Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Summary

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

Abstract

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 glycolysis^1-3,4-5, 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.

Protocol

1. Surgery Preparation

1. 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 confound^4-5,7,8. 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 b....

Results

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.......

Discussion

All solutions used in microdialysis should be filtered immediately prior to use, using a .2 μm 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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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:
CMA 12 microdialysis probes	CMA/ Microdialysis	CMA-12-XXX	These are available in various membrane lengths and cutoffs, indicated by specific codes in the 'XXX.'
Human insulin (Humulin)	Eli Lilly	N/a
Liquid swivel	Instech	375/D/22QM	This specific swivel has very low torque and internal volume, as well as a nonreactive quartz lining.