We gratefully acknowledge support from the National Institute of Health (DC010809), National Science Foundation (IOS1257150, 1856237), and the Whitney Laboratory for Marine Biosciences to J.C.L.&#160;We would like to thank past and present members of the Liao Lab for stimulating discussions.

The authors declare no competing financial interests.

The experimental protocol described provides the potential to monitor endogenous changes in sensory input across motor behaviors in an intact, behaving vertebrate. Specifically, it details an in vivo approach to performing simultaneous extracellular recordings of lateral line afferent neurons and ventral motor roots in larval zebrafish. Spontaneous afferent activity has been previously characterized in zebrafish without consideration of potential concurrent motor activity1,<sup class="x...

Afferent neurons of mechanosensory systems transmit information from hair cells to the brain during hearing and balance. Electrophysiology can reveal the sensitivity of afferent neurons through direct recordings. While whole cell patching from hair cells can be challenging, recording from downstream afferent neurons is easier and allows assessment of action potentials in response to controlled stimulations1,2,3. Stimulating hair cells lead to their deflection, which modifies mechanosensory structures, thus triggering an increase in action potentials (spikes) in afferent neurons4,5,6. In the absence of external stimuli, afferent neurons also spike spontaneously due to glutamate leak from the hair cells on to afferent post-synaptic terminals7,8, and have been shown to contribute toward maintaining sensitivity9,10. Patch clamp recording of afferent activity enables observation of hair cell sensitivity and signal dynamics that are not possible using techniques with lower temporal resolution, such as in microphonics11,12 or functional calcium imaging13,14,15. The following protocol will allow the recording of afferent activity concurrent with motor commands to reveal instantaneous changes in hair cell sensitivity.
Zebrafish (Danio rerio) use hair cells contained in neuromasts that compose the lateral line system to detect water movement relative to their body, which is translated into neural signals essential for navigation16,17,18, predator avoidance, prey capture19,20, and schooling21. Water flow can also be self-generated by the motions of swimming22,23,24, respiration22,25,26, and feeding27. These behaviors comprise repetitive movements that can fatigue hair cells and impair sensing. Therefore, it is critical that the lateral line system differentiates between external (exafferent) and self-generated (reafferent) flow stimuli. A corollary discharge attenuates self-generated flow signals during locomotion in zebrafish. This inhibitory predictive motor signal is relayed via descending neurons to the sensory receptors to modify the input or interrupt the processing of the reafferent feedback28,29. Seminal work contributing to our early understanding of this feedforward system relied on in vitro preparations where the connectivity and endogenous activity of the neural circuit were not maintained28,30,31,32,33,34,35. This protocol describes an approach to preserving an intact neural circuit where endogenous feedback dynamics are maintained thus enabling better understanding of the corollary discharge in vivo.
The protocol outlined here describes how to monitor posterior lateral line afferent neuron and motor neuron activity simultaneously in larval zebrafish. Characterizing afferent signal dynamics before, during, and after motor commands provides insights into real-time, endogenous feedback from the central nervous system that modulates hair cell sensitivity during locomotion. This protocol outlines what materials will need to be prepared prior to experiments and then describes how to paralyze and prepare zebrafish larvae. The protocol will describe how to establish a stable loose patch recording of afferent neurons and extracellular ventral root (VR) recordings of motor neurons. Representative data that can be obtained using this protocol are presented from an exemplar individual and analysis was performed on multiple replicates of the experimental protocol. Pre-processing of data is performed using custom written scripts in MATLAB. Overall, this in vivo experimental paradigm is poised to provide a better understanding of sensory feedback during locomotion in a model vertebrate hair cell system.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mL beaker</td>
			<td>PYREX</td>
			<td>1000</td>
			<td>resceptacle for etchant</td>
		</tr>
		<tr>
			<td>10x water immersion objective</td>
			<td>Olympus</td>
			<td>UMPLFLN10xW</td>
			<td>low magnification for positioning larvae and recording electrode</td>
		</tr>
		<tr>
			<td>40x water immersion objective</td>
			<td>Olympus</td>
			<td>LUMPLFLN40XW</td>
			<td>higher magnification for position electrode tip and establishing patch-clamp</td>
		</tr>
		<tr>
			<td>abfload.m</td>
			<td></td>
			<td>supplemental coding file</td>
			<td>custom written MATLAB script for converting raw electrophysiology recordings to .mat files</td>
		</tr>
		<tr>
			<td>AffVR_preprocess.m</td>
			<td></td>
			<td>supplemental coding file</td>
			<td>custom written MATLAB script for preprocessing recording data</td>
		</tr>
		<tr>
			<td>BNC coaxial cables</td>
			<td>ThorLabs</td>
			<td>2249-C-12</td>
			<td>connecting amplifier and digitizer channels; require 4</td>
		</tr>
		<tr>
			<td>borosilicate glass capillaries w/ filament</td>
			<td>Warner Instruments</td>
			<td>G150F-3</td>
			<td>inner diameter: 0.86, outer diameter: 1.50; capillary glass used to form recording electrodes</td>
		</tr>
		<tr>
			<td>burst_detect</td>
			<td></td>
			<td>supplemental coding file</td>
			<td>custom written MATLAB function necessary to run AffVR_preprocess.m</td>
		</tr>
		<tr>
			<td>computer</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>any computer should work</td>
		</tr>
		<tr>
			<td>DC Power Supply</td>
			<td>Tenma</td>
			<td>72-420</td>
			<td>used for electrically etching dissection pins</td>
		</tr>
		<tr>
			<td>electrophysiology digitizer</td>
			<td>Axon Instruments, Molecular Devices</td>
			<td>Axon DigiData 1440A</td>
			<td>enables acquisition of patch-clamp data</td>
		</tr>
		<tr>
			<td>filament</td>
			<td>Sutter Instrument Company</td>
			<td>FB255B</td>
			<td>2.5 mm box filament used in micropipette puller</td>
		</tr>
		<tr>
			<td>fine forceps</td>
			<td>Fine Science Tools</td>
			<td>Dumont #5 (0.05 x 0.02 mm) Item No. 11295-10</td>
			<td>used to manipulate larvae and insert pins</td>
		</tr>
		<tr>
			<td>fixed stage DIC microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td>microscope used to visualize and establish patch-clamp recordings</td>
		</tr>
		<tr>
			<td>flexible, tapered pipette tip</td>
			<td>Fisher Scientific</td>
			<td>02-707-169</td>
			<td>flexible tips enable insertion into recording electrode to dispense extracellular solution at the tip</td>
		</tr>
		<tr>
			<td>FluoroDish</td>
			<td>World Precision Instruments Inc.</td>
			<td>FD3510-100</td>
			<td>cover glass bottomed dish recording dish</td>
		</tr>
		<tr>
			<td>KimWipe</td>
			<td>KimTech</td>
			<td>34155</td>
			<td>task wipe used for wicking away excess fluid from larvae</td>
		</tr>
		<tr>
			<td>Kwik-Gard</td>
			<td>World Precision Instruments Inc.</td>
			<td>710172</td>
			<td>self-mixing sylgard elastomer</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td>R2020b</td>
			<td>command line software for preprocessing data</td>
		</tr>
		<tr>
			<td>microelectrode amplifier</td>
			<td>Axon Instruments, Molecular Devices</td>
			<td>MultiClamp 700B</td>
			<td>patch clamp amplifier for dual channel recordings</td>
		</tr>
		<tr>
			<td>microforge</td>
			<td>Narishige</td>
			<td>MF-830 microforge</td>
			<td>to polish recording electrode</td>
		</tr>
		<tr>
			<td>micromanipulator control unit</td>
			<td>Siskiyou</td>
			<td>MC1000-eR/T</td>
			<td>4-axis dial coordinator for controlling micromanipulator</td>
		</tr>
		<tr>
			<td>micropipette puller</td>
			<td>Sutter Instrument Company</td>
			<td>Flaming/Brown P-97</td>
			<td>for pulling capillary glass into recording electrodes</td>
		</tr>
		<tr>
			<td>microscope control unit</td>
			<td>Siskiyou</td>
			<td>MC1000e</td>
			<td>positions the microscope around the fixed stage and preparation</td>
		</tr>
		<tr>
			<td>motorized micromanipulator</td>
			<td>Siskiyou</td>
			<td>MX7600</td>
			<td>positions the headstage and attached recording electrode for patch-clamp recording</td>
		</tr>
		<tr>
			<td>MultiClamp Commander</td>
			<td>Molecular Devices</td>
			<td>2.2.2</td>
			<td>downloadable from Axon MultiClamp 700B Commander download page</td>
		</tr>
		<tr>
			<td>optical air table</td>
			<td>Newport Corporation</td>
			<td>VH3036W-OPT</td>
			<td>breadboard isolation table to float microscope and minimize vibrations during recordings</td>
		</tr>
		<tr>
			<td>pCLAMP</td>
			<td>Molecular Devices</td>
			<td>10.7.0</td>
			<td>downloadable from Axon pCLAMP 10 Electrophysiology Data Acquisition &#38; Analysis Software Download page</td>
		</tr>
		<tr>
			<td>permanent ink marker</td>
			<td>Sharpie</td>
			<td>order from amazon.com</td>
			<td>for marking the leading edge side of the VR electrode to ensure proper orientation when inserting into pipette holder</td>
		</tr>
		<tr>
			<td>petri-dish</td>
			<td>Falcon</td>
			<td>35-3001</td>
			<td>used to immerse larvae in paralytic</td>
		</tr>
		<tr>
			<td>pipette holder</td>
			<td>Molecular Devices</td>
			<td>1-HL-U</td>
			<td>hold recording electrode and connect to the headstage</td>
		</tr>
		<tr>
			<td>pneumatic transducer</td>
			<td>Fluke Biomedical Instruments</td>
			<td>DPM1B</td>
			<td>for controlling recording electrode internal pressure</td>
		</tr>
		<tr>
			<td>potassium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>221473-25G</td>
			<td>etchant for etching dissection pins</td>
		</tr>
		<tr>
			<td>silicone tubing</td>
			<td>Tygon</td>
			<td>14-169-1A</td>
			<td>tubing to connect pneumatic transducer to pipette holder</td>
		</tr>
		<tr>
			<td>spike_detect</td>
			<td></td>
			<td>supplemental coding file</td>
			<td>custom written MATLAB function necessary to run AffVR_preprocess.m</td>
		</tr>
		<tr>
			<td>stereomicroscope</td>
			<td>Carl Zeiss</td>
			<td>Stemi 2000-C</td>
			<td>used to visualize pin tips and during preparation of larvae</td>
		</tr>
		<tr>
			<td>straight edge razor blade</td>
			<td>Canopus</td>
			<td>order from amazon.com</td>
			<td>cuts the tungsten wire while making dissection pins</td>
		</tr>
		<tr>
			<td>swimbout_detect</td>
			<td></td>
			<td>supplemental coding file</td>
			<td>custom written MATLAB function necessary to run AffVR_preprocess.m</td>
		</tr>
		<tr>
			<td>syringe</td>
			<td>Becton Dickinson Compoany</td>
			<td>309602</td>
			<td>filled with extracellular solution to inject into recording electrodes</td>
		</tr>
		<tr>
			<td>transfer pipette</td>
			<td>Sigma-Aldrich</td>
			<td>Z135003-500EA</td>
			<td>single use, non-sterile pipette for transfering larvae</td>
		</tr>
		<tr>
			<td>tricaine methanesulfonate</td>
			<td>Syndel</td>
			<td>12854</td>
			<td>pharmaceutical aneasthetic used to euthanize larvae with high dosage.</td>
		</tr>
		<tr>
			<td>tungsten wire</td>
			<td>World Precision Instruments Inc.</td>
			<td>715500</td>
			<td>0.002 inch, 50.8 &#956;m diameter; used to make dissection pins</td>
		</tr>
		<tr>
			<td>vacuum filtration unit</td>
			<td>Sigma-Aldrich</td>
			<td>SCGVU11RE</td>
			<td>single use, sterile, vacuum filtration units used to sterilize extracellular solution used for electrophysiology electrode ringer</td>
		</tr>
		<tr>
			<td>voltage-clamp current-clamp headstage</td>
			<td>Molecular Devices</td>
			<td>CV-7B</td>
			<td>supplied with MultiClamp 700B amplifier used as left and right headstages</td>
		</tr>
		<tr>
			<td>&#945;-bungarotoxin</td>
			<td>ThermoFisher</td>
			<td>B1601</td>
			<td>for immobilizing the larvae prior to recording</td>
		</tr>
	</tbody>
</table>

activity of posterior lateral line afferent neurons during swimming in zebrafish

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates reafferent cues that animals must disentangle from relevant sensory cues of the surrounding environment. For example, when a fish swims, flow generated from body undulations is detected by the mechanoreceptive neuromasts, comprising hair cells, that compose the lateral line system. The hair cells then transmit fluid motion information from the sensor to the brain via the sensory afferent neurons. Concurrently, corollary discharge of the motor command is relayed to hair cells to prevent sensory overload. Accounting for the inhibitory effect of predictive motor signals during locomotion is, therefore, critical when evaluating the sensitivity of the lateral line system. We have developed an in vivo electrophysiological approach to simultaneously monitor posterior lateral line afferent neuron and ventral motor root activity in zebrafish larvae (4-7 days post fertilization) that can last for several hours. Extracellular recordings of afferent neurons are achieved using the loose patch clamp technique, which can detect activity from single or multiple neurons. Ventral root recordings are performed through the skin with glass electrodes to detect motor neuron activity. Our experimental protocol provides the potential to monitor endogenous or evoked changes in sensory input across motor behaviors in an intact, behaving vertebrate.

After zebrafish larvae are properly immobilized and the posterior lateral line afferent ganglion and VR recording is achieved, activity in both afferent and motor neurons can be measured simultaneously. Recording channels are displayed using gap-free recording protocols (step 1.4) for continuous monitoring of afferent and VR activity. In real-time, decreases in spontaneous afferent spike rate can be observed concurrent with VR activity indicative of fictive swim bouts (Figure 1E). We found t...

Watch this Scientific Journal Video about Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish at JoVE.com

Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish

We describe a protocol to monitor changes in the afferent neuron activity during motor commands in a model vertebrate hair cell system.

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates ...

activity-posterior-lateral-line-afferent-neurons-during-swimming

Research

JoVE Journal

Neuroscience

3.7K Views.  University of Florida, The Whitney Laboratory for Marine Bioscience. We describe a protocol to monitor changes in the afferent neuron activity during motor commands in a model vertebrate hair cell system.

Video: Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

Larval zebrafish are emerging as a model for describing the development and function of simple neural circuits. Due to their external fertilization, rapid development, and translucency, zebrafish are particularly well suited for optogenetic approaches to investigate neural circuit function. In this approach, light-sensitive ion channels are expressed in specific neurons, enabling the experimenter to activate or inhibit them at will and thus assess their contribution to specific behaviors. Applying these methods in larval zebrafish is conceptually simple but requires the optimization of technical details. Here we demonstrate a procedure for expressing a channelrhodopsin variant in larval zebrafish somatosensory neurons, photo-activating single cells, and recording the resulting behaviors. By introducing a few modifications to previously established methods, this approach could be used to elicit behavioral responses from single neurons activated up to at least 4 days post-fertilization (dpf). Specifically, we created a transgene using a somatosensory neuron enhancer, CREST3, to drive the expression of the tagged channelrhodopsin variant, ChEF-tdTomato. Injecting this transgene into 1-cell stage embryos results in mosaic expression in somatosensory neurons, which can be imaged with confocal microscopy. Illuminating identified cells in these animals with light from a 473 nm DPSS laser, guided through a fiber optic cable, elicits behaviors that can be recorded with a high-speed video camera and analyzed quantitatively. This technique could be adapted to study behaviors elicited by activating any zebrafish neuron. Combining this approach with genetic or pharmacological perturbations will be a powerful way to investigate circuit formation and function.

Optogenetic techniques have made it possible to study the contribution of specific neurons to behavior. We describe a method in larval zebrafish for activating single somatosensory neurons expressing a channelrhodopsin variant (ChEF) with a diode-pumped solid state (DPSS) laser and recording the elicited behaviors with a high-speed video camera.

Larval  zebrafish are emerging as a model for describing the development and function  of simple neural circuits. Due to their  external fertilization, ...

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express1 . We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B and C). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons2, 3 .The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property4-6, the frequency of which is modulated by visual cues from conspecifics2 and the neuropeptide kisspeptin 15. In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein7 to show you how to identify neurons and monitor their electrical activity in the whole brain preparation6.

In this video, we will demonstrate how to record electrical activity from identified single neurons in a whole brain preparation, which preserves complex neural circuits. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with a fluorescent protein for identification in the intact brain preparation.

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be ...

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

A Large Lateral Craniotomy Procedure for Mesoscale Wide-field Optical Imaging of Brain Activity

The craniotomy is a commonly performed procedure to expose the brain for in vivo experiments. In mouse research, most labs utilize a small craniotomy, typically 3 mm x 3 mm. This protocol introduces a method for creating a substantially larger 7 mm x 6 mm cranial window exposing most of a cerebral hemisphere over the mouse temporal and parietal cortices (e.g., bregma 2.5 - 4.5 mm, lateral 0 - 6 mm). To perform this surgery, the head must be tilted approximately 30&#176; and much of the temporal muscle must be retracted. Due to the large amount of bone removal, this procedure is intended only for acute experiments with the animal anesthetized throughout the surgery and experiment.
The main advantage of this innovative large lateral cranial window is to provide simultaneous access to both medial and lateral areas of the cortex. This large unilateral cranial window can be used to study the neural dynamics between cells, as well as between different cortical areas by combining multi-electrode electrophysiological recordings, imaging of neuronal activity (e.g., intrinsic or extrinsic imaging), and optogenetic stimulation. Additionally, this large craniotomy also exposes a large area of cortical blood vessels, allowing for direct manipulation of the lateral cortical vasculature.

This protocol presents a method for creating a large unilateral craniotomy over the temporal and parietal regions of the mouse cerebral cortex. This is especially useful for real time imaging over an expansive area of a cortical hemisphere.

The craniotomy is a commonly performed procedure to expose the brain for in vivo experiments. In mouse research, most labs utilize a small craniotomy, ...

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the different organ systems from the periphery towards the central nervous system. As such, the increased activity or &#39;sensitization&#39; of mesenteric afferent nerves has been allocated an important role in the pathophysiology of visceral hypersensitivity and abdominal pain syndromes.
Mesenteric afferent nerve activity can be measured in vitro in an isolated intestinal segment that is mounted in a purpose-built organ bath and from which the splanchnic nerve is isolated, allowing researchers to directly assess nerve activity adjacent to the gastrointestinal segment. Activity can be recorded at baseline in standardized conditions, during distension of the segment or following the addition of pharmacological compounds delivered intraluminally or serosally. This technique allows the researcher to easily study the effect of drugs targeting the peripheral nervous system in control specimens; besides, it provides crucial information on how neuronal activity is altered during disease. It should be noted however that measuring afferent neuronal firing activity only constitutes one relay station in the complex neuronal signaling cascade, and researchers should bear in mind not to overlook neuronal activity at other levels (e.g., dorsal root ganglia, spinal cord or central nervous system) in order to fully elucidate the complex neuronal physiology in health and disease.
Commonly used applications include the study of neuronal activity in response to the administration of lipopolysaccharide, and the study of afferent nerve activity in animal models of irritable bowel syndrome. In a more translational approach, the isolated mouse intestinal segment can be exposed to colonic supernatants from IBS patients. Furthermore, a modification of this technique has been recently shown to be applicable in human colonic specimens.

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Mesenteric afferent nerves convey information from the gastrointestinal tract towards the brain regarding normal homeostasis as well as pathophysiology. Gastrointestinal afferent nerve activity can be assessed by mounting isolated intestinal segments with attached afferent nerves into an organ bath, isolating the nerve, and assessing basal as well as stimulated activity.

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the ...

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory bowel diseases and diabetes. Here, we describe a protocol for the in vitro characterization of the electrophysiological properties of colonic afferents in rats. The colorectum, with the intact pelvic ganglion (PG) attached, is removed from the rat; superfused with carbogenated Krebs solution in the recording chamber; and cannulated at the oral and anal ends to allow for distension. A fine nerve bundle emanating from the PG is identified, and the multiunit afferent nerve activity is recorded using a suction electrode. Distension of the colonic segment elicits gradual increases in multiunit discharge. A principal component analysis is conducted to differentiate the low-threshold, the high-threshold, and the wide-dynamic range afferent fibers. Chemical sensitivity of colonic afferents can be studied through the bath or intraluminal administration of test compounds. This protocol can be modified for application to other species, such as mice and guinea pigs, and to study the differences in the electrophysiological properties of thoracolumbar/hypogastric and lumbosacral/pelvic afferents of the descending colon in normal and pathological conditions.

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Abnormal sensory function underlies visceral pain and other symptoms of functional and inflammatory bowel diseases. A protocol for the electrophysiological recording of the colonic afferent nerves in an ex vivo rat colorectum preparation is presented here.

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...