Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

Summary

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

Abstract

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static^1,2 or record extracellular neuronal activity during a movement.^3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

Protocol

1. Animal Preparation

1. Anesthetize rat with sodium pentobarbital (50mg/kg IP) and place on a heating pad. Shave the skin overlying the posterior skull with animal clippers. Check the animal to assure that a surgical level of level of anesthesia has been obtained by testing for the absence of a withdrawal reflex and vocalization when the toes are pinched as well as the absence of a palpebral reflex. Check the level of anesthesia every 15 minutes and maintain a surgical level of anesthetisa by injections of sodium pentobarbital 15mg/kg every 45 minutes.
2. Use aseptic technique and make an incision in the inguinal region just distal to the crease forme....

Discussion

The method illustrated here is a powerful technique which provides important insight into the function of single neurons and how the response of individual neurons contributes to neuronal circuits.⁹ This knowledge is fundamental to understanding sensorimotor function. The greatest strength of this technique is that is allows determination of a large number of parameters about a neuron including physiology, morphology and synaptic morphology and distribution. When combined with other techniques such as retrogra.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
electromagnetic vibrator	Ling Dynamic Systems	V101
signal generator	Feedback Systems	PFG605	capable of producing trapezoidal output signal
electrode glass	Sutter Instrument Co.	AF100-68-10	with filament
electrode puller	Sutter Instrument Co.	Model P-2000 or P-80
biotinamide	Vector Laboratories	SP-1120	stored at 4°C
Texas Red avidin DCS	Vector Laboratories	A-2016
tetramethlyrhodamine	Molecular Probes, Life Technologies	D-3308	3000 molecular weight, lysine fixable
mouse anti-synaptophysin antibody	Chemicon International	MAB5258
fluorescent Nissl stain	Neurotrace, Life Technologies	N-21480
electrode tester	Winston Electronics	BL-1000-B	to measure electrode impedance
electrometer	Axon Instruments	Axoprobe 1A, Axoclamp 2B