Total 56 Sprague Dawley (SD, 275-562g, males) were used in this study. Nine animals were used for behavioral responses after the construction of Parkinson’s disease (PD) model. Twenty-three and twenty-four animals were used for extracellular neural recording and immunohistochemistry, respectively. The animals were housed in a facility which stably maintained temperature (22-25℃), humidity (40-60%), ventilation (10-15 times/hour), static pressure difference (>5mmAq), and noise level (<60dB). In addition, a 12:12 hours light-dark cycle was provided to the animals while they were housed. The animals were taken out of the housing facility only during an experiment, and they were re-housed in the facility as the experiment was completed. All procedures and principles of laboratory animal care were approved by the Animal Ethics Committee at Inha University (INHA 200110-681). The current study was reported in accordance with ARRIVE guidelines, and all methods were performed in accordance with relevant guidelines and regulations.
Parkinson’s Disease model
The construction of a PD model was initiated by intraperitoneally injecting desipramine (12.5mg/kg, Enzo, New York, US), which was known as a selective inhibitor of norepinephrine transporter for the restricted effects on dopaminergic neurons by 6-hydroxydopamine (6-OHDA, Sigma, St. Louis, US). By injecting the desipramine, thus, the induced toxicity by 6-OHDA affected a targeted lesion with a high selection. After the desipramine injection, the animal was anesthetized by an intramuscular injection of a mixed solution of Ketamine (1 µl/g) and Xylazine (0.33 µl/g). Once the animal was fully anesthetized, its head was fixed using a motorized stereotaxic apparatus (Neurostar, Tubingen, Germany). Then, its skull was surgically exposed to open a hole for 6-OHDA injection at a location (2 mm lateral and 2.5 mm posterior directions) away from Bregma. The target area of 6-OHDA injection was the medial forebrain bundles (mfb) at the right side, which was located ranged from 8.5 to 9.5 mm ventral direction from the hole. Using a 26-gauge syringe (Hamilton, Reno, US), 6-OHDA (8.0 μg/μL) was injected at the speed of 8 μL/min. Completed the toxic administration, the hole and the skull were sutured, and the animal had a rest at least for 1 week before its evaluation.
Model Evaluation
PD model evaluation was performed mainly by immunohistochemistry and rota-rod test. Due to the decrease of the positive tyrosine hydroxylase (TH+) in the substantia nigra (SN) induced by 6-OHDA administration, the immunohistochemistry was conducted to detect TH+. At the beginning, the whole brains were removed and fixated in a formalin solution with 10% neutral buffer (Sigma, St. Louis, US). After completing the fixation, the area containing SN was obtained from the brains, and its dissection was performed (slice thickness: 2.5-3 μm). Then, the prepared sections were stained using the primary antibody of Rabbit anti-Tyrosine hydroxylase (MilliporeSigma, Burlington, US), and they were incubated overnight at 4°C. The stained sections were imaged by a motorized microscope (Olympus, Japan), and the captured images were stored in a magnification of 400 times (ocular: x10 and objective: x40) of the actual size. For the comparison of the affected with the unaffected area, the detected TH+ was estimated by its different density by counting the stained target neurons.
Extracellular neural recording & Galvanic Vestibular Stimulation
The extracellular neural recording in the vestibular nucleus (VN) was performed based on our previous studies. The neural recording was initiated by defining a center on the skull. Generally, the center was positioned at 3.0 mm posterior from the lambda, and the VN was positioned at 3.0 mm posterior and 2.0 mm lateral away from the center. Once the recording position was defined, an animal was anesthetized by injecting the solution (see Parkinson’s Disease model), and its head was fixed in the motorized stereotaxic apparatus (NEUROSTAR, Germany). Then, the animal’s skull was surgically exposed, and a recording hole (2-2.5 mm diameter) was opened. Through the hole, a recording electrode (5M𝝮, A-M system, US) was inserted to search a neuronal response, and all neuronal responses were continuously tested using kinetic head movements, such as horizontal head rotation and linear head transmission. Once the vestibular responses were confirmed based on the responses to the kinetic stimuli, galvanic vestibular stimulation (GVS) was applied. GVS (100μA) was applied through the stimulation electrode located between the temporal muscles and bones, and the electrodes were placed at both left and right sides. Their corresponding polarities were positive and negative, respectively. The neuronal signals were filtered (bandpass 0.5-3 kHz) and recorded (sampling rate: 40 kHz) using OmniPlex D system (Plexon, US).
Analysis of Neuronal Signals
The neuronal signals were analyzed based on the instantaneous firing rates (IFR), which were the reciprocal number of inter-spike interval (ISI). Their comparison was made by the averaged IFR for 10-second resting period. To specify the toxic (6-OHDA) effects, the neurons were grouped as the canal- and the otolith-related units, and each group was again subdivided into regular and irregular responses, depending on the neuronal discharge regularity. The discharge regularity was calculated using the normalized coefficient of variance (CV*), and its computational function was as like below;
where µ is the mean of ISI and CV is the coefficient of variance. Based on the calculated CV*, all neurons were divided into regular and irregular units. For a statistical analysis, two-sample t-test was applied. By comparing two sub-groups in four different groups (canal & regular, canal & irregular, otolith & regular, and otolith & irregular), the toxic effects were examined.
Immunohistochemistry (IHC) & Assessment of glutamate receptors
The effects of GVS were assessed by the change in the number of glutamate receptors, focusing on ꭤ-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors. The overall process was similar as prepared in staining brain tissues (see Model Evaluation) by applying the antibodies of the glutamate receptors. In summary, all animals’ brains were removed after completing stimulation, and resulted in five groups (control, PD model, PD model with 100μA, PD model with 500μA, and PD model with 1000μA). To provide the designed amount of GVS, all animals involved in the assessment of glutamate receptors underwent GVS-only under anesthesia. The methods of stimulation and anesthesia were similar as indicated in the extracellular neuronal recording (see Extracellular neural recording & Galvanic Vestibular Stimulation). To assess the effects of GVS in PD models, different amounts of GVS were applied. A set of GVS was composed of 20-time stimuli with the same stimulating duration (3 seconds) and frequency (once every 60 seconds). A 10-minute interval was provided between two stimulating sets, and total three sets of GVS were applied with different amplitudes (100, 500, and 1000 μA). Based on the given conditions of GVS, the relevant electrical charges were 0.018, 0.09, and 0.18 coulombs for 100, 500, and 1000 μA, repectively. The electrical charge was calculated as follows;
where, q was the total electrical charge, and I(t) was the current of GVS. The interval between t1 and t2 was the stimulating duration.
The brains from five groups were prepared for the immunohistochemistry of AMPA and NMDA receptors. In 4% formaldehyde solution, the portion of VN was fixated and paraffinized. The prepared paraffin block was sliced by 2.5-3 µm, and the selected slices were used for antibody staining. Using GluR1 (Sigma-Aldrich, US) and NR1 (Sigma-Aldrich, US), AMPAr and NMDAr were stained. The prepared slices were rinsed, and their endogenous peroxidase activity was blocked (3% hydrogen peroxide solution). Then, specific antigen retrieval solutions were applied (Tris-EDTA, pH 9.0, 95°C, 50 min & citrate solution, pH 6.0, 95°C, 50 min) for each subtype, and stored at 4°C overnight. Staining the subtypes of receptors, their images were taken by a motorized microscope (Olympus, Japan) with a magnification of 400 times (ocular: x10 and objective: x40). The specific location of VN was confirmed in the images, and the number of receptors was manually counted with several independent repetition.