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.

1. Animal Preparation
<ol>
<li>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 sod...

<ol>
	<li>Cuellar, C. A., Tapia, J. A., Juarez, V., Quevedo, J., Linares, P., Marinez, L., Manjarrez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation+of+sinusoidal+electrical+waves+along+the+spinal+cord+during+a+fictive+motor+task.">Propagation of sinusoidal electrical waves along the spinal cord during a fictive motor task.</a> J. Neurosci. 29, 798-810 (2010).</li><li>Frigon, A., Gossard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+specialized+rhythm-generating+mechanisms+in+the+adult+mammalian+spinal+cord.">Evidence for specialized rhythm-generating mechanisms in the adult mammalian spinal cord.</a> J. Neurosci. 30, 7061-7071 (2010).</li><li>Wang, W., Chan, S. S., Heldman, D. A., Moran, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+cortical+representation+of+hand+translation+and+rotation+during+reaching.">Motor cortical representation of hand translation and rotation during reaching.</a> J. Neurosci. 30, 958-962 (2010).</li><li>Ma, C., He, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+investigating+cortical+control+of+stand+and+squat+in+conscious+behavioral+monkeys.">A method for investigating cortical control of stand and squat in conscious behavioral monkeys.</a> J. Neurosci. Meth. 192, 1-6 (2010).</li><li>Dessem, D., Donga, R., Luo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary-+and+secondary-like+jaw-muscle+spindle+afferents+have+characteristic+topographic+distributions.">Primary- and secondary-like jaw-muscle spindle afferents have characteristic topographic distributions.</a> J. Neurophysiol. 77, 2925-2944 (1997).</li><li>Dessem, D., Moritaini, A., Ambalavanar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nociceptive+craniofacial+muscle+primary+afferent+neurons+synapse+in+both+the+rostral+and+caudal+brain+stem.">Nociceptive craniofacial muscle primary afferent neurons synapse in both the rostral and caudal brain stem.</a> J. Neurophysiol. 98, 214-223 (2007).</li><li>Luo, P., Dessem, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inputs+from+identified+jaw-muscle+spindle+afferents+to+trigeminothalamic+neurons+in+the+rat:+a+double-labeling+study+using+retrograde+HRP+and+intracellular.">Inputs from identified jaw-muscle spindle afferents to trigeminothalamic neurons in the rat: a double-labeling study using retrograde HRP and intracellular.</a> J. Comp. Neurol. 353, 50-66 (1995).</li><li>Dessem, D., Luo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jaw-muscle+spindle+afferent+feedback+to+the+cervical+spinal+cord+in+the+rat.">Jaw-muscle spindle afferent feedback to the cervical spinal cord in the rat.</a> J. Comp. Neurol. 128, 451-459 (1999).</li><li>Luo, P., Wong, R., Dessem, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projection+of+jaw-muscle+spindle+afferents+to+the+caudal+brainstem+in+rats+demonstrated+using+intracellular+biotinamide.">Projection of jaw-muscle spindle afferents to the caudal brainstem in rats demonstrated using intracellular biotinamide.</a> J. Comp. Neurol. 358, 63-78 (1995).</li><li>Luo, P., Dessem, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+anatomy+of+physiologically+identified+jaw-muscle+spindle+afferent+terminations+onto+retrogradely+labeled+jaw-elevator+motoneurons+in+the+rat.">Ultrastructural anatomy of physiologically identified jaw-muscle spindle afferent terminations onto retrogradely labeled jaw-elevator motoneurons in the rat.</a> J. Comp. Neurol. 406, 384-401 (1999).</li><li>Hassani, O. K., Henny, P., Lee, M. G., Jones, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+neurons+intermingled+with+orexin+and+MCH+neurons+in+the+lateral+hypothalamus+discharge+maximally+during+sleep.">GABAergic neurons intermingled with orexin and MCH neurons in the lateral hypothalamus discharge maximally during sleep.</a> Eur. J. Neurosci. 32, 448-457 (2010).</li><li>Inokawa, H., Yamada, H., Matsumoto, N., Muranishi, M., Kimura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Juxtacellular+labeling+of+tonically+active+neurons+and+phasically+active+neurons+in+the+rat+striatum.">Juxtacellular labeling of tonically active neurons and phasically active neurons in the rat striatum.</a> Neuroscience. 168, 395-404 (2010).</li></ol>

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...

<table border="1">
	<tbody>
 	<tr>
 	<th>Name of reagent or equipment</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments</th>
 </tr>
 <tr>
 	<td>electromagnetic vibrator</td>
 <td>Ling Dynamic Systems</td>
 <td>V101</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>signal generator</td>
 <td>Feedback Systems</td>
 <td>PFG605</td>
 <td>capable of producing trapezoidal output signal</td>
 </tr>

 <tr>
 	<td>electrode glass</td>
 <td>Sutter Instruments</td>
 <td>AF100-68-10</td>
 <td>with filament</td>
 </tr>

 <tr>
 	<td>electrode puller</td>
 <td>Sutter Instruments</td>
 <td>Model P-2000 or P-80</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 	<td>biotinamide</td>
 <td>Vector Laboratories</td>
 <td>SP-1120</td>
 <td>stored at 4&deg;C</td>
 </tr>

 <tr>
 	<td>Texas Red avidin DCS</td>
 <td>Vector Laboratories</td>
 <td>A-2016</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 	<td>tetramethlyrhodamine</td>
 <td>Molecular Probes</td>
 <td>D-3308</td>
 <td>3000 molecular weight, lysine fixable</td>
 </tr>

 <tr>
 	<td>mouse anti-synaptophysin antibody</td>
 <td>Chemicon</td>
 <td>MAB5258</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 	<td>fluorescent Nissl stain</td>
 <td>Neurotrace, Molecular Probes</td>
 <td>N-21480</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 	<td>electrode tester</td>
 <td>Winston Electronics</td>
 <td>BL-1000-B</td>
 <td>to measure electrode impedance</td>
 </tr>

 <tr>
 	<td>electrometer</td>
 <td>Axon Instruments</td>
 <td>Axoprobe 1A, Axoclamp 2B</td>
 <td>&nbsp;</td>
 </tr> 
 </tbody>
	
</table>

physiological, morphological and neurochemical characterization of neurons modulated by movement

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.

Watch this Scientific Journal Video about Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement at JoVE.com

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

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.

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

physiological-morphological-neurochemical-characterization-neurons

Research

JoVE Journal

Neuroscience

13.7K visualizações.  University of Maryland. A técnica é descrita para quantificar a resposta fisiológica em vivo dos neurônios de mamíferos durante o movimento e correlacionar a fisiologia do neurônio com a morfologia neuronal, fenótipo neuroquímico e microcircuitos sináptica.