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

Matthew  J. Fischl; Catherine J. C. Weisz

doi:10.3791/61664

JoVE Journal > Neuroscience

Neuroscienze

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

Published: August 18, 2020

doi:

10.3791/61664

Matthew J. Fischl¹, Catherine J. C. Weisz¹

¹Section on Neuronal Circuitry, National Institute on Deafness and Other Communication Disorders,NIH

Summary

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.

Abstract

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 “wedge slice” 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.

Introduction

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 µm 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 (>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 (“patching”). 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 “auditory nerve root” 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°. Here we use a commercially available 2-piece magnetic stage where the “stage” is a metal disc with a curved bottom placed in a concave magnetic “stage base.” 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.

Protocol

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.

Prepare internal solutions for patch-clamping.
1. 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.
2. 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.
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.
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.
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.
Mount an appropriate blade in the vibratome following the manufacturer’s instructions. Chill vibratome slicing chamber by surrounding it with ice.

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.

Euthanize (e.g., CO₂ asphyxiation) and decapitate the animal using approved institutional procedures.
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.
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.
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.
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.
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.
Place the preparation in a glass Petri dish filled with cold slicing solution. Place the dish under a dissecting microscope. Gently bubble with carbogen.
Trim the facial nerve close to the brainstem and expose the vestibulocochlear nerve.
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.
Once both nerve roots are free, remove the meninges and vasculature from the ventral surface of the brainstem near the trapezoid body.
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.

3. Block and mount brain on stage (magnetic disc)

Prepare the surface of the brain to fix to the stage by blocking the brain at the level of the optic chiasm.
1. 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.
2. 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˚ from vertical so that the tips exit the dorsal surface of the brain caudal to the optic chiasm.
3. Cut along the forceps using the razor blade.
Glue the brain to the surface of the stage.
1. Prepare a small block (~1 cm³) of 4% agar for supporting the brain.
2. 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.
3. 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.
4. 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).

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.

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.
Fill the chamber with ice cold slicing solution and bubble with carbogen.
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.
Once the slices are symmetrical, shift the stage ~15˚ (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.
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.
Shift the stage back 15˚ to the original position.
Move the blade away from the tissue and spin the stage base 90° 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.
Move the blade back away from the tissue and spin the stage base back so that the ventral surface is facing it.
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 °C).
NOTE: The facial nerve should be visible on both hemispheres of the slice on the rostral surface (see Figure 1B).

5. Electrophysiology set-up and recording

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 °C) ACSF bubbled with carbogen.
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.
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.
Move the field of view back to the VNTB to choose an MOC neuron to target for patch clamp electrophysiology.
Fill a recording pipette with appropriate internal solution for the proposed experiment.
Patch and record from the MOC neuron in the whole-cell configuration. Compensate membrane capacitance and series resistance if required.
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.
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.

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.

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 °C to cryoprotect.
Re-section the slice on a freezing microtome (40-70 mm) and collect serial sections in a 24 well plate in PBS.
Mount sections on gelatin coated slides and let dry completely. Place slides in slide carriage.
Prepare cresyl violet solutions
1. Prepare 1% cresyl violet acetate by mixing 5 g cresyl violet acetate in 500 mL dH₂O
2. 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.
3. Combine 1% cresyl violet acetate with the acetate buffer 1:1 for 0.5% cresyl violet in acetate buffer. Filter before use.
4. Prepare 95% and 70% ethanol by diluting 100% ethanol with appropriate volumes of dH₂O
Perform cresyl violet staining protocol. Move the slide carriage through solution trays, blotting excess solution on a paper towel between trays: xylene – 5 min; 95% ethanol – 3 min; 70% ethanol – 3 min; dH₂O – 3 min; 0.5% cresyl violet solution – 8-14 min monitoring frequently until nuclear staining becomes dark purple; dH₂O – 3 min; 70% ethanol – 3 min; 95% ethanol – 1-2 min; 100% ethanol – dip slides twice; xylene – 5 min; xylene: 25 min until mounting is performed.
CAUTION: Use xylenes only under a fume hood.
Remove slides from xylene one at a time and immediately place cover slips on slides using mounting medium. Allow mounting medium to dry (overnight).
Image sections.

7. Biocytin labeling for anterograde tracing of axons in live, unfixed tissue

Prepare a wedge slice as above (Steps 2-4).
Transfer the slice to interface paper (~1 cm²). Under a dissecting microscope, locate the CN on the thick side of the slice.
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.
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.
Place the slice in an incubation chamber. Allow the slice to incubate for 2-4 h at 35 °C to allow for the uptake and transport of the biocytin. After incubation, rinse the slice in ACSF to remove any biocytin particles.
Place slice in fixative (4% PFA in PBS) overnight. Rinse 3x for 10 min in PBS.
Cryoprotect slice in 30% sucrose in PBS overnight at 4 °C or until the slice sinks.
Resect the tissue to produce transverse sections on a freezing microtome at 70-100 mm.
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).
Image the tissue.

Representative Results

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 µm) 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 ± 0.43 ms, median ± median absolute deviation (MAD), range 4.26-5.93 ms, n = 8; VAS stimulation: 1.98 ± 0.28 ms, median ± 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 – 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’s output and eventual impact on the cochlea.

Figure 1: Wedge slice schematic and example image. (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. Please click here to view a larger version of this figure.

Figure 2: Cresyl violet stained sections from a wedge slice that was re-sectioned at 50 µm. Every other section was imaged. Sections are numbered rostral –> 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. Please click here to view a larger version of this figure.

Figure 3: Axons of ascending input to MOC neurons from the contralateral cochlear nucleus remain intact in the wedge slice. (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 µm) 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 µm. (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 µm. (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 µm. Please click here to view a larger version of this figure.

Figure 4: Electrical stimulation of afferent inputs in voltage clamp yields multipeak postsynaptic currents in MOC neurons. (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 < 0.05. Please click here to view a larger version of this figure.

Discussion

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 (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.

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

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

Materials

Experimental Preparations
Agar, powder	Fisher Scientific	BP1423500	4% agar block used to stabilize brain tissue during vibratome sectioning
AlexaFluor Hydrazide 488	Invitrogen	A10436	Fluorophore used in internal solution to confirm successful MOC neuron patch
Analytical Balance	Geneses Scientific (Intramalls)	AV114	Weighing chemicals
Double edged razor blade	Ted Pella	121-6	Vibratome cutting blade
Kynurenic acid (5g)	Sigma Aldrich	K3375-5G	Slicing ACSF additive used to reduce neuron activity during dissection and slicing in order to improve tissue health for patch clamping
pH Meter	Fisher Scientific (Intramalls)	13-620-451	Solution pH tester
Plastic petri dishes 100mm dia X 20mm	Fisher Scientific (Intramalls)	12-556-002	4% Agar Prep
Stirring Hotplate	Fisher Scientific (Intramalls)	11-500-150	Heating for 4% Agar preparation

Dissection and Slicing
Biocytin	Sigma Aldrich	B4261-250MG	Chemical used for axonal tracing (conjugated to Streptavidin 488)
Dissecting Microscope	Amscope	SM-1BN	For precision dissection during brain removal
Dumont #5 Forceps	Fine Science Tools	11252-20	Fine forceps dissection tool
Economy tweezers #3	WPI	501976	Forceps dissection tool
Glass Petri Dish 150mm dia x 15mm H	Fisher Scientific (Intramalls)	08-747E	Dissection dish
Interface paper (203 X 254mm PCTE Membrane 10um)	Thomas Scientific	1220823	Slice incubation/biocytin application
Leica VT1200S Vibratome	Leica	1491200S001	Vibratome for wedge slice sectioning
Mayo scissors	Roboz	RS-6872	Dissection tool
Single-edged carbon steel blades	Fisher Scientific (Intramalls)	12-640	Razor blade for dissection
Specimen disc, orienting	Leica	14048142068	Specialized vibratome stage for reproducible tilting
Spoonula	FisherSci	14-375-10	Dissection tool
Super Glue	Newegg	15187	Used for glueing tissue to vibratome stage
Vannas Spring Scissors	Fine Science Tools	91500-09	Dissection tool

Electrophysiology
A1R Upright Confocal Microscope	Nikon Instruments		Electrophysiology and imaging microscope, can be any microscope compatible with electrophysiology
Electrode Borosilicate glass w/ Filament OD 1.5mm, ID 1.1mm, 10 cm long	Sutter Instrument	BF150-110-10	Patch clamping pipette glass
Electrode Filler MicroFil	WPI	CMF20G	Patch electrode pipette filler
In-line solution heater	Warner Instruments (GSAdvantage)	SH-27B	Slice perfusion system heater
Multi-Micromanipulator Systems	Sutter Intruments	MPC-200 with MP285	Micromanipulators for patch clamp and stimulation electrode placement
P-1000 horizontal pipette puller for glass micropipettes	Sutter instruments	FG-P1000	Patch clamp pipetter puller
Patch-clamp amplifier and Software	HEKA	EPC-10 / Patchmaster Next	Can be any amplifier/software
Recording Chamber	Warner Instruments	RC26G	Slice "bath" during recording
Recording Chamber Harp	Warner Instruments	640253	Stablizes slice during electrophysiology recording
Slice Incubation Chamber	Custom Build		Heated, oxygenated holding chamber for slices during recovery after slicing
Stimulus isolation unit	A.M.P.I.	Iso-Flex	Stimulus isolation unit for electrophysiology
Syringe 60CC	Fischer Scientific (Intramalls)	14-820-11	Electrophysiology perfusion fluid handling
Temperature controller	Warner Instruments (GSAdvantage)	TC-324C	Slice perfusion system temperature controller
Tubing 1/8 OD 1/16 ID	Fischer Scientific (Intramalls)	14-171-129	Electrophysiology perfusion fluid handling
Tugsten concentric bipolar microelectrode	WPI	TM33CCINS	Stimulating electrode for electrophysiology

Histology
24 well Plate	Fisher Scientific (Intramalls)	12-556006	Histology slice collection and immunostaining
Alexa Fluor 488 Streptavidin	Jackson Immuno labs	016-540-084	Secondary antibody for biocytin visualization
Corning Orbital Shaker	Sigma	CLS6780FP	Shaker for immunohistochemistry agitation
Cresyl Violet Acetate	Sigma Aldrich (Intramalls)	C5042-10G	Cellular stain for histology
Disposable Microtome Blades	Fisher Scientific	22-210-052	Sliding microtome blade
Filter-syringe Nalgene 4mm Cellulose Acetate 0.2um	Fisher Scientific (Intramalls)	09-740-34A	Syringe filter for filling recording pipettes with internal solution
Fluoromount-G Slide Mounting Medium	Fisher Scientific	OB100-01	Immunohistochemistry fluorescence mounting medium
glass slide staining dish with rack	Fisher Scientific (Intramalls)	08-812	Cresyl Violet staining chamber
Microm HM450 Sliding Microtome	ThermoFisher	910020	Freezing microtome for histology
Microscope Cover Glasses: Rectangles 50mm X 24mm	Fisher Scientific (Intramalls)	12-543D	Histochemistry slide cover glass
Permount mounting medium	Fisher Scientific	SP15-100	Cresyl violet section mounting medium
Superfrost Slides	Fisher Scientific	22-034980	Histology slides

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Fischl, M. J., Weisz, C. J. C. In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity. J. Vis. Exp. (162), e61664, doi:10.3791/61664 (2020).