A case report of refractory otalgia after Ramsay Hunt syndrome successfully treated by applying pulsed radiofrequency to the great auricular nerve: A CARE-compliant article.

RATIONALE: Ramsay Hunt syndrome is a type of herpes zoster infection involving geniculate ganglion and facial nerve. Unilateral facial palsy, otalgia, and painful vesicular rash on the auricle and external auditory canal are the typical symptoms. Although postherpetic neuralgia (PHN) is a devastating complication of herpes zoster infection, PHN following Ramsay Hunt syndrome has rarely been reported. PATIENT CONCERNS: A 55-year-old immunocompetent female patient visited our pain clinic, for left-sided refractory otalgia (PHN) that persisted for 3 months after she was diagnosed with Ramsay Hunt syndrome. Although facial palsy and tinnitus had recovered within 2 to 4 weeks after symptom onset, the patient had been experiencing a persistent and severe otalgia radiating to mandibular angle, temporal and upper cervical area of neuropathic nature. DIAGNOSES: The patient's pain persisted despite conservative medication and administration of ultrasound-guided stellate ganglion block, facial nerve block, and great auricular nerve block several times. INTERVENTIONS: The patient was treated with the application of ultrasound-guided pulsed radiofrequency (PRF) to the great auricular nerve. OUTCOMES: The patient experienced significant pain reduction more than 50% on a numeric rating scale after 2 weeks of PRF treatment. LESSONS: Chronic otalgia might be a type of PHN after Ramsay Hunt syndrome with cervical nerve involvement. PRF treatment to the great auricular nerve can be a therapeutic option for refractory otalgia following Ramsay Hunt syndrome.

The Effect of Auricular Acupressure for Chronic Low Back Pain in Elders: A Randomized Controlled Study.

Chronic low back pain (CLBP) is a major problem throughout the world and getting worse because of population increase and aging. The cost of treatment increases as the population of people with CLBP increases. This study aimed to examine the effect of auricular acupressure (AA) on pain and disability in elders with chronic low back pain. The randomized, single-blinded, and placebo-controlled study was conducted on 51 elders with CLBP in South Korea from June 2019 to August 2019. The experimental group (n = 26) received AA on low back pain-related points, whereas the placebo control group (n = 25) received AA on points unrelated to low back pain. Participants received 6 weeks of AA in weekly cycles. Collected data were analyzed using IBM SPSS Statistics, version 25. Statistically significant differences between the 2 groups emerged in the visual analog scale (P < .001), pain threshold (P < .001), and Oswestry Disability Index (P < .001). This study showed that 6 weeks of AA improved CLBP and pain-related disability. Therefore, AA can be used as a noninvasive and self-managed alternative intervention for CLBP in older adults.

Vagus nerve stimulation boosts the drive to work for rewards.

Interoceptive feedback transmitted via the vagus nerve plays a vital role in motivation by tuning actions according to physiological needs. Whereas vagus nerve stimulation (VNS) reinforces actions in animals, motivational effects elicited by VNS in humans are still largely elusive. Here, we applied non-invasive transcutaneous auricular VNS (taVNS) on the left or right ear while participants exerted effort to earn rewards using a randomized cross-over design (vs. sham). In line with preclinical studies, acute taVNS enhances invigoration of effort, and stimulation on the left side primarily facilitates invigoration for food rewards. In contrast, we do not find conclusive evidence that acute taVNS affects effort maintenance or wanting ratings. Collectively, our results suggest that taVNS enhances reward-seeking by boosting invigoration, not effort maintenance and that the stimulation side affects generalization beyond food reward. Thus, taVNS may enhance the pursuit of prospective rewards which may pave avenues to treat motivational deficiencies.

Preliminary Effectiveness of Auricular Point Acupressure on Chemotherapy-Induced Neuropathy: Part 2 Laboratory-Assessed and Objective Outcomes.

PURPOSE: To manage chemotherapy-induced neuropathy (CIN), this paper explores reliable and valid objectives measures to evaluate the treatment effects of auricular point acupressure (APA). DESIGN/METHOD: This study was a repeated-measures one-group design. Participants received four weeks of APA to manage their CIN. The laboratory-assessed and objective outcomes included quantitative sensory testing, grip and pinch strength, and inflammatory biomarkers. Wilcoxon matched pairs signed-rank tests were conducted to determine change scores of outcomes at pre- vs. post- and pre- vs. 1-month follow-up. Spearman's rho correlation coefficient was used to examine the linear association of score changes of all objective study outcomes. RESULTS: Comparing pre-and-post APA, (1) the mean score of the monofilament for all lower extremity sites tested decreased after APA, indicating sensory improvement; (2) the suprathreshold pinprick stimuli mean scores on the upper extremities increased, except the scores from the index finger and thumb; (3) the pain tolerance of thumb and trapezius areas increased; (4) decreasing IL1ß (p = .05), IFNγ (p = .02), IL-2 (p = .03), IL-6 (p = .05), IL-10 (p = .05), and IP10/CXCL10 (p = .04) were observed pre-post APA. Conditional pain modulation was significantly (p< .05) associated with pain intensity (r = 0.55), tingling (r = 0.59); and IL1ß concentration (r = 0.53) pre-post APA. The sustained effects of 4-week APA were observed at the 1-month follow-up. CONCLUSIONS: Our study findings demonstrated the promising effectiveness of APA in the management of CIN, and these treatment effects can be assessed using reliable and valid objective measures. CLINICAL IMPLICATIONS: If the efficacy of APA to manage CIN is confirmed in a larger sample, APA has the potential to be a scalable treatment for CIN because it is a reproducible, standardized, and easy-to-perform intervention.

Development of a training paradigm for voluntary control of the peri-auricular muscles: a feasibility study.

BACKGROUND: Spinal cord injury (SCI) can lead to severe and permanent functional deficits. In humans, peri-auricular muscles (PAMs) do not serve any physiological function, though their innervation is preserved in even high level SCI. Auricular control systems provide a good example of leveraging contemporary technologies (e.g., sEMG controlled computer games) to enable those with disabilities. Our primary objective is to develop and test the effectiveness of an auricular muscle training protocol to facilitate isolated and coordinated, bilateral voluntary control that could be used in individuals without volitional control of the vestigial PAMs. METHODS: Seventeen non-disabled persons were screened; 13 were eligible and 10 completed the entire protocol. The facilitation phase, included one session of sub-motor threshold, sensory electrical stimulation followed by neuromuscular electrical stimulation paired with ear movement feedback for up to 8 additional sessions. Participants progressed to the skill acquisition phase where they dawned an auricular control device that used sEMG signals to control movements of a cursor through three levels of computer games, each requiring increasingly more complex PAM coordination. RESULTS: The 10 who completed the protocol, finished the facilitation phase in 3 to 9 sessions and achieved some level of voluntary auricle movement that ranged between 1 and 5 mm. Qualitative analysis of longitudinal post-session auricular movement, revealed two subgroups of learners. Six successfully completed all 3 games-the "Learners". Two were partially successful in game completion and two were unable to complete a single game--"Poor/Non-Learners". Quantitative analysis revealed a significant group difference in auricular amplitude for both facilitation and skill phases (p < .05), and a significant relationship between performance in the two phases (R2 = 0.84, p = 0.004). CONCLUSION: Sixty percent of those who completed the facilitation phase were able to learn and demonstrate functional voluntary control of the vestigial PAMs. Those who progressed the fastest through facilitation were also those who were most proficient in skill acquisition with the device. There was considerable variability in progression through the two-phase protocol, with 20% deemed Poor/Non-Learners and unable to complete even the most basic game following training. There were no serious adverse events. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02358915 , first posted February 9, 2015.

Preliminary Effectiveness of Auricular Point Acupressure on Chemotherapy-Induced Neuropathy: Part 1 Self-Reported Outcomes.

PURPOSE: To reduce chemotherapy-induced neuropathy (CIN)-a significant challenge among cancer patients following chemotherapy-we explored the effects of auricular point acupressure (APA), which involves needleless, acupuncture-like stimulation on specific ear points. DESIGN/METHOD: This pilot study examined the effects of a 4-week APA intervention in the management of CIN. Descriptive analysis was used to examine the changes in study outcomes. RESULTS: Fifteen participants were enrolled. Two participants dropped out because they developed new medical conditions. Thirteen participants completed the study (87% retention rate). Study participants had more severe symptoms in their lower extremities (i.e., toes, feet, soles) than in their upper extremities (i.e., fingers, wrists, elbows). After the 4-week APA intervention, the mean percentage change scores ranged from 38% (tingling) to 49% (numbness); compared to pre-intervention, the therapeutic effects of APA were sustained at the 1-month follow-up. Function in both upper and lower extremities improved after the APA intervention (≥28%) and continued to improve at the 1-month follow-up (≥36%). CONCLUSIONS: Preliminary results from this small sample provide initial evidence of the effectiveness of APA on CIN. Future studies should confirm these results using a larger sample, a comparative sham control, and an examination of the underlying physiological mechanisms of the anti-CIN effects of APA. CLINICAL IMPLICATIONS: APA may provide an inexpensive and effective complementary approach for the self-management of CIN. Once the seeds have been taped to the patient's ear by the provider, patients are empowered to self-manage their CIN in their own environment.

White matter lesions and sudden sensorineural hearing loss.

Aim of this paper is to investigate the presence of White Matter Lesions (WMLs) in subjects affected by Sudden Sensorineural Hearing Loss (SSNHL) and possibly to evaluate the significance of WMLs in SSNHL patients. A total of 64 patients (cases) affected by SSNHL were included in this case-control study. Hearing tests were performed at SSNHL onset, after 7 days and after 30 days. Cerebral MRI sequences were performed to rule out retrocochlear pathology, and WMLs were evaluated if present. MRI control group included 32 subjects, without hearing loss, affected by pituitary adenoma, who underwent cerebral MRI of follow-up. WML presence in those affected by SSNHL resulted having a similar distribution to that of the control group; however, we observed complete hearing recovery in 42,9% of patients without WML and in 11,6% of patients with WML >1 (p = 0,017*). The incidence of WML in patients with SSNHL was not different compared to that of the control group; however, MRI could have a prognostic role for SSNHL patients, as the presence of WMLs can been linked to a poorer hearing recovery rate.

First Implant-Induced Changes in Rostral Brainstem Impair Second Implant Outcomes in Sequential Bilateral Cochlear Implant Children With Long Inter-Implant Delay.

HYPOTHESIS: Long-term unilateral use of a cochlear implant (CI) induces abnormal maturation of the rostral brainstem innervating the contralateral ear. BACKGROUND: In sequential bilateral CI children with long inter-implant delay, both sides of auditory cortices were dominantly activated by the first CI, but mechanisms of this abnormal development of the auditory system remain unclear. METHODS: Fifteen sequential bilateral CI children with long delay (mean ±â€ŠSD, 28.7 ±â€Š12.1 mo) underwent electrically evoked auditory brainstem response (EABR) testing using each of the first and second CI (CI1 and CI2, respectively), immediately after the second implantation. CI1 and CI2 were implanted at age of 23.7 ±â€Š9.6 and 53.1 ±â€Š12.1 months (mean ±â€ŠSD), respectively. Apical-to-basal difference in electrically evoked interwave III-V latencies (eIII-eV slope) which decreases with implant use was compared between the CI1 and CI2 sides. Their speech perception scores were evaluated 3 years after the second implantation. RESULTS: eIII-eV slopes evoked by the CI2 showed less mature pattern than those by the CI1. This CI2 versus CI1 difference in eIII-eV slopes, however, reduced as the inter-implant delay was prolonged, suggesting CI1-induced maturation of the rostral brainstem innervating the second ear before the second implantation. The smaller CI2 versus CI1 difference in eIII-eV slopes at the second implantation was correlated to poorer outcomes using the CI2 than the CI1. CONCLUSIONS: In this population, long-term unilateral CI use induced re-organization of the rostral brainstem innervating the second ear, which affected hearing outcomes using the CI2. Evaluation of eIII-eV slopes at the second implantation may be useful to predict hearing outcomes with CI2.

A synaptic theory of internal delays.

Neurons in the medial superior olive perform a coincidence analysis between inputs from the two ears, as predicted by Jeffress [J. Comp. Psychol. 41, 35-39 (1948)]. Jeffress also correctly predicted inputs to express a range of internal delays for which he invoked axonal delay lines. These, however, cannot explain that the inputs of many binaural neurons differ by a combination of a time delay and a phase shift. This study proposes an alternative source of internal delay. An interaural asymmetry in the activation threshold of the inner hair cell synapses is shown to reproduce the main features of internal delays of binaural neurons.

Management of Great Auricular Neuralgia Confirmed by Electrophysiologic Examination: A Case Report.

The great auricular nerve (GAN) is a sensory branch of the cervical plexus originating from the C2 and C3 nerve roots that innervates the external ear, mandibular angle, and parotid gland. Since idiopathic GAN neuralgia is a rare condition and branches of the GAN overlap with other cervical and cranial nerves, its diagnosis is challenging and can be confused with other facial neuralgias. This article describes the case of a 55-year-old woman with intractable unilateral periauricular and lateral head pain. No significant findings were found on cervical and brain imaging. At first, the patient was suspected to be suffering from trigeminal neuralgia or great occipital neuralgia; however, the symptoms persisted despite pharmacotherapy, cervical plexus and medial branch block, and repetitive transcranial magnetic stimulation. On the basis of an electrophysiologic examination, the patient was diagnosed as having GAN lesions. Pain subsided immediately after ultrasound-guided GAN block with local anesthetics and steroids. These findings indicate that electrophysiologic studies are helpful for accurately diagnosing patients with unclear pain in the periauricular and lateral head.

Evidence of a Right Ear Advantage in the absence of auditory targets.

The Right Ear Advantage effect (REA) was explored in a white noise speech illusion paradigm: binaural white noise (WN) could be presented i) in isolation (WN condition), ii) overlapped to a voice pronouncing the vowel /a/ presented in the left ear (LE condition), iii) overlapped to a voice pronouncing the vowel /a/ presented in the right ear (RE condition). Participants were asked to report in which ear the voice has been perceived. The voice could be female or male, and it could be presented at 4 different intensities. Participants carried out the task correctly both in LE and in RE conditions. Importantly, in the WN condition the "right ear" responses were more frequent with respect to both the chance level and the "left ear" responses. A perceptual REA was confirmed both in LE and RE conditions. Moreover, when the voice was presented at low intensities (masked by WN), it was more frequently reported in the right than in the left ear ("illusory" REA). A positive correlation emerged between perceptual and illusory REA. Potential links of the REA effects with auditory hallucinations are discussed.

What does a butterfly hear? Physiological characterization of auditory afferents in Morpho peleides (Nymphalidae).

Many Nymphalidae butterflies possess ears, but little is known about their hearing. The tympanal membrane of butterflies typically comprises distinct inner and outer regions innervated by auditory nerve branches NII and NIII and their respective sensory organs. Using the Blue Morpho butterfly (Morpho peleides) as a model, we characterized threshold and suprathreshold responses of NII and NIII. Both are broadly tuned to 1-20 kHz with best frequencies at 1-3 kHz, but NIII is significantly more sensitive than NII. The compound action potentials (CAPs) of both branches increase their first peak amplitudes and areas in response to higher sound levels. NII and NIII differed in their suprathreshold CAP responses to sound frequencies, with stronger responses to 1-3 and 4-6 kHz, for NIII and NII respectively; results that are consistent with tympanal membrane mechanics. These results indicate that butterflies are capable of amplitude and frequency discrimination. Both auditory branches responded to playbacks of the flight and calls of predatory birds. We propose that the ears of butterflies, like those of many vertebrate prey such as some rabbits and lizards, function primarily in predator risk assessment.

Corneal Neurotization With a Great Auricular Nerve Graft: Effective Reinnervation Demonstrated by In Vivo Confocal Microscopy.

PURPOSE: We describe the first case of minimally invasive corneal neurotization with the great auricular nerve (GAN) to treat unilateral neurotrophic keratopathy. We assessed corneal sensation and reinnervation by esthesiometry and confocal microscopy over 12 months of follow-up, and we provide a detailed description of the surgical technique. METHODS: Corneal neurotization was successfully achieved with the ipsilateral GAN in a 58-year-old woman. Cochet-Bonnet esthesiometry and in vivo confocal microscopy were performed before and after corneal neurotization, to monitor the recovery of corneal sensation and corneal reinnervation by subbasal nerve fibers. RESULTS: Neurotrophic keratopathy was a complication of the surgical treatment of meningioma. Before surgery, the patient had no corneal sensation or corneal innervation. Six months after surgery, confocal microscopy confirmed regrowth of a large number of nerve fibers in the subepithelial space of the cornea. Nine months after surgery, a central esthesiometry score of 10-mm was attained. CONCLUSIONS: Corneal neurotization leads to reinnervation of the cornea and recovery of ocular sensation in adults. The GAN is suitable for use in corneal neurotization because of its anatomical proximity and the low level of associated morbidity. Confocal microscopy demonstrated the occurrence of corneal reinnervation, which preceded the recovery of corneal sensation.

Whole-Mount Adult Ear Skin Imaging Reveals Defective Neuro-Vascular Branching Morphogenesis in Obese and Type 2 Diabetic Mouse Models.

Obesity and type 2 diabetes are frequently associated with peripheral neuropathy. Though there are multiple methods for diagnosis and analysis of morphological changes of peripheral nerves and blood vessels, three-dimensional high-resolution imaging is necessary to appreciate the pathogenesis with an anatomically recognizable branching morphogenesis and patterning. Here we established a novel technique for whole-mount imaging of adult mouse ear skin to visualize branching morphogenesis and patterning of peripheral nerves and blood vessels. Whole-mount immunostaining of adult mouse ear skin showed that peripheral sensory and sympathetic nerves align with large-diameter blood vessels. Diet-induced obesity (DIO) mice exhibit defective vascular smooth muscle cells (VSMCs) coverage, while there is no significant change in the amount of peripheral nerves. The leptin receptor-deficient db/db mice, a severe obese and type 2 diabetic mouse model, exhibit defective VSMC coverage and a large increase in the amount of smaller-diameter nerve bundles with myelin sheath and unmyelinated nerve fibers. Interestingly, an increase in the amount of myeloid immune cells was observed in the DIO but not db/db mouse skin. These data suggest that our whole-mount imaging method enables us to investigate the neuro-vascular and neuro-immune phenotypes in the animal models of obesity and diabetes.

The Efficacy of Magnetic Resonance Imaging for the Diagnosis of Superior Semicircular Canal Dehiscence.

OBJECTIVE: This study aimed to evaluate the efficacy of magnetic resonance imaging (MRI) for diagnosing superior semicircular canal dehiscence (SSCD). MATERIALS AND METHODS: The radiological records of patients who were admitted to our clinic with complaints of otologic and neuro-otologic symptoms between October 2014 and December 2015 were retrospectively reviewed. Among these patients, those who underwent both computed tomography and MRI and were reported to have SSCD in the temporal bone on at least one side were included in the study group. MRI records of patients with a confirmed diagnosis were then assessed for the presence of SSCD. RESULTS: The left and right semicircular canals of 52 patients were evaluated in this study. The sensitivity and specificity of MRI in the diagnosis of SSCD was 89.06% and 90%, respectively. The positive and negative predictive values were 93.44% and 83.72%, respectively. CONCLUSION: The use of multiplanar reformats and angulation techniques during MRI assessment of patients with neuro-otologic symptoms can improve the diagnostic process for patients with SSCD. This may allow early diagnosis of the disease by using just one imaging method, which would also reduce the costs per patient during the diagnosis period.

A novel approach for regional anaesthesia of the auricular region in horses: an anatomic and imaging study.

OBJECTIVE: To investigate the anatomy of the ear innervation in horses and develop a new approach for the block of the internal and external pinna and acoustic meatus. STUDY DESIGN: Prospective, descriptive, cadaveric study. ANIMALS: A total of 25 heads and necks from horse cadavers. METHODS: Phase I: anatomical dissection and dye deposition on 19 heads. With the head positioned in lateral recumbency, 10 mL methylene blue (1:1 sterile water) for the internal auricular and 2 mL for the great auricular nerve block were injected in 21 ears (seven left, 10 right and two bilaterally). The anatomy of all the ears (bilaterally) and the accuracy of dye deposition (successful nerve staining defined as > 2 cm) was assessed following dissection. Occurrence of intraparotid, intravascular, or intraneural injection was recorded. Phase II: the same technique and volumes were applied, bilaterally, using a lidocaine 2%:iohexol solution mixture (3:1) in six different cadavers. Computed tomography was performed and the needle position and spread of the mixture evaluated and recorded. RESULTS: Phase I: Inter- and intraindividual differences were found in the anatomy of the great auricular nerve, but not in the internal and lateral auricular nerve branches and the caudal auricular nerve. No auriculotemporal nerve was found. Nerve staining was successful in 95% of the cases for the great auricular nerve, 85% of the cases for the internal auricular nerve, and 100% of the cases for the lateral auricular branch and caudal auricular nerve. Failure was observed in horses with abundant periauricular fat. No intravascular, intraparotid or intraneural injections occurred. Phase II: A 100% success rate was recorded from computed tomography images. CONCLUSIONS AND CLINICAL RELEVANCE: The described approach seems suitable for blockade of the sensory innervation of the equine ear.

Optimization of Transcutaneous Vagus Nerve Stimulation Using Functional MRI.

OBJECTIVE/HYPOTHESIS: Vagus nerve stimulation (VNS) is an established therapy for drug-resistant epilepsy, depression, and a number of other disorders. Transcutaneous stimulation of the auricular branch of the vagus nerve (tVNS) has been considered as a non-invasive alternative. Several functional magnetic resonance imaging (fMRI) studies on the effects of tVNS used different stimulation parameters and locations in the ear, which makes it difficult to determine the optimal tVNS methodology. The present study used fMRI to determine the most effective location for tVNS. MATERIALS AND METHODS: Four stimulation locations in the ear were compared: the inner tragus, inferoposterior wall of the ear canal, cymba conchae, and earlobe (sham). Thirty-seven healthy subjects underwent two 6-min tVNS stimulation runs per electrode location (monophasic rectangular 500 µs pulses, 25 Hz). General linear model was performed using SPM; region-of-interest analyses were performed for the brainstem areas. RESULTS: Stimulation at the ear canal resulted in the weakest activation of the nucleus of solitary tract (NTS), the recipient of most afferent vagal projections, and of the locus coeruleus (LC), a brainstem nucleus that receives direct input from the NTS. Stimulation of the inner tragus and cymba conchae activated these two nuclei as compared to sham. However, ROI analysis showed that only stimulation of the cymba conchae produced a significantly stronger activation in both the NTS and LC than did the sham stimulation. CONCLUSIONS: These findings suggest that tVNS at the cymba conchae properly activates the vagal pathway and results in its strongest activation, and thus may be the optimal location for tVNS therapies applied to the auricle.

Coupled ears in lizards and crocodilians.

Lizard ears are coupled across the pharynx, and are very directional. In consequence all auditory responses should be directional, without a requirement for computation of sound source location. Crocodilian ears are connected through sinuses, and thus less tightly coupled. Coupling may improve the processing of low-frequency directional signals, while higher frequency signals appear to be progressively uncoupled. In both lizards and crocodilians, the increased directionality of the coupled ears leads to an effectively larger head and larger physiological range of ITDs. This increased physiological range is reviewed in the light of current theories of sound localization.

Sound source localization and segregation with internally coupled ears: the treefrog model.

Acoustic signaling plays key roles in mediating many of the reproductive and social behaviors of anurans (frogs and toads). Moreover, acoustic signaling often occurs at night, in structurally complex habitats, such as densely vegetated ponds, and in dense breeding choruses characterized by high levels of background noise and acoustic clutter. Fundamental to anuran behavior is the ability of the auditory system to determine accurately the location from where sounds originate in space (sound source localization) and to assign specific sounds in the complex acoustic milieu of a chorus to their correct sources (sound source segregation). Here, we review anatomical, biophysical, neurophysiological, and behavioral studies aimed at identifying how the internally coupled ears of frogs contribute to sound source localization and segregation. Our review focuses on treefrogs in the genus Hyla, as they are the most thoroughly studied frogs in terms of sound source localization and segregation. They also represent promising model systems for future work aimed at understanding better how internally coupled ears contribute to sound source localization and segregation. We conclude our review by enumerating directions for future research on these animals that will require the collaborative efforts of biologists, physicists, and roboticists.

Neuroanatomical Tracing Techniques in the Ear: History, State of the Art, and Future Developments.

The inner ear has long been at the cutting edge of tract tracing techniques that have shaped and reshaped our understanding of the ear's innervation patterns. This review provides a historical framework to understand the importance of these techniques for ear innervation and for development of tracing techniques in general; it is hoped that lessons learned will help to quickly adopt transformative novel techniques and their information and correct past beliefs based on technical limitations. The technical part of the review presents details of our protocol as developed over the last 30 years. We also include arguments as to why these recommendations work best to generate the desired outcome of distinct fiber and cell labeling, and generate reliable data for any investigation. We specifically focus on two tracing techniques, in part developed and/or championed for ear innervation analysis: the low molecular multicolor dextran amine tract tracing technique and the multicolor tract tracing technique with lipophilic dyes.