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 sodium pentobarbital 15mg/kg every 45 minutes.</li>
<li>Use aseptic technique and make an incision in the inguinal region just distal to the crease formed by the abdomen and inner thigh and insert a cannula (1mm diameter, Clay Adams) into the femoral vein, and femoral artery. Make a midline incision in the submandibular region, reflect the infrahyoid muscles. Make a small incision in the trachea and insert a cannula (2mm diameter) to allow ventilation. Tye a suture around the trachael cannual to secure it in place.Monitor systemic diastolic and systolic blood pressure via the arterial cannula. In addition to monitoring the withdrawal and palpebral reflex now monitor blood pressure to assess the level of anesthesia. Administer additional anesthesia when needed via the venous cannula.</li> 
<li>Place the rat into a stereotaxic frame and perform a craniotomy to expose the cerebellum. Attach the tracheal cannula to a rodent ventilator. Ventilate the animal with a volume of 2 cm3 at a rate of 100/min with humidified air. A positive end expiratory pressure of 1 cm H2O should be maintained to prevent lung collapse. Every 15 minutes hyperinflate the lungs to prevent atelectasis. Then cover the surface of the brain with warmed mineral oil (30&deg;C). Be careful to avoid the large venous sinus located directly under the junction between the parietal and interparietal bones.</li>
<li>Apply cyanoacrylate glue to a rod coupled to an electromagnetic vibrator and attach the rod in the diastema of the jaw. Move the mandible using command signals to the vibrator from either a A/D computer output or a signal generator.</li>
<li>Place a siliver-silver chloride grounding electrode under the skin adjacent to the craniotomy.</li>
</ol>
2. Electrode preparation 
<ol>
<li>Fabricate microelectrodes from quartz or aluminosilicate glass using a horizontal microelectrode puller.</li>
<li>Fill the microelectrode with 5-10% biotinamide dissolved in 0.25M KCl and 0.5M Tris HCl buffer (pH 7.6) or 2% tetramethlyrhodamine in saline (pH 6). Check electrode impedance with an electrode tester. To record from large diameter axons make electrodes with an impedance of 60-80M &Omega;, for small axons and interneurons make electrodes with impedance of 80-150M&Omega;.</li>
<li>Place the microelectrode into the head stage of the electrometer. Visualize the electrode through a small telescope (20X with an enclosed reticle) which is fixed in position behind the animal's head. Using the telescope is important because it allows electrodes to be stereotaxically positioned with great precision.</li>
</ol>
3. Electrophysiological recording and intracellular staining 
<ol>
<li>Make a small opening in the pia mater and overcompensate the capacitance feedback so that when the electrode touches the brain a feedback signal is produced. This allows accurate location of the surface of the brain.</li>
<li>Produce repeated mandibular displacement and advance the electrode into the brain with a stepping motor.</li>
<li>Recognize neuronal impalements by sudden drops in DC potential and identify intrasomatic neuronal impalements by ongoing synaptic activity. Buzzing the electrode with overcompensation of capacitance or tapping rarely produce a successful cell penetration. Because of the high impedance of the electrodes, neuronal responses are rarely observed prior to penetration.</li>
<li>After you impale a neuron and the penetration is deemed stable, characterize the neuronal response using ramp and hold and sinusoidal jaw movement.5 Map the receptive field of the neuron by probing the skin around the head and intra oral cavity with a non-conductive probe such as a wooden stick. If relevant to the study, examine the response of the neuron to other functionally relevant stimuli such as muscle contraction and noxious stimuli.6 Primary afferent nociceptive neuronal receptors respond to nociceptive stimuli. General anesthesetics such as sodium pentobarbital do not block axonal conduction in primary afferent neurons but rather depress synaptic transmission. Thus axonal conduction in the primary afferent neuronal axon is preserved.</li>
<li>Inject current (DC, 1-4nA) for a total injection time of 15-70nA minutes. Monitor electrode penetration during current injection and discontinue if membrane potential becomes more positive than -30mV.</li>
</ol>
4. Tissue processing 
<ol>
<li>Euthanize the animal by overdose of pentobarbital (140mg/kg IV) and perfuse with a vascular rinse (0.9% NaCl 38&deg;C containing 500 units of heparin and 1ml 2% xylocaine followed by 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4).</li>
<li>Remove the brain and section it at 50-100&mu;m using a vibratome in either the frontal, sagittal or horizontal plane. Secions are collected free floating in 25&deg;C PBS.</li>
<li>Process the brain for DAB by incubating in 1-2% normal goat serum and 1% Triton X-100 in 0.01M PBS followed by incubation in avidin biotin complex (1:50 Elite Vectastain). React sections using nickel-DAB with H2O2. To process for Texas Red, incubate sections in avidin-biotin complex (1:50) in PBS overnight at 4&deg;C and then incubate in 4% Texas Red avidin DCS in PBST at 4&deg;C overnight. Counterstain sections with a fluorescent Nissl stain (NeuroTrace) 20min at 23&deg;C.</li>
<li>If neuron was injected with rhodamine, visualize the injected neuron directly with a fluorescent microscope (Ex 545nm).</li>
</ol>
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. 
<ol>
<li>The method visualized here can easily be combined with retrograde neuronal labeling.7-10 To do this, anesthetize the animal and using aseptic technique, inject a neuronal tracer such as horseradish peroxidase (20% horseradish peroxidase. Sigma VI, Sigma) and 1% wheat-germ aggultinated horseradish peroxidase (Sigma) into peripheral target regions such as the masseter muscle. This neuronal tracer will be taken up into peripheral axons and transported via axonal transport to the motoneuron somata. For the masseter muscle, 15&mu;l of tracer is injected into the muscle using a sterile 10 microliter microsyringe. Animals are then placed on a heating pad and monitored until recovered from anesthesia and then placed in their cages for recovery. After 24h, anesthetize animals and physiologically characterize neurons and intracellularly stain them. Then perfuse the animal as described above and process tissue sections for the presence of HRP using tetramethylbenzidine (TMB) as the chromagen and sodium tungstate as the stabilizer and intensified with cobalt. Place sections in 1% sodium tungstate dissolved in 0.1M PBS (pH 6.5) and 0.0007% TMB dissolved in absolute ethanol and acetone at 15&deg;C for 20 min. Then react tissue sections by adding 1.0 ml of 0.3% H2O2 per 100ml of incubation solution for 60 min. Rinse tissue in 0.1M PBS (pH 6.5) and place in 0.05% diaminobenzidine (pH 7.4), 0.02% cobalt, and 0.01% H2O2 in 0.1M PBS (pH 7.4) for 10 min. at 37&deg;C. Process the tissue for visualization of the neuron injected with biotinamide as described and visualize the relationships between the labeled sensory neuron and a variety of motoneurons.</li>
<li>The technique shown here can also readily be combined with immnocytochemistry.6 As an example, immunocytochemically process the brain for synaptophysin to accurately locate synapses within labeled neurons (Fig. 3). To do this, incubate brain sections in mouse anti-synaptophysin antibody (1:10,000) for 2 days at 4&deg;C and then incubate in anti-mouse FITC (1:400) for 1h at 23&deg;C.</li> 
<li>Neurons labeled with fluorescent markers or processed for fluorescent imaging are ideal for confocal imaging. Figure 3 is an example of an optical section obtained with confocal microscopy through a axon bouton. (Fig. 3). Generate animations of labeled neurons by acquiring multiple optical sections through the labeled neuron (Fig. 4).</li>
<li>Neurons labeled with this method can be used for quantitative colocalization analysis. To do this, use a publicly available software macro in conjunction with NIH Image (found at http://phy.ucsf.edu/&deg;idl/colocalization.htm). Localize synaptophysin within single axon terminals by combining intracellular labeling with synaptophysin immunocytochemistry.6</li>
</ol>
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.
<img src="/files/ftp_upload/2650/2650fig1.jpg" alt="Figure 1"/> 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.
<img src="/files/ftp_upload/2650/2650fig2.jpg" alt="Figure 2"/> 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.
<img src="/files/ftp_upload/2650/2650fig3.jpg" alt="Figure 3"/> 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. <a href="https://www.jove.com/files/ftp_upload/2650/collateral4.avi">Download a high resolution video of Figure 4 here</a> <a href="https://www.jove.com/files/ftp_upload/2650/medres.mov">Download a medium resolution video of Figure 4 here</a>

<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>

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.

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

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JoVE Journal

Neuroscience

13.7K 조회수.  University of Maryland. 기술은 운동하는 동안 포유류의 뉴런의 생체내 생리적 반응을 수량과의 연결 형태, neurochemical 표현형 및 시냅스 microcircuitry와 신경 세포의 생리 신원을 확인하도록 설명되어 있습니다.

비디오: Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

Eye Movement Monitoring of Memory

Explicit (often verbal) reports are typically used to investigate memory (e.g. "Tell me what you remember about the person you saw at the bank yesterday."), however such reports can often be unreliable or sensitive to response bias 1, and may be unobtainable in some participant populations. Furthermore, explicit reports only reveal when information has reached consciousness and cannot comment on when memories were accessed during processing, regardless of whether the information is subsequently accessed in a conscious manner. Eye movement monitoring (eye tracking) provides a tool by which memory can be probed without asking participants to comment on the contents of their memories, and access of such memories can be revealed on-line 2,3. Video-based eye trackers (either head-mounted or remote) use a system of cameras and infrared markers to examine the pupil and corneal reflection in each eye as the participant views a display monitor. For head-mounted eye trackers, infrared markers are also used to determine head position to allow for head movement and more precise localization of eye position. Here, we demonstrate the use of a head-mounted eye tracking system to investigate memory performance in neurologically-intact and neurologically-impaired adults. Eye movement monitoring procedures begin with the placement of the eye tracker on the participant, and setup of the head and eye cameras. Calibration and validation procedures are conducted to ensure accuracy of eye position recording. Real-time recordings of X,Y-coordinate positions on the display monitor are then converted and used to describe periods of time in which the eye is static (i.e. fixations) versus in motion (i.e., saccades). Fixations and saccades are time-locked with respect to the onset/offset of a visual display or another external event (e.g. button press). Experimental manipulations are constructed to examine how and when patterns of fixations and saccades are altered through different types of prior experience. The influence of memory is revealed in the extent to which scanning patterns to new images differ from scanning patterns to images that have been previously studied 2, 4-5. Memory can also be interrogated for its specificity; for instance, eye movement patterns that differ between an identical and an altered version of a previously studied image reveal the storage of the altered detail in memory 2-3, 6-8. These indices of memory can be compared across participant populations, thereby providing a powerful tool by which to examine the organization of memory in healthy individuals, and the specific changes that occur to memory with neurological insult or decline 2-3, 8-10.

Eye movement monitoring (or eye tracking) reveals where in space the eyes linger, when and for how long. Here, we demonstrate how eye tracking can be used to investigate the integrity of memory in multiple participant populations, without requiring verbal, or otherwise explicit, reports.

Explicit (often verbal) reports are typically used to investigate memory (e.g. "Tell me what you remember about the person you saw at the bank ...

Coherence between Brain Cortical Function and Neurocognitive Performance during Changed Gravity Conditions

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and focused on psychological aspects. Until now, objective observation of neurophysiological parameters has not been carried out - undoubtedly because the technical and methodological means have not been available -, investigations into the neurophysiological effects of weightlessness are in their infancy (Schneider et al. 2008).
While imaging techniques such as positron emission tomography (PET) and magnetic resonance imaging (MRI) would be hardly applicable in space, the non-invasive near-infrared spectroscopy (NIRS) technique represents a method of mapping hemodynamic processes in the brain in real time that is both relatively inexpensive and that can be employed even under extreme conditions. The combination with electroencephalography (EEG) opens up the possibility of following the electrocortical processes under changing gravity conditions with a finer temporal resolution as well as with deeper localization, for instance with electrotomography (LORETA).
Previous studies showed an increase of beta frequency activity under normal gravity conditions and a decrease under weightlessness conditions during a parabolic flight (Schneider et al. 2008a+b). Tilt studies revealed different changes in brain function, which let suggest, that changes in parabolic flight might reflect emotional processes rather than hemodynamic changes. However, it is still unclear whether these are effects of changed gravity or hemodynamic changes within the brain. Combining EEG/LORETA and NIRS should for the first time make it possible to map the effect of weightlessness and reduced gravity on both hemodynamic and electrophysiological processes in the brain. Initially, this is to be done as part of a feasibility study during a parabolic flight. Afterwards, it is also planned to use both techniques during medium- and long-term space flight.
It can be assumed that the long-term redistribution of the blood volume and the associated increase in the supply of oxygen to the brain will lead to changes in the central nervous system that are also responsible for anaemic processes, and which can in turn reduce performance (De Santo et al. 2005), which means that they could be crucial for the success and safety of a mission (Genik et al. 2005, Ellis 2000).
Depending on these results, it will be necessary to develop and employ extensive countermeasures. Initial results for the MARS500 study suggest that, in addition to their significance in the context of the cardiovascular and locomotor systems, sport and physical activity can play a part in improving neurocognitive parameters. Before this can be fully established, however, it seems necessary to learn more about the influence of changing gravity conditions on neurophysiological processes and associated neurocognitive impairment.

The effect of weightlessness and hypergravity on both hemodynamic and electrophysiological processes in the brain is going to be followed during parabolic flight by EEG and NIRS techniques. 
A feasibility study of a more complex experiment, which is planned to carry out during medium- and long-term space flight.

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and ...

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique known as electron probe microanalysis (EPMA) are described. Intramitochondrial calcium is a particular focus because of the critical role that mitochondrial calcium overload plays in neurodegenerative diseases. The method is based on the analysis of X-rays generated in an electron microscope (EM) by interaction of an electron beam with the specimen. In order to maintain the native distribution of diffusible elements in electron microscopy specimens, EPMA requires &#34;cryofixation&#34; of tissue followed by the preparation of ultrathin cryosections. Rapid freezing of cultured cells or organotypic slice cultures is carried out by plunge freezing in liquid ethane or by slam freezing against a cold metal block, respectively. Cryosections nominally 80 nm thick are cut dry with a diamond knife at ca. -160 &#176;C, mounted on carbon/pioloform-coated copper grids, and cryotransferred into a cryo-EM using a specialized cryospecimen holder. After visual survey and location mapping at &#8804;-160 &#176;C and low electron dose, frozen-hydrated cryosections are freeze-dried at -100 &#176;C for ~30 min. Organelle-level images of dried cryosections are recorded, also at low dose, by means of a slow-scan CCD camera and subcellular regions of interest selected for analysis. X-rays emitted from ROIs by a stationary, focused, high-intensity electron probe are collected by an energy-dispersive X-ray (EDX) spectrometer, processed by associated electronics, and presented as an X-ray spectrum, that is, a plot of X-ray intensity vs. energy. Additional software facilitates: 1) identification of elemental components by their &#34;characteristic&#34; peak energies and fingerprint; and 2) quantitative analysis by extraction of peak areas/background. This paper concludes with two examples that illustrate typical EPMA applications, one in which mitochondrial calcium analysis provided critical insight into mechanisms of excitotoxic injury and another that revealed the basis of ischemia resistance.

This paper describes the application of cryoanalytical electron microscopy to the quantitative measurement of total calcium content and distribution at subcellular resolution in physiologically defined biological specimens.

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing patterns. The inclusion of biocytin/neurobiotin in the recording electrode permits post-hoc recovery of morphological details, which are necessary to determine the dendritic arborization and the regions targeted by the axons of the recorded neurons. However, given the presence of morphologically similar neurons with distinct neurochemical identities and functions, immunohistochemical staining for cell-type-specific proteins is essential to definitively identify neurons. To maintain network connectivity, brain sections for physiological recordings are prepared at a thickness of 300 &#181;m or greater. However, this thickness often hinders immunohistological postprocessing due to issues with antibody penetration, necessitating the resectioning of the tissue. Resectioning of slices is a challenging art, often resulting in the loss of tissue and morphology of the cells from which electrophysiological data was obtained, rendering the data unusable. Since recovery of morphology would limit data loss and guide in the selection of neuronal markers, we have adopted a strategy of recovering cell morphology first, followed by secondary immunostaining. We introduce a practical approach to biocytin filling during physiological recordings and subsequent serial immunostaining for the recovery of morphology, followed by the restaining of sections to determine the neurochemical identity. We report that sections that were filled with biocytin, fixed with paraformaldehyde (PFA), stained, and coverslipped can be removed and restained with a second primary antibody days later. This restaining involves the removal of the coverslip, the washing of sections in a buffer solution, and the incubation of primary and secondary antibodies to reveal the neurochemical identity. The method is advantageous for eliminating data loss due to an inability to recover morphology and for narrowing down the neurochemical markers to be tested based on morphology.

This protocol presents a method for the morphological recovery of neurons patched during electrophysiological recordings using biocytin filling and subsequent immunohistochemical postprocessing. We show that thick biocytin-filled sections that were stained and coverslipped can be restained with a second primary antibody days&#160;or months later.

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing ...