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">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/pubmed?term=((Hippocampal+memory+processes+are+modulated+by+insulin+and+high-fat-induced+insulin+resistance%5BTitle%5D)+AND+%22Neurobiology+of+Learning+and+Memory%22%5BJournal%5D)">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/pubmed?term=((Regional+cerebral+glucose+phosphorylation+and+blood+flow+after+insertion+of+a+microdialysis+fiber+through+the+dorsal+hippocampus+in+the+rat%5BTitle%5D)+AND+%22Journal+of+Neurochemistry%22%5BJournal%5D)">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/pubmed?term=((Cellular+reactions+to+implantation+of+a+microdialysis+tube+in+the+rat+hippocampus%5BTitle%5D)+AND+%22Acta+Neuropathologica%22%5BJournal%5D)">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/pubmed?term=((Decreases+in+rat+extracellular+hippocampal+glucose+concentration+associated+with+cognitive+demand+during+a+spatial+task%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((Hippocampal+memory+processes+are+modulated+by+insulin+and+high-fat-induced+insulin+resistance%5BTitle%5D)+AND+%22Neurobiol.+Learn+Mem%22%5BJournal%5D)">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/pubmed?term=((A+microdialysis+method+allowing+characterization+of+intercellular+water+space+in+humans%5BTitle%5D)+AND+%22American+Journal+of+Physiology%22%5BJournal%5D)">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/pubmed?term=((Extracellular+glucose+concentrations+in+the+rat+hippocampus+measured+by+zero-net-flux%3A+effects+of+microdialysis+flow+rate%5BTitle%5D)+AND+%22%22%5BJournal%5D)">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. obert <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+neurobiological+basis+of+spontaneous+alternation%5BTitle%5D)+AND+%22Neuroscience+%26+Biobehavioral+Reviews%22%5BJournal%5D)">The neurobiological basis of spontaneous alternation.</a> Neuroscience & Biobehavioral Reviews. 26 (1), 91(2002).</li>
<li>Richman, C., Dember, W., Kim, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spontaneous+alternation+behavior+in+animals%3A+A+review%5BTitle%5D)+AND+%22Current+Psychology%22%5BJournal%5D)">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/pubmed?term=((Modulation+of+memory+with+septal+injections+of+morphine+and+glucose%3A+effects+on+extracellular+glucose+levels+in+the+hippocampus%5BTitle%5D)+AND+%22Physiol.+Behav%22%5BJournal%5D)">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/pubmed?term=((Memory+modulation+across+neural+systems%3A+intra-amygdala+glucose+reverses+deficits+caused+by+intraseptal+morphine+on+a+spatial+task+but+not+on+an+aversive+task%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">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/pubmed?term=((Stimulus-dependent+changes+of+extracellular+glucose+in+the+rat+hippocampus+determined+by+in+vivo+microdialysis%5BTitle%5D)+AND+%22Physiol.+Behav%22%5BJournal%5D)">Stimulus-dependent changes of extracellular glucose in the rat hippocampus determined by in vivo microdialysis.</a> Physiol. Behav. 98 (4), 467(2009).</li>
</ol>

No conflicts of interest declared.

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...

<table><tbody><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;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:&lt;/p&gt;</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 &#039;XXX.&#039;</td></tr><tr><td>Human insulin (Humulin)</td><td>Eli Lilly</td><td>N/a</td><td></td></tr><tr><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

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...

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

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

The overall aim of this procedure is to measure hippocampal metabolism during behavioral testing. This is accomplished by surgically preparing a well-handled animal with a microdialysis guide cannula. On the day of testing, a microdialysis probe is inserted into the cannula and the PERFUSE eight flow is confirmed.Next, the probe is allowed to equilibrate while the animal is sitting in a control chamber. Ultimately, appropriate analytical methods, for instance, a glucose oxidase, NADP, N-A-D-P-H reaction and flu metric analysis. If measuring glucose is used to show the level of metabolism and or neurochemical release throughout the testing period, One of the most difficult aspects of this technique is the care needed in handling the microdialysis tubing and also often chew it or tangle the lines if care isn't taken.So the first time I used hippocampal microdialysis during behavioral testing was doing my graduate work, and we wanted to check whether the existing models bring glucose supply. Were in fact compatible with the known psychological data regarding enhancement of memory by glucose. This methodology allowed us to show that in fact, the existing models of glucose applied to the brain were completely wrong.Following standard surgical procedures, prepare the animal, expose the skull and drill holes for cannula placement. Insert a cannula at predetermined coordinates and then slowly lower it to the target depth. Once correctly positioned, secure the cannula in place with dental cement.Use a sterile surgical suture to close the wound if needed. Lastly, a style is used to maintain cannular patency. Next, prepare the PERFUSE eight to be used for microdialysis.It should be noted that accurate fluid composition is essential for optimal results. On the day of testing bovine serum albumin BSA should be added and fully dissolved. After preparation, the PERFUSE eight should be filtered through a 0.2 micron filter.Next, prepare the treatment that will be administered to the brain, if any, using an aliquot of the prepared A ECF. Here insulin is prepared for delivery into the hippocampus. A fresh microdialysis probe and line should be prepared the day before testing.Create two separate lines for inflow and outflow. Use PE 50 tubing to connect two one meter long pieces of FEP tubing and ensure that there is minimal dead space between the lines. Tubing should be trimmed as shown to create slanted ends for ease of insertion into the PE 50 connectors.Connect the inflow tubing to a one milliliter Hamilton syringe filled with sterile filtered deionized water and attach the probe to the other end to allow the animal to move freely while taking measurements. Connect the inflow and outflow lines to a two channel swivel. Next, connect the outflow tubing to the pump and run it at 1.5 microliters per minute until you see deionized water exiting the outflow tube on the probe.Turn the pump off and connect the other line between the outflow tube and a sample collection tube. Run five milliliters through the tube and place the probe in a vial containing sterile deionized water overnight, ensuring that the probe tip always remains wet. 24 hours before testing, pre-Roe the test subject by removing the dummy stylet and inserting a microdialysis probe that is used only for this purpose.For 10 minutes afterwards, replace the dummy style it and put the rat back into its cage. Alternatively, use a fresh probe in this stage and leave in place without removal until taking measurements. The following day.On the day of Microdialysis, fill the Hamilton syringe and scintillation vial with filtered A ECF and allow the pump to equilibrate for one hour. If needed, fill a second Hamilton syringe with prepared treatment and place into the syringe pump. The key to success in this procedure is to remain calm and to handle the animal really extensively.So remain calm, move slowly and precisely and above all practice. When the equilibration is complete, remove the dummy stylet and gently insert the equilibrated microdialysis probe into the rat's brain via the cannula. Place the rat into a clear plastic box that contains some of the rat's home bedding.Make sure to counterbalance the tubing. Attach a 1.6 milliliter micro centrifuge tube filled with water to the tubing outside the cage in such a way that gravity keeps the microdialysis lines un kinked but not taught. Allow the probe and rat to equilibrate for two hours.At the start of this period, confirm the flow of Perfuse eight is unblocked. To do this, collect PERFUSE eight outflow over a defined period and weigh the sample to confirm that the expected volume is exiting the system before collecting samples. Ensure that the correlation between sample dialysis and collection is accurately calculated.Here we have a 30 microliter volume between the probe and collection point at a rate of 1.5 microliters per minute. There is a 20 minute lag from when the sample exits the brain until it is collected. After the equilibration period, collect at least three samples while the rat is at rest in the home chamber.To establish a stable baseline of measurements Before behavioral testing, carefully plan out the timing of delivery of your treatment to the brain. So in this setup where we have a volume of 30 microliters between the syringe tip and the probe going into the animal's brain, we change the treatment syringe to the profuse eight treatment solution 20 minutes prior to. We want that treatment to arrive at the hippocampus To change syringes.Simply disconnect the inflow line from the control perfuse eight syringe and rapidly connect to the treatment perfuse eight syringe. This should take no more than five seconds to avoid interruption to the PERFUSE eight flow. Take care not to introduce air bubbles that may reduce proficiency at the appropriate time period.After treatment delivery, gently move the rat into the behavioral testing apparatus of choice. Here, spontaneous alternation is measured on a forearm plus shaped maze. Allow the animal to freely explore the maze for 20 minutes.Record the sequence and timing of arm Aries either using a video recording or by hand to collect samples. During this period, move the outflow tubing to a new collection tube. Every five minutes hold the tubing such that it moves freely.It should not hinder the rat's movement or move in front of the animal and distracted remain still so that your movements do not impact the rat's behavior. Following testing, remove the rat gently from the maze and return it to the control chamber. Continue microdialysis sample collection for at least four samples to cover the period of recovery from task performance.After collecting the desired samples, gently remove the probe from the animal's head and place into a storage vial. Return the animal to its home cage and observe closely for any post experimental change in health or behavior. Finally, flush the tubing by switching to a syringe containing a solution of catho to prevent microbe growth.This example shows a task associated dip in hippocampal ECF glucose. The gray box represents the time the animal spent in the maze. The purple line shows hippocampal glucose in animals with no maze testing, while the red line represents animals performing the forearm spontaneous alternation task.The orange line shows measurements in animals performing the easier three arm version of this task. The changes in hippocampal, glucose and lactate after administration of insulin are seen here. Note that these changes are seen without any behavioral testing, and hence reflect the impact of insulin alone under baseline conditions.When performing this procedure, it's really important to remain calm. Rats can pick up on the experiment of stress and they'll become agitated, and that can both make it much more difficult and probably confound your results. This combination of techniques has allowed for accurate neurochemical investigation of behavioral processes.Similar techniques are now also used in human patients. For example, patients with epilepsy that have electrodes implanted to localize their seizure focus may also have microdialysis cannula implanted to permit metabolic measurements.

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Research

JoVE Journal

Neuroscience

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Learn hippocampal insulin microinjection and in vivo microdialysis for measuring brain metabolism during spatial memory testing in behaving animals. This method enables real-time neurochemical analysis.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

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