All experimental procedures and the laboratory animal care in this study were verified and approved by Inha University Animal Ethics Committee. All animals (SD rats, male) were provided by an animal provision company (Oriental Bio corp., Korea).
Unilateral labyrinthectomy (UL) was performed by intratympanic injection of ferric chloride (FeCl3) using rodents (SD rats, male). For the injection, a syringe (0.5 mm-diameter needle) was inserted through the tympanic membrane until its tip encountered the middle-ear ossicles. Once positioned, the needle was slightly pulled back, and the injection was conducted. To estimate a proper amount of the FeCl3 solution (mixture of 0.97 g FeCl3 and 1.4 ml saline), we initially used saline to assess the volume of the middle ear cavity. An animal was anesthetized by the intramuscular injection of a mixed solution (1.3 ml/kg) of Ketamine (1 µl/g) and Xylazine (0.33 µl/g). Following the anesthesia, saline was injected until it was emerged back through the tympanic membrane. Once the backward flow of saline was observed, the injection was ceased, and the injected volume was measured. Determining the averaged amount based on multiple measurements, the same volume of FeCl3 solution was applied to an animal for a UL model. After constructing the UL model, the animal was in a resting stage until it was recovered from the anesthesia.
Both tests for static and dynamic symptoms were conducted immediately after the animals were fully awaken, which was approximately 0.5-1 hour later from the moment of FeCl3 injection. Also, the behavioral tests generally lasted for an hour to complete both types of tests. Thus, all behavioral tests were finalized within 2 hours after FeCl3 injection.
The static symptoms were observed in the absence of the head movements. Spontaneous nystagmus (SN), skewed deviation (SD), head deviation (HD), and paw distance (PW) were used as the critical indicators for the unilateral vestibular dysfunction. SN was the rhythmical ocular movement with a rapid phase toward the opposite side of the lesion. SD was the horizontal misalignment by the ocular displacements, defined as the tilted degree between a horizontal line and the imaginary line by both eyes. HD was the vertical misalignment of the head, defined as the tilted degree between a vertical line and the imaginary line by the head. PW was estimated by the distance between two front or hind paws. For the PW measurement, the animal was placed in a transparent box right after its awakening, and the paw positions were identified for at least 30 sec. Using the recorded positions, PW was calculated before and after UL.
The dynamic symptoms were examined in the animals’ free head movements. Three different behavioral responses, such as rota-rod test, tail-hanging, and rotational direction, were used for identifying the unilateral vestibular dysfunctions. The rota-rod test has been commonly used to estimate the animal’s motor function by measuring the time that the animal was maintaining on the rolling rod (5.9 cm/sec). Using the tail-hanging test, it was examined if the body was spun, which rarely happen before UL. In an open space, the animal’s directional movement was also examined to identify the rotational direction, depending on the side of the vestibular lesion. All behavioral tests were conducted to identify the unilateral vestibular dysfunction after UL.
Extracellular neural recording
Following the behavioral tests, the animal was re-anesthetized by the same method (see Animal preparation). Once the animal was fully anesthetized, it was placed on the motorized stereotaxic apparatus (NEUROSTAR, Germany) to fix its head. The overall neural recording process followed our previous methodological approach [19-21] for approximately 3 hours. In short, surgically removed its scalp, the superior surface of the skull was exposed. On the surface, the lambda was designated as a center, and the hole (2.0 mm diameter) for a recording electrode (5 M𝝮, A-M system, US) was opened (generally, 3.0 mm posterior and 2.0 mm lateral away from the center). Due to the recording position, the right side of the animal was defined as the ipsilateral direction. The neuronal activities were explored by advancing the recording electrode, and they were tested by the kinetic stimuli, such as a horizontal rotation and the linear translation following the x- axis (ipsi- & contra-lateral). Based on the responses to the kinetic stimulation, the neurons were classified as a pure otolith or a convergent (otolith+canal-related) neuron. The pure otolith neuron showed a neuronal response only to the linear translation while the convergent neuron responded to both kinetic stimuli. The neuronal responses to the stimulation were recorded with the sampling rate of 40 kHz in OmniPlex D system (Plexon, TX) after amplified and filtered (bandpass 0.5-3 kHz). All the recordings were performed under the anesthetized condition.
Behavioral tests were composed of four types of static symptom and three types of dynamic symptom tests. For PW, the measured distances were executed off-line using a user code written in MATLAB (MathWorks, USA). The distances were presented in a bar chart to identify a possible width by two feet, and the normal width was assessed by measuring the largest and the smallest values (1-15cm for front & 1-10cm for hind). The final value was presented by a range, instead of averaged values. As briefly explained in Behavioral test, SD and HD were examined by the misalignments compared with the horizontal and vertical lines, and they were assessed by the altered lines in the ocular and the head displacements, respectively. The tail-hanging, the rotational motion, and SN were confirmed based on the observation, and the rota-rod test was analyzed using the measured times (mean ± standard deviation).
The directional preference was determined by the neuronal response to the kinetic stimulation following the x-axis (passive inter-aural translation), which was originated from the utricle. During the repeated right- or leftward linear translation, IFR of the neuron was evaluated; if IFR increased as the head was linearly translated to the right direction, the neuron was identified as ipsi-preferred unit. The contra-preferred neuron increased its IFR as the head was linearly translated to the left direction. In addition, the neuronal information responding to the horizontal rotation was examined to demonstrate if the recording response was originated from a pure or a convergent neuron.
To assess a statistical significance, Binomial Cumulative Distribution (BCD) function was applied. As all compared groups were one or the other, BCD tested if the different numbers in two groups were statistically similar. Based on this test, the bias was determined between the groups with different directional preference; the ipsi- and the contra-preferred neurons.