Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

Summary

This paper details the use of fixed-potential amperometric recordings using carbon fiber electrodes and enzymatic biosensor technology to measure the release of dopamine and glutamate with high temporal resolution during natural rewarding behavior in the female hamster.

Abstract

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or manipulations; a powerful tool in elucidating underlying mechanisms and circuitry. While the technique of microdialysis has been used for decades to measure nearly any analyte of interest in the brain, this technique is limited in temporal resolution. Alternatively, fast scan cyclic voltammetry is both temporally precise and extremely sensitive; however, because this technically difficult method relies on the electroactivity of the analyte of interest, the possibility to detect nonelectroactive substances (e.g., the neurotransmitter glutamate) is eliminated. This paper details the use of a turn-key system that combines fixed-potential amperometry and enzymatic biosensing to measure both electroactive and nonelectroactive neurotransmitters with temporal precision. The pairing of these two powerful techniques allows for the measurement of both tonic and phasic neurotransmission with relative ease, and permits recording of multiple neurotransmitters simultaneously. The aim of this manuscript is to demonstrate the process of measuring dopamine and glutamate neurotransmission in vivo using a naturally rewarding behavior (i.e., sexual behavior) in female hamsters, with the ultimate goal of displaying the technical feasibility of this assay for examining other behaviors and experimental paradigms.

Introduction

The ability to measure neurotransmitter release in awake behaving animals allows researchers to link specific behaviors with spatial and temporal patterns of neurotransmission-a powerful tool to investigate mechanisms and circuitry underlying both natural and operant behaviors in real-time. Historically, microdialysis has been employed to measure both electrically reactive and nonreactive substances in the extracellular milieu of the brain¹. This technique uses a continuous flow of an aqueous solution of similar ionic composition to the extracellular fluid, through a microdialysis probe composed of a small shaft with a tip made of a semipermeab....

Protocol

All procedures described here were approved by the Institutional Animal Care and Use Committee (IACUC) of The University of Minnesota, and are in accordance with The Guide for the Care and Use of Laboratory Animals¹³.

1. Animals and Cannulation Surgery

1. Obtain Syrian hamsters from a common animal supplier at approximately 55 days of age.
   NOTE: Although the age of animals will vary due to constraints of various experimental paradigms, for sexual behavior it is important to obtain sexually mature animals that are all approximately the same age.
2. House animals in a controlled temperature (22 &....

Results

Using the electrochemical and behavioral coding methodology described above, this lab has begun to characterize both tonic and phasic fluctuations in both dopamine and glutamate during in vivo recordings of sexual behavior. Due to the temporally-precise manner of this methodology, we can more accurately characterize neurotransmission during sexual behavior; as well as ascribe specific changes in release patterns to corresponding tonic changes during mating bouts, and correspondin.......

Discussion

Although relatively straightforward, some issues can arise when employing this technique. First, the stereotaxic placement of the probes must be precise: unlike microdialysis that samples a wider radius of the extracellular milieu surrounding the probe, this technique only allows the measurement of a neurotransmitter that comes into direct contact with the probe. Second, in the case of the carbon fiber recording, due to the small width of fiber, breakage can occur, and the probe must be inserted with deliberate care. In .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Nembutal	Oak Pharmaceuticals Inc.	76478-501-50	Pentobarbital sodium injection, USP. This lab uses 8.5 mg/100 g body weight, injected intraperotineally.
Loxicom analgesic	Norbrook Laboratories	6451603670	NSAID antinflammatory and analgesic used for post-operative pain control. Generic: meloxicam.
Enroflox antibiotic	Norbrook Laboratories	5552915411	Fluoroquinolone antibiotic for post-operative infection prevention. Generic: Enrofloxacin.
Beuthanasia-D	Merck Animal Health	00061047305	Pentobarbital Sodium, Phenytoin Sodium euthanasia agent.
Bone screws	Pinnacle Technologies, Inc.	8111-16	1/8" bone screw (Pkg. of 16) used to affix skull cap to skull.
Dental acrylic (Bosworth Duz-All)	Bosworth	166261C	Self curing dental acrylic is used in construction of a skull cap to affix cannula and head mount to skull.
Hardware biosensor setup	Pinnacle Technologies, Inc.	8400-K2	Pinnacle offers complete hardware kits for new users of our tethered biosensor system for rats. Kits include a commutator, preamplifier, and data conditioning and acquisition system
Base video computer package	Pinnacle Technologies, Inc.	9000-K1	The base computer package includes a preconfigured computer with ample hard disk storage, a high-definition monitor, a keyboard and mouse, an uninterruptible power supply, and all necessary cables.
Video EQ700 EverFocus camera  package	Pinnacle Technologies, Inc.	9000-K10	EQ700 night vision capable box camera with independent IR source was obtained as part of Pinnacle video computer package. Dome camera (9000-K9) and HD camera (9000-K11) options are also available.
Sirenia Acquisition software	Pinnacle Technologies, Inc.	Free--available to download from pinnaclet.com	Sirenia Acquisition provides a single platform for recording data from any Pinnacle hardware system. The software features synchronization of all data streams, user-configurable settings, data consolidation, and multiple export options. In addition, the software includes basic review and analysis modules for biosensor recordings. Sirenia delivers free ll-in-one software that is ideal for data acquisition and review.
Tethered rat in vitro calibration kit	Pinnacle Technologies, Inc.	7000-K2-T-BAS	In order to relate the current changes measured by a biosensor to actual changes in analyte concentration, it is necessary to calibrate the biosensor prior to implantation into the animal. The process also confirms the integrity and selectivity of the sensors. Calibration kit includes 20 mL jacketed beaker (#7058), 1/2" by 1/8" magnetic stir bar (#7059), right angle clamp (#7056), 2 prong single-adjustment clamp (#7055), 4-channel calibration preamplifer (#7053), and calibration holder (#7051).
Stir plate	Corning	6795-410D	Corning digital Stirrer, 5" x 7", 120 VAC used to spin magnetic stirrer in jacketed beaker during in vitro calibration of glutamate biosensors.
Water bath capable of closed loop circulation	PolyScience	8006A11B	PolyScience 8006A11B 6L Standard Digital Heated Circulating Bath, 120VAC water bath was used with plastic tubing to heat jacketed beaker to physiological temperature.
Carbon fiber sensor with BASi rat cannulae	Pinnacle Technology, Inc.	7002-CFS	Carbon fiber electrode used for recording dopamine neurotransmission.
Ag/AgCl reference electrode	Pinnacle Technology, Inc.	7065	Necessary for carbon fiber recordings.
Glutamate biosensors	Pinnacle Technology, Inc.	7001	Enzymatic biosensor probe used for recording glutamatergic neurotransmission.
BASi guide cannulae	Pinnacle Technologies, Inc.	7030	Guide cannulae implanted into brain region of interest to guide probe.
BASi cannula plastic headpiece for rats	Pinnacle Technologies, Inc.	7011	Headmount stabilizes probe and attaches to potentiostat.