Name: in vitro wedge slice preparation for mimicking in vivo neuronal circuit connectivity
Uploaded: 2020-08-18T14:00:00.000+00:00
Duration: 10 min 31 s
Description: Watch this Scientific Journal Video about In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity at JoVE.com

This research was supported by the Intramural Research Program of the NIH, NIDCD, Z01 DC000091 (CJCW).

All experimental procedures were approved by the National Institute of Neurological Disorders and Stroke/National Institute on Deafness and Other Communication Disorders Animal Care and Use Committee.
1. Experimental preparations
NOTE: Details regarding slice preparation including slicing solution, slicing temperature, slice incubation temperature and apparatus (etc.) are specific for brainstem preparation performed in this experiment. Slice incubation details can be altered per laboratory experience.
<ol>
	<li>Prepare internal solutions for patch-clamping.
	<ol>
		<li>Prepare voltage clamp solution containing (in mM) 76 Cs-methanesulfonate, 56 CsCl, 1 MgCl2, 1 CaCl2, 10 HEPES, 10 EGTA, 0.3 Na-GTP, 2 Mg-ATP, 5 Na2-phosphocreatine, 5 QX-314, and 0.01 Alexa Fluor-488 hydrazide. Adjust the pH to 7.2 with CsOH.</li>
		<li>Prepare current clamp solution containing (in mM) 125 K-gluconate, 5 KCl, 1 MgCl2, 0.1 CaCl2, 10 HEPES, 1 EGTA, 0.3 Na-GTP, 2 Mg-ATP, 1 Na2-phosphocreatine, and 0.01 Alexa Fluor-488 hydrazide. Adjust the pH to 7.2 with KOH.</li>
	</ol>
	</li>
	<li>Prepare 100 mL of 4% agar by adding 4 g of agar to 100 mL hot (near boiling) water. Place on heated stir plate to maintain temperature and stir until completely dissolved. Pour into 100 mm plastic Petri dishes to approximately 1 cm depth and let cool. Refrigerate until needed.</li>
	<li>Prepare 1 L artificial cerebrospinal fluid (ACSF) containing in mM: 124 NaCl, 1.2 CaCl2, 1.3 MgSO4, 5 KCl, 26 NaHCO3, 1.25 KH2PO4, and 10 dextrose. Bubble with carbogen (5% CO2 / 95% O2) for at least 10 min, then adjust final pH to 7.4 with 1 M NaOH if needed. Maintain oxygenation and pH of solution by bubbling continuously with carbogen throughout experiment.</li>
	<li>Prepare 200 mL slicing solution by adding 1 mM kynurenic acid to ACSF. Sonicate solution in a sonicating water bath for 10 min until kynurenic acid is dissolved. Continuously bubble with carbogen and place on ice. 
	CAUTION: Use appropriate personal protective equipment when handling kynurenic acid.</li>
	<li>Mount an appropriate blade in the vibratome following the manufacturer&#8217;s instructions. Chill vibratome slicing chamber by surrounding it with ice.</li>
</ol>
2. Brain removal with intact auditory nerve root for stimulation
NOTE: Mice for these experiments were obtained by crossing ChAT-IRES-Cre transgenic mice on a C57BL/6J background with tdTomato reporter mice (Ai14). Mice used for histology and electrophysiology were post-hearing onset (P14-P23), which is around P12 in mice. Neurons expressing tdTomato in the ventral nucleus of the trapezoid body (VNTB) have been previously characterized as MOC neurons in this mouse line57.
<ol>
	<li>Euthanize (e.g., CO2 asphyxiation) and decapitate the animal using approved institutional procedures.</li>
	<li>Using a razor blade, cut the skin at the midline of the skull from the nose to the back of the neck. Peel back skin to expose the skull.</li>
	<li>Using small scissors, make an incision in the skull through the midline starting at the base (caudal end near spinal cord) of the skull and continuing towards the nose.</li>
	<li>At the lambda suture, make cuts in the skull from the midline, lateral toward the ear on both sides. Peel back the skull to expose the brain.</li>
	<li>Starting at the rostral end, gently lift the brain away from the skull with a small lab spatula or blunt forceps. Cut the optic nerve and continue to gently work the brain backwards, exposing the ventral surface.</li>
	<li>Cut the trigeminal nerves by pinching them with fine forceps near the ventral surface of the brainstem. 
	NOTE: Do this carefully as the vestibulocochlear nerve lies just below this and needs to be intact for eventual stimulation.</li>
	<li>Place the preparation in a glass Petri dish filled with cold slicing solution. Place the dish under a dissecting microscope. Gently bubble with carbogen.</li>
	<li>Trim the facial nerve close to the brainstem and expose the vestibulocochlear nerve.</li>
	<li>Using fine forceps, push the tips into the foramina where the vestibulocochlear nerve exits the skull as far as possible and pinch the nerve to sever it, leaving the nerve root attached to the brainstem. Repeat this on the other side.</li>
	<li>Once both nerve roots are free, remove the meninges and vasculature from the ventral surface of the brainstem near the trapezoid body.</li>
	<li>Free the brain completely from the skull by pinching the remaining cranial nerves and connective tissue taking care to preserve the remaining spinal cord if possible.</li>
</ol>
3. Block and mount brain on stage (magnetic disc)
<ol>
	<li>Prepare the surface of the brain to fix to the stage by blocking the brain at the level of the optic chiasm.
	<ol>
		<li>With the ventral surface up, stabilize the brain using a blunt tool to gently immobilize the spinal cord so that the brain does not tilt during the following step.</li>
		<li>At the level of the optic chiasm, use open forceps to create the plane for blocking the brain by inserting through the brain down to the bottom of the dish. Insert the forceps at an angle of approximately 20&#730; from vertical so that the tips exit the dorsal surface of the brain caudal to the optic chiasm.</li>
		<li>Cut along the forceps using the razor blade.</li>
	</ol>
	</li>
	<li>Glue the brain to the surface of the stage.
	<ol>
		<li>Prepare a small block (~1 cm3) of 4% agar for supporting the brain.</li>
		<li>Place a small drop of glue on the stage and spread it into a rectangle so both the brain and agar block can be glued down.</li>
		<li>Using forceps, carefully lift the brain and gently dab the excess liquid using the edge of a paper towel. Place the blocked surface onto the glue, ventral surface will be towards the blade during slicing.</li>
		<li>Push the agar block gently against the dorsal surface of the brain to support it during slicing and to ensure proper brain positioning (i.e., angle).</li>
	</ol>
	</li>
</ol>
4. Slice brain to create wedge slice
NOTE: Prepare a brain slice using vibratome that has the cochlear nerve root on the thick side and medial olivocochlear (MOC) neurons and the medial nucleus of the trapezoid body (MNTB) on the thin side.
<ol>
	<li>Place the magnetic disc with attached brain onto the stage base and place it in the slicing chamber with the ventral surface of the brain oriented towards the blade.</li>
	<li>Fill the chamber with ice cold slicing solution and bubble with carbogen.</li>
	<li>Lower the blade into the solution and cut slices caudal to the region of interest to make sure the slices are symmetrical. If the slices appear asymmetrical, tilt the stage slightly to obtain symmetry. 
	NOTE: Blade speeds between 0.05-0.10 mm/s were effective for cutting healthy slices and may vary depending on animal age and brain region.</li>
	<li>Once the slices are symmetrical, shift the stage ~15&#730; (corresponding to approximately 3 concentric rings on the stage base) to one side. 
	NOTE: Shift the stage away from the auditory nerve root that you want to preserve in the slice.</li>
	<li>Continue slicing carefully until the auditory nerve root is close to the surface on one side, and the facial nerve can be seen at the surface of the other side.</li>
	<li>Shift the stage back 15&#730; to the original position.</li>
	<li>Move the blade away from the tissue and spin the stage base 90&#176; so that the lateral edge of the thin side is facing the blade. Lower the blade several hundred microns and then slowly bring the blade close to the edge of the tissue. Repeat this until the blade touches the lateral edge. Lower the blade to the desired thickness of the thin edge of the slice, here an additional two hundred microns. 
	NOTE: The resulting slice is ideally ~300 mm thick at the level of the ventral nucleus of the trapezoid body (VNTB) on the side where patch clamping will take place.</li>
	<li>Move the blade back away from the tissue and spin the stage base back so that the ventral surface is facing it.</li>
	<li>Make the cut that designates the rostral surface of the wedge slice. Transfer the slice to a piece of interface paper (1 cm2) caudal surface down. Move the slice to the incubation chamber or other suitable incubation apparatus for recovery (30 min at 35 &#176;C). 
	NOTE: The facial nerve should be visible on both hemispheres of the slice on the rostral surface (see Figure 1B).</li>
</ol>
5. Electrophysiology set-up and recording
<ol>
	<li>Place the wedge slice in the recording chamber and secure slice with a harp or stabilizing system. Perfuse the tissue continuously at a rate of 7-10 mL/min with warm (35 &#176;C) ACSF bubbled with carbogen.</li>
	<li>Identify genetically labeled MOC neurons in the VNTB using epifluorescence with 561 nm emission filters for patch-clamp recordings. Flip slice if there are no potentially patchable cells.</li>
	<li>Using DIC optics, focus on the auditory nerve root on the thick side of the slice and use a micromanipulator to move the bipolar tungsten stimulating electrode down to the auditory nerve root and gently into the surface of the tissue. 
	NOTE: Suction electrodes have been used in auditory nerve stimulation experiments in other labs. Theta glass electrodes, or optical stimulation methods can be employed if applicable to other specific preparations.</li>
	<li>Move the field of view back to the VNTB to choose an MOC neuron to target for patch clamp electrophysiology.</li>
	<li>Fill a recording pipette with appropriate internal solution for the proposed experiment.</li>
	<li>Patch and record from the MOC neuron in the whole-cell configuration. Compensate membrane capacitance and series resistance if required.</li>
	<li>Adjust electrical stimulation amplitude of the auditory nerve root to obtain consistent postsynaptic events in the MOC neuron. 
	NOTE: It may be necessary to move the stimulation electrode.</li>
	<li>Run appropriate stimulation protocols to observe evoked synaptic currents in MOC (voltage clamp) or action potential patterns (current clamp). 
	NOTE: The wedge slice preparation can be used with any typical patch-clamp tools such as loose patch recordings, pharmacology, optogenetics, calcium imaging, neurotransmitter uncaging, etc.</li>
</ol>
6. Histological confirmation of brainstem nuclei
NOTE: This is done with cresyl violet staining, in fixed, re-sectioned wedge slice. This method allows for visualization of nuclei which are contained in the slice.
<ol>
	<li>After preparing a wedge slice, submerge slice in fixative (4% PFA in PBS) overnight. Rinse the slice 3x for 10 min in PBS (room temperature on a shaker), then place in 30% sucrose in PBS overnight at 4 &#176;C to cryoprotect.</li>
	<li>Re-section the slice on a freezing microtome (40-70 mm) and collect serial sections in a 24 well plate in PBS.</li>
	<li>Mount sections on gelatin coated slides and let dry completely. Place slides in slide carriage.</li>
	<li>Prepare cresyl violet solutions
	<ol>
		<li>Prepare 1% cresyl violet acetate by mixing 5 g cresyl violet acetate in 500 mL dH2O</li>
		<li>Prepare acetate buffer by first preparing 90 mL solution A (540 mL glacial acetic acid + 89.46 mL dH2O) and 10 mL solution B (136 mg sodium acetate in 10 mL dH2O). Combine solution A and solution B yielding the acetate buffer.</li>
		<li>Combine 1% cresyl violet acetate with the acetate buffer 1:1 for 0.5% cresyl violet in acetate buffer. Filter before use.</li>
		<li>Prepare 95% and 70% ethanol by diluting 100% ethanol with appropriate volumes of dH2O</li>
	</ol>
	</li>
	<li>Perform cresyl violet staining protocol. Move the slide carriage through solution trays, blotting excess solution on a paper towel between trays: xylene &#8211; 5 min; 95% ethanol &#8211; 3 min; 70% ethanol &#8211; 3 min; dH2O &#8211; 3 min; 0.5% cresyl violet solution &#8211; 8-14 min monitoring frequently until nuclear staining becomes dark purple; dH2O &#8211; 3 min; 70% ethanol &#8211; 3 min; 95% ethanol &#8211; 1-2 min; 100% ethanol &#8211; dip slides twice; xylene &#8211; 5 min; xylene: 25 min until mounting is performed. 
	CAUTION: Use xylenes only under a fume hood.</li>
	<li>Remove slides from xylene one at a time and immediately place cover slips on slides using mounting medium. Allow mounting medium to dry (overnight).</li>
	<li>Image sections.</li>
</ol>
7. Biocytin labeling for anterograde tracing of axons in live, unfixed tissue
<ol>
	<li>Prepare a wedge slice as above (Steps 2-4).</li>
	<li>Transfer the slice to interface paper (~1 cm2). Under a dissecting microscope, locate the CN on the thick side of the slice.</li>
	<li>Carefully remove excess ACSF from the area surrounding the slice by twisting up a corner of a tissue paper to draw the ACSF away from the tissue. This prevents the biocytin from spreading to surrounding areas of the slice which could lead to uptake into cells outside the CN.</li>
	<li>With fine forceps, select a small crystal of biocytin and place it on the surface of the CN. Gently press the crystal into the tissue to promote contact with neurons and subsequent uptake into somata. Repeat this step to cover the desired region of interest, in this case CN regions containing T-stellate and GBC neurons.</li>
	<li>Place the slice in an incubation chamber. Allow the slice to incubate for 2-4 h at 35 &#176;C to allow for the uptake and transport of the biocytin. After incubation, rinse the slice in ACSF to remove any biocytin particles.</li>
	<li>Place slice in fixative (4% PFA in PBS) overnight. Rinse 3x for 10 min in PBS.</li>
	<li>Cryoprotect slice in 30% sucrose in PBS overnight at 4 &#176;C or until the slice sinks.</li>
	<li>Resect the tissue to produce transverse sections on a freezing microtome at 70-100 mm.</li>
	<li>Process tissue using standard immunohistochemical methods with a fluorescently conjugated streptavidin. 
	NOTE: Additional immunohistochemistry can be performed on the sections if helpful for labeling presynaptic cell bodies, axons, receptors, or other synaptic molecules important for circuit visualization (i.e., primary antibody steps should not adversely affect biocytin secondary visualization).</li>
	<li>Image the tissue.</li>
</ol>

The authors have nothing to disclose.

The slicing procedure described here termed a wedge slice is powerful for maintaining intact presynaptic neuronal circuitry, but with the accessibility of brain slice experimentation for analysis of neuronal function. Great care must be taken in several initial steps in order to maximize utility of the preparation for circuit analysis. The dimensions of the wedge should be confirmed using histological examination, which is integral for confirmation that both presynaptic nuclei and their axonal projections are contained within the prepared wedge slice. Slice geometry may require modification if projections are severed or few axons reach the target nuclei. More generally, the finishing cut on the vibratome for the wedge slice is critically important. Optimal wedge slice preparation will require a combination of consistent use of vibratome configurations including use of concentric circle markings on the stage base, along with adjustment of settings based on known brain landmarks. After optimization of slice geometry, we recorded consistent PSCs in MOC neurons evoked by electrically stimulating the auditory nerve root in 8 of 18 wedge slices. In our previous work we were able to evoke inhibitory PSCs via direct stimulation of MNTB axons in approximately 60% of MOC neurons57, suggesting that our success rate here is only a modest reduction given the long range of the inputs and necessity for polysynaptic circuit activation. When preparing the slice, it is advisable to err on the thicker side as a decrease in visibility due to a thicker slice is favorable over an unusable section which is incomplete or lacks circuit connectivity. Any slice configuration or dimensions can be used, as long as the slice fits under the recording microscope objective, is accessible by patch and stimulating electrodes (or other probes or equipment), and is thin enough for optically-based patch-clamping at the postsynaptic cell of interest. Rapid, gentle dissection and proper incubation and recovery conditions are also important to maintain viability of the circuit for patch-clamp experiments. Specific to our auditory brainstem preparation, the brain must be removed very carefully from the skull in order to preserve intact and functional auditory nerve roots. Stretching or tearing the nerve will impact the ability to stimulate the fibers and elicit activity in auditory neurons. Due to the larger volume of tissue in the slice, modification of traditional slicing solutions, temperatures, incubation details, and perfusion systems may improve the health of the slice. Here we employ slight modifications to our normal slice preparation. These include shorter recovery incubation times (30 minutes vs. 60 minutes) and faster flow rates in the slice chamber perfusion system.
Once the slice dimensions and incubation details have been determined, the function and connectivity of different components of circuitry within the slice should be demonstrated. In our preparation, we ensure that both excitatory and inhibitory inputs, hypothesized to originate in the cochlear nucleus with T-stellate and GBC (via the MNTB), are present as expected. Alternative stimulation methods such as suction electrodes for the auditory nerve, or optical stimulation methods such as optogenetics or focal neurotransmitter uncaging may also increase circuit activation or allow for cell-type specific activation when paired with genetic targeting of cell subtypes.
While this slicing method will hopefully be useful for many systems and circuits, some of the limitations of standard thin-slice sections are also relevant to this preparation. Generally, it may be difficult to preserve circuits with less planar projection patterns, as axons would likely be severed. Activating the circuit at the cranial nerves, as done here to mimic auditory inputs, may not be feasible in many circuits. As with other slice preparations, the network effects of any pharmacology must be considered. For example, bath application of glutamate receptor blockers to isolate inhibitory (GABA- or glycinergic) or other modes of transmission cannot be used with polysynaptic circuit activation when glutamate is necessary for activation of neurons upstream of the patched target neuron. This is true in our case as both AN/CN and GBC/MNTB synapses are glutamatergic, therefore, all transmission would be eliminated at MOC neurons with bath application of glutamate receptor blockers. Additionally, application of GABA or glycine receptor blockers to eliminate MNTB-MOC synaptic responses would have the unintended consequence of eliminating intrinsic inhibitory connectivity within the CN that may shape the patterns of afferent inputs to MOC neurons. Focal application of receptor blockers, with pressure ejection or iontophoresis, could be used to restrict pharmacological function.
Finally, the main limitation of this, and any, in vitro technique is that although this preparation maximizes activation of monaural ascending auditory circuitry, the rest of the nervous system, including peripheral receptors that encode stimuli, is absent. This includes the cochlea itself, excitatory inputs from the other ear66, commissural CN connections67,68,69,70, and descending cortical71,72,73,74 and collicular75,76 projections and modulatory inputs77,78,79,80 known to influence activity of CN and SOC nuclei. While it is possible that a portion of descending IC projections are maintained it would be impossible to include both cortical projections and commissural CN projections due to slice geometry. Hence, we focus on the ascending auditory circuitry from the cochlea with the current experiments. The minimal thickness of the slice on the thin side also reduces the ability to perform binaural polysynaptic circuit analyses, which is an advantage of symmetrical thick slice preparations10,11. Additionally, we are unable to stimulate the auditory nerve with sound to evoke natural patterns of circuit activity. Auditory nerve responses are tonotopically varied, jittery, and plastic81,82,83,84, making it difficult to perfectly simulate with our electrical stimulation method. This is a major drawback of in vitro experimentation in the auditory system. Tonotopic restriction of our stimulation is not possible since stimulating the entire AN root will elicit spiking in AN fibers across the tonotopic gradient. Accurately mimicking the diversity of AN fiber responses (i.e., low vs. high spontaneous rate fibers) to an electrical stimulus pattern is also not possible. It is also difficult to precisely match the dynamic intensity coding of multiple AN fibers at the CN. However, we are able to use our electrophysiology software to produce a variety of stimulation patterns aimed to mimic appropriate auditory nerve output during different acoustic stimuli (e.g. short, loud sounds, quiet, prolonged sounds or sounds in background noise) by varying the stimulus frequency both between electrical stimulus protocols and also within an individual protocol to approximate combined AN inputs&#160;(modeled in ref.85). Monitoring MOC output during these experiments will test our hypotheses regarding what stimulus patterns may favor inhibition or excitation at MOC neurons.
Despite the limitations described above, a wedge slice preparation method has benefits compared to in vivo and typical in vitro slice physiology methods and can be used to approach in vivo circuit activation as closely as possible in the slice for cells that are difficult to access. In vivo whole-cell recordings in the auditory brainstem have been rare due to difficulties accessing this area surgically86. Instead the slice was prepared to include ascending inputs to MOC neurons beginning with the auditory nerve, which is stimulated directly to activate the entire monaural ascending circuit. We demonstrate activation of both excitatory and inhibitory synaptic inputs, and responses to these inputs provide valuable information about timing of synaptic inputs as they reach the MOC neurons. This provides a platform for high throughput experimentation where we can employ a large repertoire of in vitro electrophysiology tools such as calcium or voltage imaging, neurotransmitter uncaging, and both intracellular (via the patch pipette) and extracellular (via bath application or iontophoresis) pharmacology. The preparation should also offer an increase in throughput over thick slice preparations due to better visibility of target neurons using DIC optics, which blur with increased tissue thickness, especially in the ventral brainstem. Overall, this technique provides improvements in targeting and throughput over in vivo methods, and better opportunities for circuit analysis than traditional slice physiology methods.

Observation of activity of neural circuits is ideally performed with native sensory inputs and feedback, and intact connectivity between brain regions, in vivo. However, performing experiments that give single-cell resolution of neural circuit function is still limited by technical challenges in the intact brain. While in vivo extracellular electrophysiology or multiphoton imaging methods can be used for investigating activity in intact nervous systems, interpreting how different inputs integrate or measuring subthreshold synaptic inputs remains difficult. In vivo whole-cell recordings overcome these limitations but are challenging to perform, even in brain regions which are easily accessed. Technical challenges of single-cell resolution experiments are further amplified in certain neuron populations that are located deep in the brain, or in spatially diffuse populations that require either genetic tools to locate cells in vivo (e.g., genetic expression of channelrhodopsin paired with optrode recording) or post-hoc histochemical identification after recording site labeling (e.g. with neurotransmission-specific markers). Being located diffusely near the ventral surface of the brainstem, medial olivocochlear (MOC) neurons suffer from the above limitations1, making them extremely difficult to access for in vivo experimentation.
Brain slices (~100-500 &#181;m thickness) have long been used to study brain circuitry, including auditory brainstem circuitry, because of the physical segregation of connected neurons that are contained within the same slice2,3,4,5,6,7,8,9. Experiments using much thicker slices (&#62;1 mm) have been employed in other labs to understand how bilateral inputs integrate in areas of the superior olivary complex (SOC) including the medial superior olive10,11. These slices were prepared such that axons of the auditory nerve (AN) remained intact within the slice and were electrically stimulated to initiate synaptic neurotransmitter release in the CN, mimicking activity of first order auditory neurons as they would respond to sound. One major disadvantage of these thick slices is visibility of neurons for patch-clamp electrophysiological recordings (&#8220;patching&#8221;). Patching becomes increasingly difficult as the numerous axons in this area become myelinated with age12,13,14,15, making the tissue optically dense and obscuring neurons even in a typical, thin brain slice. Our goal is to create in vitro preparations that more closely resemble the circuit connectivity of in vivo recordings, but with the high-throughput and high-resolution recording abilities of visually guided patch-clamp electrophysiology in brain slices.
Our lab investigates the physiology of neurons of the auditory efferent system, including MOC neurons. These cholinergic neurons provide efferent feedback to the cochlea by modulating the activity of outer hair cells (OHCs)16,17,18,19,20. Previous studies have shown that this modulation plays a role in gain control in the cochlea21,22,23,24,25,26 and protection from acoustic trauma27,28,29,30,31,32,33. In mice, MOC neurons are diffusely located in the ventral nucleus of the trapezoid body (VNTB) in the auditory brainstem1. Our group has utilized the ChAT-IRES-Cre mouse line crossed with the tdTomato reporter mouse line to target MOC neurons in brainstem slices under epifluorescent illumination. We showed that MOC neurons receive afferent inhibitory input from the ipsilateral medial nucleus of the trapezoid body (MNTB), which is excited, in turn, by axons from globular bushy cells (GBC) in the contralateral cochlear nucleus (CN)34,35,36,37,38. Additionally, MOC neurons likely receive their excitatory input from T-stellate cells in the contralateral CN39,40,41. Taken together, these studies show MOC neurons receive both excitatory and inhibitory inputs derived from the same (contralateral) ear. However, the presynaptic neurons, and their axons converging on MOC neurons, are not quite close enough to each other to be fully intact in a typical coronal slice preparation. To investigate how integration of synaptic inputs to MOC neurons affects their action potential firing patterns, with a focus on newly described inhibition, we developed a preparation in which we could stimulate the diverse afferents to MOC neurons from one ear in the most physiologically realistic way possible, but with the technical benefits of in vitro brain slice experiments.
The wedge slice is a modified thick slice preparation designed for investigation of circuit integration in MOC neurons (schematized in Figure 1A). On the thick side of the slice, the wedge contains the severed axons of the auditory nerve (termed &#8220;auditory nerve root&#8221; hereafter) as they enter the brainstem from the periphery and synapse in the CN. The auditory nerve root can be electrically stimulated to evoke neurotransmitter release and synaptic activation of cells of the fully intact CN42,43,44,45,46. This stimulation format has several benefits for circuit analysis. First, instead of directly stimulating the T-stellate and GBC axons that provide afferent input to the MOC neurons, we stimulate the AN to allow activation of intrinsic circuits abundant in the CN. These circuits modulate the output of CN neurons to their targets throughout the brain, including MOC neurons46,47,48,49,50,51. Second, the polysynaptic activation of afferent circuits from the AN through the CN upstream of MOC neurons allows for more natural activation timing and for plasticity to occur at these synapses as they would in vivo during auditory stimulation. Third, we can vary our stimulation patterns to mimic AN activity. Finally, both excitatory and inhibitory monaural projections to MOC neurons are intact in the wedge slice, and their integration can be measured at an MOC neuron with the precision of patch-clamp electrophysiology. As a whole, this activation scheme provides a more intact circuit to the MOC neurons compared to a typical brain slice preparation. This brainstem wedge slice can also be used to investigate other auditory areas which receive inhibitory input from ipsilateral MNTB including the lateral superior olive, superior olivary nucleus and medial superior olive10,11,52,53,54,55,56. Beyond our specific preparation, this slicing method can be used or modified to evaluate other systems with the benefits of maintaining connectivity of long-range inputs and improving visualization of neurons for a variety of single-cell resolution electrophysiology or imaging techniques.
This protocol requires the use of a vibratome stage or platform which can be tilted approximately 15&#176;. Here we use a commercially available 2-piece magnetic stage where the &#8220;stage&#8221; is a metal disc with a curved bottom placed in a concave magnetic &#8220;stage base.&#8221; The stage can then be shifted to adjust the slice angle. Concentric circles on the stage base are used to estimate the angle reproducibly. The stage and stage base are placed in the slicing chamber, where the magnetic stage base can also be rotated.

<table><tbody><tr><td>&lt;strong&gt;Experimental Preparations&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Agar, powder</td><td>Fisher Scientific</td><td>BP1423500</td><td>4% agar block used to stabilize brain tissue during vibratome sectioning</td></tr><tr><td>AlexaFluor Hydrazide 488</td><td>Invitrogen</td><td>A10436</td><td>Fluorophore used in internal solution to confirm successful MOC neuron patch</td></tr><tr><td>Analytical Balance</td><td>Geneses Scientific (Intramalls)</td><td>AV114</td><td>Weighing chemicals</td></tr><tr><td>Double edged razor blade</td><td>Ted Pella</td><td>121-6</td><td>Vibratome cutting blade</td></tr><tr><td>Kynurenic acid (5g)</td><td>Sigma Aldrich</td><td>K3375-5G</td><td>Slicing ACSF additive used to reduce neuron activity during dissection and slicing in order to improve tissue health for patch clamping</td></tr><tr><td>pH Meter</td><td>Fisher Scientific (Intramalls)</td><td>13-620-451</td><td>Solution pH tester</td></tr><tr><td>Plastic petri dishes 100mm dia X 20mm</td><td>Fisher Scientific (Intramalls)</td><td>12-556-002</td><td>4% Agar Prep</td></tr><tr><td>Stirring Hotplate</td><td>Fisher Scientific (Intramalls)</td><td>11-500-150</td><td>Heating for 4% Agar preparation</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Dissection and Slicing&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Biocytin</td><td>Sigma Aldrich</td><td>B4261-250MG</td><td>Chemical used for axonal tracing (conjugated to Streptavidin 488)</td></tr><tr><td>Dissecting Microscope</td><td>Amscope</td><td>SM-1BN</td><td>For precision dissection during brain removal</td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11252-20</td><td>Fine forceps dissection tool</td></tr><tr><td>Economy tweezers #3</td><td>WPI</td><td>501976</td><td>Forceps dissection tool</td></tr><tr><td>Glass Petri Dish 150mm dia x 15mm H</td><td>Fisher Scientific (Intramalls)</td><td>08-747E</td><td>Dissection dish</td></tr><tr><td>Interface paper (203 X 254mm PCTE Membrane 10um)</td><td>Thomas Scientific</td><td>1220823</td><td>Slice incubation/biocytin application</td></tr><tr><td>Leica VT1200S Vibratome</td><td>Leica</td><td>1491200S001</td><td>Vibratome for wedge slice sectioning</td></tr><tr><td>Mayo scissors</td><td>Roboz</td><td>RS-6872</td><td>Dissection tool</td></tr><tr><td>Single-edged carbon steel blades</td><td>Fisher Scientific (Intramalls)</td><td>12-640</td><td>Razor blade for dissection</td></tr><tr><td>Specimen disc, orienting</td><td>Leica</td><td>14048142068</td><td>Specialized vibratome stage for reproducible tilting</td></tr><tr><td>Spoonula</td><td>FisherSci</td><td>14-375-10</td><td>Dissection tool</td></tr><tr><td>Super Glue</td><td>Newegg</td><td>15187</td><td>Used for glueing tissue to vibratome stage</td></tr><tr><td>Vannas Spring Scissors</td><td>Fine Science Tools</td><td>91500-09</td><td>Dissection tool</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Electrophysiology&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>A1R Upright Confocal Microscope</td><td>Nikon Instruments</td><td></td><td>Electrophysiology and imaging microscope, can be any microscope compatible with electrophysiology</td></tr><tr><td>Electrode Borosilicate glass w/ Filament OD 1.5mm, ID 1.1mm, 10 cm long</td><td>Sutter Instrument</td><td>BF150-110-10</td><td>Patch clamping pipette glass</td></tr><tr><td>Electrode Filler MicroFil</td><td>WPI</td><td>CMF20G</td><td>Patch electrode pipette filler</td></tr><tr><td>In-line solution heater</td><td>Warner Instruments (GSAdvantage)</td><td>SH-27B</td><td>Slice perfusion system heater</td></tr><tr><td>Multi-Micromanipulator Systems</td><td>Sutter Intruments</td><td>MPC-200 with MP285</td><td>Micromanipulators for patch clamp and stimulation electrode placement</td></tr><tr><td>P-1000 horizontal pipette puller for glass micropipettes</td><td>Sutter instruments</td><td>FG-P1000</td><td>Patch clamp pipetter puller</td></tr><tr><td>Patch-clamp amplifier and Software</td><td>HEKA</td><td>EPC-10 / Patchmaster Next</td><td>Can be any amplifier/software</td></tr><tr><td>Recording Chamber</td><td>Warner Instruments</td><td>RC26G</td><td>Slice &quot;bath&quot; during recording</td></tr><tr><td>Recording Chamber Harp</td><td>Warner Instruments</td><td>640253</td><td>Stablizes slice during electrophysiology recording</td></tr><tr><td>Slice Incubation Chamber</td><td>Custom Build</td><td></td><td>Heated, oxygenated holding chamber for slices during recovery after slicing</td></tr><tr><td>Stimulus isolation unit</td><td>A.M.P.I.</td><td>Iso-Flex</td><td>Stimulus isolation unit for electrophysiology</td></tr><tr><td>Syringe 60CC</td><td>Fischer Scientific (Intramalls)</td><td>14-820-11</td><td>Electrophysiology perfusion fluid handling</td></tr><tr><td>Temperature controller</td><td>Warner Instruments (GSAdvantage)</td><td>TC-324C</td><td>Slice perfusion system temperature controller</td></tr><tr><td>Tubing 1/8 OD 1/16 ID</td><td>Fischer Scientific (Intramalls)</td><td>14-171-129</td><td>Electrophysiology perfusion fluid handling</td></tr><tr><td>Tugsten concentric bipolar microelectrode</td><td>WPI</td><td>TM33CCINS</td><td>Stimulating electrode for electrophysiology</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Histology&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>24 well Plate</td><td>Fisher Scientific (Intramalls)</td><td>12-556006</td><td>Histology slice collection and immunostaining</td></tr><tr><td>Alexa Fluor 488 Streptavidin</td><td>Jackson Immuno labs</td><td>016-540-084</td><td>Secondary antibody for biocytin visualization</td></tr><tr><td>Corning Orbital Shaker</td><td>Sigma</td><td>CLS6780FP</td><td>Shaker for immunohistochemistry agitation</td></tr><tr><td>Cresyl Violet Acetate</td><td>Sigma Aldrich (Intramalls)</td><td>C5042-10G</td><td>Cellular stain for histology</td></tr><tr><td>Disposable Microtome Blades</td><td>Fisher Scientific</td><td>22-210-052</td><td>Sliding microtome blade</td></tr><tr><td>Filter-syringe Nalgene 4mm Cellulose Acetate 0.2um</td><td>Fisher Scientific (Intramalls)</td><td>09-740-34A</td><td>Syringe filter for filling recording pipettes with internal solution</td></tr><tr><td>Fluoromount-G Slide Mounting Medium</td><td>Fisher Scientific</td><td>OB100-01</td><td>Immunohistochemistry fluorescence mounting medium</td></tr><tr><td>glass slide staining dish with rack</td><td>Fisher Scientific (Intramalls)</td><td>08-812</td><td>Cresyl Violet staining chamber</td></tr><tr><td>Microm HM450 Sliding Microtome</td><td>ThermoFisher</td><td>910020</td><td>Freezing microtome for histology</td></tr><tr><td>Microscope Cover Glasses: Rectangles 50mm X 24mm</td><td>Fisher Scientific (Intramalls)</td><td>12-543D</td><td>Histochemistry slide cover glass</td></tr><tr><td>Permount mounting medium</td><td>Fisher Scientific</td><td>SP15-100</td><td>Cresyl violet section mounting medium</td></tr><tr><td>Superfrost Slides</td><td>Fisher Scientific</td><td>22-034980</td><td>Histology slides</td></tr></tbody></table>

in vitro wedge slice preparation for mimicking in vivo neuronal circuit connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Histological examination of wedge slice 
For our investigation of auditory brainstem neuron function, the wedge slice preparation was designed to contain the auditory nerve root and CN contralateral to the MOC neurons targeted for recordings (example slice shown in Figure 1B). Initial histological examination of the preparation is important to confirm that the slice contains the nuclei necessary for circuit activation and that axonal projections are intact. Two cell types within the CN provide sound information to MOC neurons. T-stellate cells are hypothesized to provide the excitatory input to MOC neurons39,40,41,58. Globular bushy cells (GBC) excite MNTB neurons in the contralateral hemisphere (via the specialized calyx of Held synapse)34,36,37,38,59,60 which, in turn, provide inhibitory input to MOC neurons57 (schematic Figure 1A). To confirm the presence of both T-stellate cells and GBCs, we re-sectioned (to 50 &#181;m) a wedge slice that was fixed by submersion in 4% PFA and performed cresyl violet staining to label somata. In the thick side of the wedge slice (Figure 2, left hemisphere), the CN was present in nearly its full rostro-caudal extent. Additionally, the dorsal and ventral subdivisions of the CN were intact (Figure 2; arrow and arrowhead in S19). T-stellate neurons and GBCs cluster in the ventral cochlear nucleus near where the auditory nerve (Figure 2; arrow in S17) enters the CN61,62,63,64,65. The wedge slice also contains neurons of the MNTB ipsilateral to MOC neurons from which recordings are performed (thin hemisphere of the original wedge slice, right side in Figure 2). This confirms that at least part of the inhibitory input to MOC neurons is intact (Figure 2, slices 1-15, highlighted by dashed ovals in S11).
In separate experiments, we confirmed that the axons and presynaptic terminals of CN neurons were intact in the wedge slice using anterograde labeling with biocytin. First, the live wedge slice was prepared and placed on interface paper. Immediately after preparing the wedge slice, biocytin crystals were placed in the CN which allowed uptake and anterograde transport along axons during an incubation period. Then fixing and re-sectioning of the tissue (70 mm sections) was performed. Staining of sections with fluorescently labeled streptavidin was performed to visualize axons labeled with the biocytin. Confocal images of these sections show bright labeling in the CN where the crystals were placed and taken up into cell bodies (Figure 3A, left hemisphere, dashed area). Axons exiting the CN along the ventral acoustic stria (Figure 3A, white arrowheads) were clearly labeled and could be followed to their termination points. Biocytin-positive puncta surrounding contralateral MOC neurons suggest our preparation preserves synaptic contacts originating from the CN (Figure 3B). Likewise, labeled calyces of Held in the contralateral MNTB indicate axons projecting from GBCs to MNTB neurons are preserved in the wedge slice (Figure 3C). These histological examinations confirm our wedge slice contains both the cell bodies and axonal projections of the afferent input circuitry to MOC neurons, which, therefore, allows us to measure postsynaptic responses evoked by stimulation of the auditory nerve and subsequent propagation of activity through ascending circuitry.
Synaptic physiology in wedge slice 
Integration of excitatory and inhibitory synaptic inputs critically shapes neuronal activity. We recently described inhibitory inputs to MOC neurons from neurons of the MNTB57, but the effect of integration of these inputs with excitatory inputs on MOC neuron activity is unknown. In a wedge slice from a ChAT-IRES-Cre x tdTomato mouse, voltage-clamp recordings were performed from an MOC neuron. Current was applied via a bipolar tungsten stimulating electrode driven by a stimulus isolation unit to evoke neurotransmitter release from presynaptic axons. First the ventral acoustic stria (VAS) at the midline was electrically stimulated to activate T-stellate axons directly and MNTB neurons via GBC axon stimulation (Figure 4A), to measure the latency to post-synaptic responses in a recording configuration that mimics typical thin-slice experiments (Figure 4B), example traces, grey, holding potential -60 mV). In separate experiments, the auditory nerve root was stimulated to activate monaural ascending circuitry and post-synaptic responses were measured at MOC neurons as described above. Electrical stimulation in either location evoked a fast-electrical artifact followed by multipeaked current responses (example responses from AN stimulation in Figure 4C, black traces, holding potential -60 mV). We compared onset latency measures of the first postsynaptic current (PSC) evoked with direct stimulation of the VAS with those evoked with auditory nerve stimulation and found a significantly longer latency to AN stimulation events. This was attributed to the synaptic delay incurred at the AN/CN synapse (AN stimulation: 5.27 &#177; 0.43 ms, median &#177; median absolute deviation (MAD), range 4.26-5.93 ms, n = 8; VAS stimulation: 1.98 &#177; 0.28 ms, median &#177; MAD, range 0.75-3.46 ms, n = 17; Wilcoxon Signed Ranks Test, p = 0.014, Figure 4D). These results confirm that stimulation of the auditory nerve root results in synaptic activation of CN neurons and subsequent circuit activity, more closely representing in vivo &#8211; like timing than direct stimulation of T-stellate or GBC/MNTB axons.
With our cesium based, high [Cl-] internal solution used in voltage clamp, excitatory (glutamatergic) and inhibitory (GABA and glycinergic) PSCs are both inward at resting membrane potential (-60 mV) and therefore indistinguishable. While evoking circuit activity in the AN-stimulating configuration, we electrically isolated the presumed inhibitory input by shifting the holding potential to 0 mV, the approximate reversal potential for AMPA mediated glutamatergic currents. In our example neuron, outward current responses were observed at 0 mV (Figure 4Ci, red traces) indicative of chloride conductances. These are likely to be GABA- or glycinergic synaptic responses. These data demonstrate the utility of the wedge slice to activate both excitatory and inhibitory inputs to MOC neurons by stimulating the auditory nerve root, with activation of subsequent afferent circuitry. Further, diverse patterns of post-synaptic responses were evoked by AN stimulation, suggesting that even under conditions of identical stimulation of AN axons, activity of the entire circuit is dynamic and complex. This experimental paradigm allows for a detailed analysis of how complex auditory stimuli propagate through the brainstem and integrate at MOC neurons, determining the MOC efferent system&#8217;s output and eventual impact on the cochlea.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61664/61664fig01.jpg" /> 
Figure 1: Wedge slice schematic and example image.&#160;(A) Schematic of the medial olivocochlear feedback circuit. Blue arrows indicate the afferent ascending pathway to MOC neurons and black arrows indicate the descending feedback pathway from MOC neurons to the base of outer hair cells (OHC). (B) Brightfield image of a wedge slice with labels of the auditory nerve root (ANR) and cochlear nucleus (dashed outline) on the thick side. Asterisk indicates the approximate location of the ventral nucleus of the trapezoid body where MOC neurons are targeted for patch-clamping on the thin side of the wedge slice. Dashed black lines indicate the facial nerves which can be seen in both hemispheres of the slice on the rostral surface. IHC - inner hair cell, GBC - globular bushy cell, SPN - superior paraolivary nucleus, MNTB - medial nucleus of the trapezoid body, VNTB - ventral nucleus of the trapezoid body, LSO - lateral superior olive. <a href="https://www.jove.com/files/ftp_upload/61664/61664fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61664/61664fig02.jpg" /> 
Figure 2: Cresyl violet stained sections from a wedge slice that was re-sectioned at 50 &#181;m.&#160;Every other section was imaged. Sections are numbered rostral --&#62; caudal. Wedge slices tended to contain the entirety of the cochlear nucleus (CN) including both the dorsal CN (arrow in S19) and ventral CN (arrowhead in S19), auditory nerve root (open arrowhead in S17) and much of the MNTB (dark area near ventral surface in S3-S15, highlighted with dashed ovals in S11). D and V on scale bar represent dorsal and ventral in slice orientation. <a href="https://www.jove.com/files/ftp_upload/61664/61664fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61664/61664fig03.jpg" /> 
Figure 3: Axons of ascending input to MOC neurons from the contralateral cochlear nucleus remain intact in the wedge slice.&#160;(A) The rostral-most section from a wedge slice taken from a P23 ChAT-IRES-Cre x tdTomato (red fluorescence) mouse that was re-sectioned (70 &#181;m) and processed for biocytin visualization. Confocal image is a tiled, maximum intensity projection z-stack. Axons in the ventral acoustic stria are highlighted by white arrowheads. Dashed outline indicates the small portion of the cochlear nucleus remaining in this rostral-most slice. Scale bar 500 &#181;m. (B) Confocal image of a ChAT-IRES-Cre x tdTomato positive neuron in the VNTB with biocytin positive puncta in the surrounding neuropil. Scale bar 50 &#181;m. (C) Biocytin labeled axons shown crossing the midline and terminating in the contralateral MNTB as calyces of Held. Vertical dashed line represents the midline of the slice. Scale bar 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/61664/61664fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61664/61664fig04.jpg" /> 
Figure 4: Electrical stimulation of afferent inputs in voltage clamp yields multipeak postsynaptic currents in MOC neurons.&#160;(A) Schematic of wedge slice with recording set-up for both ventral acoustic stria (VAS) stimulation (grey stimulating electrode) and auditory nerve (AN) stimulation (black stimulating electrode) of afferent inputs to MOC. (B) Examples of postsynaptic currents (PSCs) from an individual P17 neuron evoked with a single electrical stimulus near the midline at -60 mV. (C) PSCs evoked during AN stimulation at -60 mV in a P15 neuron. (Ci) Example PSCs in the same cell as C evoked at 0 mV holding potential (the approximate reversal potential for AMPA mediated currents in our recording setup, red). (D) Population data for quantification of latency to first PSC for VAS and AN stimulation. Boxes: quartiles, line inset: median, square inset: mean, whiskers: median absolute deviation. * p &#60; 0.05. <a href="https://www.jove.com/files/ftp_upload/61664/61664fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity at JoVE.com

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...

in-vitro-wedge-slice-preparation-for-mimicking-vivo-neuronal-circuit

Nanyang Technological University

Research

JoVE Journal

Neuroscience

5.4K Views.  NIH. Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

Video: In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

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 ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell resolution using electrophysiology, microscopy, and pharmacology. However, a major consideration in the design of in vitro experiments is the extent to which different slice preparations recapitulate naturalistic patterns of neural activity as observed in vivo. In the intact brain, the hippocampal network generates highly synchronized population activity reflective of the behavioral state of the animal, as exemplified by the sharp-wave ripple complexes (SWRs) that occur during waking consummatory states or non-REM sleep. SWRs and other forms of network activity can emerge spontaneously in isolated hippocampal slices under appropriate conditions. In order to apply the powerful brain slice toolkit to the investigation of hippocampal network activity, it is necessary to utilize an approach that optimizes tissue health and the preservation of functional connectivity within the hippocampal network. Mice are transcardially perfused with cold sucrose-based artificial cerebrospinal fluid. Horizontal slices containing the hippocampus are cut at a thickness of 450 &#956;m to preserve synaptic connectivity. Slices recover in an interface-style chamber and are transferred to a submerged chamber for recordings. The recording chamber is designed for dual surface superfusion of artificial cerebrospinal fluid at a high flow rate to improve oxygenation of the slice. This protocol yields healthy tissue suitable for the investigation of complex and spontaneous network activity in vitro.

This protocol describes the preparation of horizontal hippocampal-entorhinal cortex (HEC) slices from mice exhibiting spontaneous sharp-wave ripple activity. Slices are incubated in a simplified interface holding chamber and recordings are performed under submerged conditions with fast-flowing artificial cerebrospinal fluid to promote tissue oxygenation and the spontaneous emergence of network-level activity.

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell ...

Preparation of Acute Slices from Dorsal Hippocampus for Whole-Cell Recording and Neuronal Reconstruction in the Dentate Gyrus of Adult Mice

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in molecular, anatomical and functional criteria suggesting a division into different sub-circuits along its rostro-caudal extent. Owing to differential connectivity and function the most fundamental distinction is made between the dorsal and the ventral hippocampus, which are preferentially involved in spatial and emotional processing, respectively. Accordingly, in vivo work regarding spatial memory formation has focused on the dorsal hippocampus.
In contrast, electro-physiological in vitro recordings have been preferentially performed on intermediate-ventral hippocampus, largely motivated by factors like slice viability and circuit integrity. To allow for direct correlation of in vivo data on spatial processing with in vitro data we have adapted previous sectioning methods to obtain highly viable transverse brain slices from the dorsal-intermediate hippocampus for long-term recordings of principal cells and interneurons in the dentate gyrus. As spatial behavior is routinely analyzed in adult mice, we have combined this transversal slicing procedure with the use of protective solutions to enhance viability of brain tissue from mature animals. We use this approach for mice of about 3 months of age. The method offers a good alternative to the coronal preparation which is frequently used for in vitro studies on dorsal hippocampus. We compare these two preparations in terms of quality of recordings and preservation of morphological features of recorded neurons.

We present a protocol to prepare acute-slices from the dorsal-intermediate hippocampus of mice. We compare this transversal preparation with coronal slicing in terms of quality of recordings and preservation of morphological features of recorded neurons.

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in ...

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...

Preparing Wedge Brain Slices to Visualize Auditory Neurons

Source: Fischl, M. J. et al., <a href="https://app.jove.com/v/61664">In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity.</a>&#160;J. Vis. Exp.&#160;(2020)
This video demonstrates the process of preparing wedge-shaped brain slices in a laboratory setting. Using a vibratome, a wedge-shaped slice from a mouse&#39;s brain is crafted with varying thickness. This slice contains the auditory nerve root positioned on the thicker end, while the hearing-related neurons are located on the thinner end.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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Neuronal Culture Techniques

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An Electrophysiological Study of Retinogeniculate and Corticogeniculate Synapses in Mouse Brain Slices

Source: Chen, X., et al. <a href="https://app.jove.com/v/59680">Electrophysiological Investigations of Retinogeniculate and Corticogeniculate Synapse Function.</a> J. Vis. Exp. (2019).
The video describes the electrophysiological recording of retinogeniculate and corticogeniculate synapses in mouse brain slices. The slices containing the dorsal lateral geniculate nucleus (dLGN) are placed in a recording chamber. A stimulating pipette is positioned on either the optic tract to stimulate retinal ganglion cells (RGCs) and investigate retinogeniculate synapses or the nucleus reticularis thalami to stimulate cortical neurons and investigate corticogeniculate synapses. Upon stimulation, the RGCs and cortical neurons release excitatory neurotransmitters, which produce synaptic currents in the LGN relay neurons. These currents are recorded using a patch-clamp recording pipette.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
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In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

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Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

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

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Preparation of Acute Slices from Dorsal Hippocampus for Whole-Cell Recording and Neuronal Reconstruction in the Dentate Gyrus of Adult Mice

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

Preparing Wedge Brain Slices to Visualize Auditory Neurons

An Electrophysiological Study of Retinogeniculate and Corticogeniculate Synapses in Mouse Brain Slices