Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

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 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.
3. 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°C). Be careful to avoid the large venous sinus located directly under the junction between the parietal and interparietal bones.
4. 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.
5. Place a siliver-silver chloride grounding electrode under the skin adjacent to the craniotomy.

2. Electrode preparation

1. Fabricate microelectrodes from quartz or aluminosilicate glass using a horizontal microelectrode puller.
2. 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 Ω, for small axons and interneurons make electrodes with impedance of 80-150MΩ.
3. 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.

3. Electrophysiological recording and intracellular staining

1. 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.
2. Produce repeated mandibular displacement and advance the electrode into the brain with a stepping motor.
3. 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.
4. 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.
5. 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.

4. Tissue processing

1. Euthanize the animal by overdose of pentobarbital (140mg/kg IV) and perfuse with a vascular rinse (0.9% NaCl 38°C containing 500 units of heparin and 1ml 2% xylocaine followed by 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4).
2. Remove the brain and section it at 50-100μm using a vibratome in either the frontal, sagittal or horizontal plane. Secions are collected free floating in 25°C PBS.
3. 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°C and then incubate in 4% Texas Red avidin DCS in PBST at 4°C overnight. Counterstain sections with a fluorescent Nissl stain (NeuroTrace) 20min at 23°C.
4. If neuron was injected with rhodamine, visualize the injected neuron directly with a fluorescent microscope (Ex 545nm).

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.

1. 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, 15μl 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°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°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.
2. 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°C and then incubate in anti-mouse FITC (1:400) for 1h at 23°C.
3. 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).
4. 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/°idl/colocalization.htm). Localize synaptophysin within single axon terminals by combining intracellular labeling with synaptophysin immunocytochemistry.⁶

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