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 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 ...

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

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

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 ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Analyzing Dendritic Morphology in Columns and Layers

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

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

Summary

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Chapters in this video

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

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

Analyzing Dendritic Morphology in Columns and Layers