Name: in vitro wedge slice preparation for mimicking in vivo neuronal circuit connectivity
Uploaded: 2020-08-18T14:00:00
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 ...

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 w...

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>Experimental Preparations</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>Dissection and Slicing</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>Electrophysiology</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 &#34;bath&#34; 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>Histology</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 typ...

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 ...

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