Department of Neural and Pain Sciences, University of Maryland
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Dessem, D. Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement. J. Vis. Exp. (50), e2650, doi:10.3791/2650 (2011).
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 static1,2 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.
1. Animal Preparation
2. Electrode preparation
3. Electrophysiological recording and intracellular staining
4. Tissue processing
5. Combining method with retrograde labeling, immunocytochemistry, confocal imaging, quantitative colocalization analysis
The success of combining this technique with other methods is largely dependent upon good intracellular labeling.
6. Representative Results:
An overview of the representative results that can be obtained using this method are illustrated in Figure 1. This single brainstem neuron was electrophysiologically recorded during movement of the mandible and, as can clearly be seen, the response of this neuron (Figure 1 lower left, light blue) was modulated during movement. This neuron was injected with biotinamide after electrophysiological characterization and subsequently processed for visualization. The reconstructed neuron (Figure 1 middle, green) can be related to an anatomical landmark, in this case the trigeminal motor nucleus designated (red outline). Based upon the neuronal response during movement and reconstruction this neuron can be identified as a secondary muscle spindle afferent neuron. Figure 2 illustrates a representative example of the physiological response of a neuron during jaw displacement. The response of the neuron is represented as instantaneous firing frequency. Note that the neuronal response closely mimics mandibular displacement indicating that this particular neuron provides sensory feedback related to mandibular position. Figure 3 is a high magnification image of an intracellularly stained axon combined with staining for synaptophysin and a Nissl stain. Note the colocalization of synaptophysin (yellow) within the axon bouton. Figure 4 is an animation of a single, physiologically characterized and intracellularly labeled neuron.
Figure 1. Overview of method. Upper left: mandibular displacement. Middle Intracellular recording (green) from single neuron (yellow). The morphology of this neuron was reconstructed after intracellular recording and injection. Red outline indicates location of the trigeminal motor nucleus. Lower left: physiological response of this neuron during jaw movement.
Figure 2. Representative physiological response of a single muscle sensory neuron recorded in vivo during movement of the mandible. Note the similarity of the neuronal response with jaw displacement.
Figure 3. Terminal axonal arborization with synaptic boutons (red swellings) of an intracellularly-stained sensory neuron which responded during muscle probing. Subsequent immunocytochemical processing for synaptophysin shows localization of synaptophysin within the axonal bouton (yellow). Green is a fluorescent Nissl stain.
Figure 4. Animation of muscle spindle primary afferent neuron axon whose physiological response was recorded in vivo during mandibular movement, The axon was then intracellularly stained and processed for visualization.
Download a high resolution video of Figure 4 here
Download a medium resolution video of Figure 4 here
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 retrograde neuronal labeling additional information such as neuronal circuitry can be characterized.7,8 Another advantage of this method is that it can be learned in steps. For instance, intracellular recording can be conducted initially, followed by intracellular staining with immunocytochemistry or retrograde neuronal labeling added after mastery of the initial method. Perhaps the greatest limitation of the technique is that only a small number of neurons can be labeled in any one experiment. Typically two to three neurons of a particular physiological type are initially labeled. Once the potential relationship between physiology and morphology is formulated, additional experiments are used to carefully check this relationship in experiments in which only a single neuron is labeled.
The most crucial step for the success of this method is maintaining electrophysiological intracellular recording stability. Recording stability will vary greatly depending upon the location within the brain of the neuron under study but a number of manipulations can be used to increase recording stability. A pneumothorax can be performed and the animal artificially ventilated to reduce respiratory pulsations. Stability can be increased further by applying a positive end expiratory pressure of about 1 cm H2O. When the region of interest within the brain is reached, stability can be enhanced by hyperventilating the animal by decreasing respiratory volume and increasing respiratory rate. Some electrophysiological studies have applied warm agar over the brain and cannulated the bladder; these procedures have not been effective in increasing neuronal recording stability in the brainstem. It is important to point out that injection times do not need to be long. Good results can be obtained with injection times of about 5 minutes. Due to the small tip size of the microelectrodes, breakage of the electrode within the brain typically does not produce a large release of tracer. Therefore the electrode can be replaced and neurons successfully recorded and stained within several hundred microns of the location of electrode breakage. If the electrode is clogged or the recording solution does not fill the tip of the electrode adequately, noise will be greatly increased and the electrode should be replaced. Testing electrode impedance prior to insertion into the brain greatly reduces unproductive electrode tracts and saves time. If you are attempting to record from a small region of the brain stereotaxic positioning is paramount. I use a telescope attached to the recording table to maintain a fixed stereotaxic zero. The telescope is very useful because the electrode can be placed into the electrode holder attached to the recording table and then viewed under magnification. This allows very accurate placement of the microelectrode and zeroing of replacement electrodes.
A number of recent studies have used juxtacellular neuronal labeling.11,12 With this method an electrode is placed in proximity to a neuron based upon the characteristics of the neuronal recording and a neuronal tracer is ejected. An obvious potential problem with this method is spurious labeling since tracer can be incorporated into the dendrites and axons of other neurons in the vicinity of the electrode. In addition, input-output relationships of the neuron cannot be determined because extracellularly recorded action potentials can be generated not only by synaptic input to the neuron but by intrinsic properties of the neuron. With the method reported here, neurons are only labeled while the microelectrode is actually within the neuron and thus there is no ambiguity concerning the attribution of neuronal activity to the stained neuron. This is particularly important when labeling axons because movement of the microelectrode by a few microns results in spurious labeling. Additionally, subthreshold events including synaptic potentials can be recorded from the impaled neuron.
Future studies could combine this method with evoked movements. For instance cortical stimulation can evoke masticatory movements and the recording stability within the brainstem should allow intracellular recording and staining of neurons during these evoked movements. Since this technique can be done with minimal surgical intervention, it may also be possible to use this method to inject substances which alter gene expression in vivo.
Experiments on animals were performed in accordance with the guidlines and regulation set forth in the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23, revised 1985) and the University of Maryland Animal Care and Use Committee.
I thank Anthony Taylor for initial training in in vivo intracellular recording and A Brown and David Maxwell for help with the initial development of the intracellular staining technique. I thank M. Silver for help with the collocalization macro. Many scholars with whom I have collaborated provided insight into the development of this technique including R. Donga, M. Moritani, P. Luo, R. Ambalavanar. This technique was developed with considerable support from NIH grants DE10132, DE15386 and RR017971.
|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|