Name: simple surgical induction of conductive hearing loss with verification using otoscope visualization and behavioral clap startle response in rat
Uploaded: 2019-10-26T12:30:00
Description: Watch this Scientific Journal Video about Simple Surgical Induction of Conductive Hearing Loss with Verification Using Otoscope Visualization and Behavioral Clap Startle Response in Rat at JoVE.com

This work was supported in part by the Hong Kong Research Grants Council, Early Career Scheme, Project #21201217 to C. L., for the project Brain mapping guided electrophysiology with applications in hearing and noise pollution research. We thank the Posgrado en Ciencias Biom&#233;dicas, the Instituto de Neurobiolog&#237;a of the Universidad Nacional Aut&#243;noma de M&#233;xico (UNAM), the Consejo Nacional de Ciencia y Tecnolog&#237;a (CONACyT) M&#233;xico for the Graduate Fellowship 578458 to FAM Manno.

The present study and procedures were approved by the animal research ethics committees of the City University of Hong Kong, the University of Hong Kong, and the Department of Health of the Hong Kong Special Administrative Region.
1. Animals
<ol>
	<li>Use Sprague-Dawley (SD) rats of two months (N = 90, 200-250 g). 
	NOTE: Rodents were provided by the accredited Laboratory Animal Unit of the University of Hong Kong.</li>
	<li>Maintain the rats under a constant 25 &#176;C temperature and ...

<ol>
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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+period+for+the+impact+of+hearing+loss+on+auditory+perception.">A sensitive period for the impact of hearing loss on auditory perception.</a> The Journal of Neuroscience. 34 (6), 2276-2284 (2014).</li><li>Finck, A., Sofouglu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+sensitivity+of+the+Mongolian+gerbil.">Auditory sensitivity of the Mongolian gerbil.</a> The Journal of Auditory Research. 6, 313-319 (1966).</li><li>Finck, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+sensitivity+of+the+Mongolian+Gerbil+(Merionesunguiculatus).">Auditory sensitivity of the Mongolian Gerbil (Merionesunguiculatus).</a> The Journal of the Acoustical Society of America. 41 (60), 1579 (1967).</li><li>Finck, A., Schneck, C. D., Hartman, A. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+conductive+hearing+loss+leads+to+cochlear+degeneration.">Chronic conductive hearing loss leads to cochlear degeneration.</a> PLoS One. 10 (11), e0142341 (2015).</li><li>Tucci, D. L., Cant, N. B., Durham, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conductive+hearing+loss+results+in+a+decrease+in+central+auditory+system+activity+in+the+young+gerbil.">Conductive hearing loss results in a decrease in central auditory system activity in the young gerbil.</a> Laryngoscope. 109, 1359-1371 (1999).</li><li>Tucci, D. L., Cant, N. B., Durham, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+conductive+hearing+loss+on+gerbil+central+auditory+system+activity+in+silence.">Effects of conductive hearing loss on gerbil central auditory system activity in silence.</a> Hearing Research. 155, 124-132 (2001).</li><li>Cook, R. D., Hung, T. Y., Miller, R. L., Smith, D. W., Tucci, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+conductive+hearing+loss+on+auditory+nerve+activity+in+gerbil.">Effects of conductive hearing loss on auditory nerve activity in gerbil.</a> Hearing Research. 164, 127-137 (2002).</li><li>Albuquerque, A. A., Rossato, M., Oliveira, J. A., Hyppolito, M. 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F., Li, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+anatomy+of+the+rat+middle+ear.">Surgical anatomy of the rat middle ear.</a> Otolaryngology-Head and Neck Surgery. 117, 438-447 (1997).</li><li>Hellstr&#246;m, S., Sal&#233;n, B., Stenfors, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+rat+middle+ear.+A+study+under+the+dissection+microscope.">Anatomy of the rat middle ear. A study under the dissection microscope.</a> Acta Anatomica (Basel). 112, 346-352 (1982).</li><li>Albiin, N., Hellstr&#246;m, S., Sal&#233;n, B., Stenfors, L. E., Wirell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vascular+supply+of+the+rat+tympanic+membrane.">The vascular supply of the rat tympanic membrane.</a> The Anatomical Record. 212, 17-22 (1985).</li><li>Li, P., Ding, D., Gao, K., Salvi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+surgical+approaches+to+ear+surgery+in+rats.">Standardized surgical approaches to ear surgery in rats.</a> Journal of Otology. 10, 72-77 (2015).</li><li>Johansson, U., Hellstr&#246;m, S., Anniko, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Round+window+membrane+in+serous+and+purulent+otitis+media.+Structural+study+in+the+rat.">Round window membrane in serous and purulent otitis media. 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A study on rat middle ear cavity.</a> Acta Oto-Laryngologica. 93, 435-440 (1982).</li><li>Shen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolds+for+tympanic+membrane+regeneration+in+rats.">Scaffolds for tympanic membrane regeneration in rats.</a> Tissue Engineering Part A. 19, 657-668 (2013).</li><li>Wang, A. 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We describe a simple surgical induction of CHL with verification using otoscope visualization and behavioral clap startle response in rat. Here we demonstrate the method on rat and previously this method has been applied to gerbils and mice. The method can easily be adopted to other rodents. Induction of CHL allows the study of a subtle form of hearing loss which manifests in auditory cortical alterations and psychophysical behavioral findings4,5,<sup ...

The prevalence of the hearing loss in children and adults is approximately 19.5%1 and 15.2%2 respectively. However, approximately 39.3% of all newborns with an abnormal hearing screening do not receive remedial treatment as reported by the Centers for Disease Control3. Hearing loss is a widely studied condition, and the rodent is a robust model to study normal hearing and hearing related disorders4,5,6,7,8,9,10,11,12,13,14,15. Hearing disorders such as conductive hearing loss (CHL) lead to an increase in the short term synaptic depression in the auditory cortex4, which results in shallower psychometric slopes associated with frequency modulation detection thresholds5. Conductive hearing loss models by surgical removal/displacement of the malleus, tympanic membrane (TM) puncture or earplug are easily employed and allow the rapid induction of the hearing loss model5,14,15,16,17,18. The goal of the present protocol and method is to demonstrate a simple and reproducible CHL model in rodents.
The present protocol is inexpensive (&#8776;USD$300 with all tools), and readily amendable to different research pursuits. The rat has had detailed assessments of middle ear anatomy19,20,21,22,23, surgical approaches24, models in otitis media25,26,27 and TM puncture regeneration16,17,18,28,29,30, making it an ideal model to study hearing loss. Here, a simple CHL induction procedure is described with verification by otoscope and behavioral assessment with clap startle response in rat, which then may be used to explore additional sequelae&#160;of hearing loss. The CHL procedure is induced by surgical puncture of the TM. Verification of the CHL procedure is performed by otoscope visualization to determine absence of the TM. Behavioral assessment is performed by a high decibel (dB) sound pressure level (SPL) hand-clap. This method has been applied previously in a variety of rodents.&#160;It is easy to replicate, produces robust psychometric differences and changes in neural physiological responses4,5,16,17,18.

<table border="0" cellpadding="0" cellspacing="0" style="width:612px;" width="610">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Latex, polyvinyl or nitrile gloves</td>
			<td style="width:95px;">AMMEX</td>
			<td style="width:123px;"></td>
			<td style="width:172px;">Use unpowdered gloves 8-mil</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Micro spring scissors (see Fig. 1b)</td>
			<td style="width:95px;">RWD Life Science</td>
			<td style="width:123px;">S11035-08</td>
			<td>8.0 cm total length, with 3.5mm cutting edge, or similar micro forceps. Standard tweezers with spring action will suffice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Otoscope mini 3000</td>
			<td style="width:95px;">HEINE&#160;</td>
			<td style="width:123px;">D-008.70.120M</td>
			<td>Standard LED otoscope will suffice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Rat or mouse</td>
			<td style="width:95px;">JAX labs</td>
			<td style="width:123px;"></td>
			<td>Any small rodent&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Small rodent cage</td>
			<td style="width:95px;">Tecniplast</td>
			<td style="width:123px;">1284L</td>
			<td>Need two cages to separate CHL rodent from hearing rodent. If rodent are in direct contact with one-another, they will startle each other. Cage dimensions 365 x 207 x 140 mm, floor area: 530 cm2/82.15 in2</td>
		</tr>
	</tbody>
</table>

simple surgical induction of conductive hearing loss with verification using otoscope visualization and behavioral clap startle response in rat

Conductive hearing loss (CHL) is a prevalent hearing impairment in humans. The goal of the protocol is to describe a simple surgical procedure for inducing CHL in rodents. The protocol demonstrates CHL by tympanic membrane puncture. Verification of CHL surgery was by otoscope examination and behavioral assessment by clap startle response, both replicable and reliable, and are simple methods to demonstrate hearing loss has occurred. The simple CHL procedure is advantageous due to its reproducibility and flexibility to different pursuits in hearing loss research. The limitations of inducing CHL by a surgical approach are associated with the learning curve to perform the surgical procedure and confidence in audiological examination. Inducing a hearing impairment by CHL allows one to readily study the neural manifestations and behavioral outcomes of hearing loss.

The simple CHL procedure was performed on 90 rats and out of this group 2 had significant bleeding and 2 did not have hearing loss the next day as assessed by behavioral clap startle. These four rats were discarded. Rats should be discarded as described by the reasons in the discussion due to complications. Inducing TM puncture and/or malleus displacement/removal (Figure 2B) elicits CHL, which results in behavioral manifestations (i.e. CHL - no response to lo...

Watch this Scientific Journal Video about Simple Surgical Induction of Conductive Hearing Loss with Verification Using Otoscope Visualization and Behavioral Clap Startle Response in Rat at JoVE.com

Simple Surgical Induction of Conductive Hearing Loss with Verification Using Otoscope Visualization and Behavioral Clap Startle Response in Rat

Here, we present a protocol to establish a replicable conductive hearing loss induction via surgical tympanic membrane puncture and verification by otoscope visualization and behavioral assessment by clap startle.

Conductive hearing loss (CHL) is a prevalent hearing impairment in humans. The goal of the protocol is to describe a simple surgical procedure for ...

The overall goal of this study is to establish a reliable hearing loss induction via surgical tympanic membrane puncture and verification by otoscope visualization and behavioral assessment by clap startle. The purpose of our practical is to develop a model for human hearing conductive disorders. We demonstrate in the practical, the conductive hearing loss surgical procedure, which is a simplification in the malleus removal, the method to verify conductive hearing loss by otoscope visualization, and a simple behavioral clap startle, which validate that conductive hearing loss has occurred.The main advantage of the procedure we described are non-invasive procedures, verification of conductive hearing loss by otoscope, and validation by clap startle. The present study and procedures were approved by the animal research ethics committee and the City University of Hong Kong, the University of Hong Kong, and the Department of Health of the Hong Kong Special Administrative Region. For the present protocol, we choose rats, but you may choose any rodent.Weigh animals to ensure proper anesthesia for IP injection. For anesthesia, it is important that animals have the proper dose. Pick up your rat by the scruff.Ensure your correct dose of ketamine and xylazine for IP injection, and inject. Make sure to perform toe pinch to verify no pain sensation. For surgical setup consists of your anesthetized rat, the microscissors, and your otoscope.Please thoroughly clean the surgical area with ethanol. Place the surgical drape on the cleaned bench. Subsequently introduce your scissors, otoscope, and rat.The size of your scissors will depend on the size of your rodent. Under otoscope, visualize the left and right ear of the rat to ensure a healthy tympanic membrane. Grab the helix and extend the external auditory canal to become obscured and blackened.Here you will introduce the microscissors. Introduce the microscissors to the center of the auditory canal, paying close attention not to nick the tissue. Listen for the pop sound, then twist to ensure displacement of the malleus.The confirmation of a successful conductive hearing loss surgery, should be made with an otoscope. Evaluate each rat ear before and after surgery with an otoscope with a small diameter speculum. When visualized with an otoscope, the differences between a normal tympanic membrane and a punctured tympanic membrane should be studied.Here, the normal tympanic membrane on the left has the head of the malleus in place and is drastically different than the punctured tympanic membrane on the right. The punctured tympanic membrane, after the conductive hearing loss surgery, has the malleus displaced and the pars tensa ruptured with the umbo of the malleus and head completely missing. Postoperative care for rodents.Place the rodent in the home cage under a warm lamp and inject with antibacterial agents and glucose. Corroborate connective hearing loss with behavioral clap startle. The CHL rat is placed next to a normal rat in two adjoining cages.These are placed in a silent room. Stand half a meter away and clap in equally spaced durations a specific number of times. Here, we choose five.Here, our representative behavioral results. Note the rat at the bottom does not jump to the loud clap sound. The rat at the top of the screen is startled by these loud clapping sounds.Please study the before and after the clap difference for these two rats. Failure to induce conductive hearing loss. A failure to induce conductive hearing loss could be caused by bleeding in one or both ears.And in this case, the rodent should be euthanized. This would indicate that the scissors did not make contact with the tympanic membrane. A no popping sound during the procedure.A no popping sound would indicate that the conductive hearing loss procedure was poor and not clean. A no popping sound would mean that your microscissors did not make direct contact with the tympanic membrane. Otoscope visualization.If, when visualizing by otoscope, the tympanic membrane appears normal, this would indicate that no CHL has occurred. A response to the loud clap sound test. This would indicate that no CHL has occurred and the rodent does not have conductive hearing loss.This would occur when a rat jumps or is startled by a loud clap sound. The main advantage of the procedure we described, are non-invasive procedures, verification of conductive hearing loss by otoscope, and validation by clap startle. After doing the practical, you can induce conductive hearing loss in a rodent model and explore the behavioral and psychophysical manifestations of this hearing disorder.

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Research

JoVE Journal

Neuroscience

Habituation and Prepulse Inhibition of Acoustic Startle in Rodents

The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated within a few milliseconds, leading to a whole body flinch in rodents1. Although startle responses are reflexive responses that can be reliably elicited, they are not stereotypic. They can be modulated by emotions such as fear (fear potentiated startle) and joy (joy attenuated startle), by non-associative learning processes such as habituation and sensitization, and by other sensory stimuli through sensory gating processes (prepulse inhibition), turning startle responses into an excellent tool for assessing emotions, learning, and sensory gating, for review see 2, 3. The primary pathway mediating startle responses is very short and well described, qualifying startle also as an excellent model for studying the underlying mechanisms for behavioural plasticity on a cellular/molecular level3.
We here describe a method for assessing short-term habituation, long-term habituation and prepulse inhibition of acoustic startle responses in rodents. Habituation describes the decrease of the startle response magnitude upon repeated presentation of the same stimulus. Habituation within a testing session is called short-term habituation (STH) and is reversible upon a period of several minutes without stimulation. Habituation between testing sessions is called long-term habituation (LTH)4. Habituation is stimulus specific5. Prepulse inhibition is the attenuation of a startle response by a preceding non-startling sensory stimulus6. The interval between prepulse and startle stimulus can vary from 6 to up to 2000 ms. The prepulse can be any modality, however, acoustic prepulses are the most commonly used.
Habituation is a form of non-associative learning. It can also be viewed as a form of sensory filtering, since it reduces the organisms' response to a non-threatening stimulus. Prepulse inhibition (PPI) was originally developed in human neuropsychiatric research as an operational measure for sensory gating7. PPI deficits may represent the interface of "psychosis and cognition" as they seem to predict cognitive impairment8-10. Both habituation and PPI are disrupted in patients suffering from schizophrenia11, and PPI disruptions have shown to be, at least in some cases, amenable to treatment with mostly atypical antipsychotics12, 13. However, other mental and neurodegenerative diseases are also accompanied by disruption in habituation and/or PPI, such as autism spectrum disorders (slower habituation), obsessive compulsive disorder, Tourette's syndrome, Huntington's disease, Parkinson's disease, and Alzheimer's Disease (PPI)11, 14, 15 Dopamine induced PPI deficits are a commonly used animal model for the screening of antipsychotic drugs16, but PPI deficits can also be induced by many other psychomimetic drugs, environmental modifications and surgical procedures.

Habituation and prepulse inhibition of startle are operational measures of sensory gating. Sensory gating is disrupted in schizophrenia, and some other mental disorders and neurodegenerative diseases. We here describe a standard protocol to assess short-term and long-term habituation as well as prepulse inhibition of acoustic startle responses in rats and mice.

The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium carbonate. Animal studies as well as human genetic studies indicate that lithium affects molecular targets that are involved in neuronal growth, survival and maturation, and notably molecules involved in Wnt signaling. Given the ethical challenge to obtaining brain biopsies for investigating dynamic molecular changes associated with lithium-response in the central nervous system (CNS), one may consider the use of neurons obtained from olfactory tissues to achieve this goal.The olfactory epithelium contains olfactory receptor neurons at different stages of development and glial-like supporting cells. This provides a unique opportunity to study dynamic changes in the CNS of patients with neuropsychiatric diseases, using olfactory tissue safely obtained from nasal biopsies. To overcome the drawback posed by substantial contamination of biopsied olfactory tissue with non-neuronal cells, a novel approach to obtain enriched neuronal cell populations was developed by combining nasal biopsies with laser-capture microdissection. In this study, a system for investigating treatment-associated dynamic molecular changes in neuronal tissue was developed and validated, using a small pilot sample of BD patients recruited for the study of the molecular mechanisms of lithium treatment response.

In this study, a novel platform to investigate intraneuronal molecular signatures of treatment response in bipolar disorder (BD) was developed and validated. Olfactory epithelium from BD patients was obtained through nasal biopsies. Then laser-capture microdissection was combined with Real Time RT PCR to investigate the molecular signature of lithium response in BD.

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

Cochlear Implant Surgery and Electrically-evoked Auditory Brainstem Response Recordings in C57BL/6 Mice

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of hearing. Improvement of the implant technology is needed if CI users are to perceive music and perform in more natural environments, such as hearing out a voice with competing talkers, reflections, and other sounds. Such improvement requires experimental animals to better understand the mechanisms of electric stimulation in the cochlea and its responses in the whole auditory system. The mouse is an increasingly attractive model due to the many genetic models available. However, the limited use of this species as a CI model is mainly due to the difficulty of implanting small electrode arrays. More details about the surgical procedure are therefore of great interest to expand the use of mice in CI research.
In this report, we describe in detail the protocol for acute deafening and cochlear implantation of an electrode array in the C57BL/6 mouse strain. We demonstrate the functional efficacy of this procedure with electrically-evoked auditory brainstem response (eABR) and show examples of facial nerve stimulation. Finally, we also discuss the importance of including a deafening procedure when using a normally hearing animal. This mouse model provides a powerful opportunity to study genetic and neurobiological mechanisms that would be of relevance for CI users.

Animal models of cochlear implants can advance knowledge of the technological bases of treating permanent sensorineural hearing loss with electrical stimulation. This study presents a surgical protocol for acute deafening and cochlear implantation of an electrode array in mice as well as the functional assessment with auditory brainstem response.

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of ...

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

Long-range Channelrhodopsin-assisted Circuit Mapping of Inferior Colliculus Neurons with Blue and Red-shifted Channelrhodopsins

When investigating neural circuits, a standard limitation of the in vitro patch clamp approach is that axons from multiple sources are often intermixed, making it difficult to isolate inputs from individual sources with electrical stimulation. However, by using channelrhodopsin assisted circuit mapping (CRACM), this limitation can now be overcome. Here, we report a method to use CRACM to map ascending inputs from lower auditory brainstem nuclei and commissural inputs to an identified class of neurons in the inferior colliculus (IC), the midbrain nucleus of the auditory system. In the IC, local, commissural, ascending, and descending axons are heavily intertwined and therefore indistinguishable with electrical stimulation. By injecting a viral construct to drive expression of a channelrhodopsin in a presynaptic nucleus, followed by patch clamp recording to characterize the presence and physiology of channelrhodopsin-expressing synaptic inputs, projections from a specific source to a specific population of IC neurons can be mapped with cell type-specific accuracy. We show that this approach works with both Chronos, a blue light-activated channelrhodopsin, and ChrimsonR, a red-shifted channelrhodopsin. In contrast to previous reports from the forebrain, we find that ChrimsonR is robustly trafficked down the axons of dorsal cochlear nucleus principal neurons, indicating that ChrimsonR may be a useful tool for CRACM experiments in the brainstem. The protocol presented here includes detailed descriptions of the intracranial virus injection surgery, including stereotaxic coordinates for targeting injections to the dorsal cochlear nucleus and IC of mice, and how to combine whole cell patch clamp recording with channelrhodopsin activation to investigate long-range projections to IC neurons. Although this protocol is tailored to characterizing auditory inputs to the IC, it can be easily adapted to investigate other long-range projections in the auditory brainstem and beyond.

Channelrhodopsin-assisted circuit mapping (CRACM) is a precision technique for functional mapping of long-range neuronal projections between anatomically and/or genetically identified groups of neurons. Here, we describe how to utilize CRACM to map auditory brainstem connections, including the use of a red-shifted opsin, ChrimsonR.

When investigating neural circuits, a standard limitation of the in vitro patch clamp approach is that axons from multiple sources are often intermixed, ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...