Eltoprazine modulated gamma oscillations on ameliorating L‐dopa‐induced dyskinesia in rats

Abstract Aim Parkinson's disease (PD) is a pervasive neurodegenerative disease, and levodopa (L‐dopa) is its preferred treatment. The pathophysiological mechanism of levodopa‐induced dyskinesia (LID), the most common complication of long‐term L‐dopa administration, remains obscure. Accumulated evidence suggests that the dopaminergic as well as non‐dopaminergic systems contribute to LID development. As a 5‐hydroxytryptamine 1A/1B receptor agonist, eltoprazine ameliorates dyskinesia, although little is known about its electrophysiological mechanism. The aim of this study was to investigate the cumulative effects of chronic L‐dopa administration and the potential mechanism of eltoprazine's amelioration of dyskinesia at the electrophysiological level in rats. Methods Neural electrophysiological analysis techniques were conducted on the acquired local field potential (LFP) data from primary motor cortex (M1) and dorsolateral striatum (DLS) during different pathological states to obtain the information of power spectrum density, theta‐gamma phase–amplitude coupling (PAC), and functional connectivity. Behavior tests and AIMs scoring were performed to verify PD model establishment and evaluate LID severity. Results We detected exaggerated gamma activities in the dyskinetic state, with different features and impacts in distinct regions. Gamma oscillations in M1 were narrowband manner, whereas that in DLS had a broadband appearance. Striatal exaggerated theta‐gamma PAC in the LID state contributed to broadband gamma oscillation, and aperiodic‐corrected cortical beta power correlated robustly with aperiodic‐corrected gamma power in M1. M1–DLS coherence and phase‐locking values (PLVs) in the gamma band were enhanced following L‐dopa administration. Eltoprazine intervention reduced gamma oscillations, theta–gamma PAC in the DLS, and coherence and PLVs in the gamma band to alleviate dyskinesia. Conclusion Excessive cortical gamma oscillation is a compelling clinical indicator of dyskinesia. The detection of enhanced PAC and functional connectivity of gamma‐band oscillation can be used to guide and optimize deep brain stimulation parameters. Eltoprazine has potential clinical application for dyskinesia.


| INTRODUC TI ON
Parkinson's disease (PD), one of the most common neurodegenerative diseases, imposes a great burden on society. The clinical spectrum of PD consists of motor symptoms such as tremors, rigidity, bradykinesia, and non-motor symptoms. 1 Pathologically, PD is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) combined with aberrant cortico-basal ganglia-thalamic (CBT) network neural activity. 2 Dopamine (DA) replacement therapy is the backbone of PD treatment. 3 Levodopa-induced dyskinesia (LID) is a major motor complication of chronic levodopa (L-dopa) treatment, reflecting the current lack of a standard and effective PD treatment.
Neuroelectrophysiological recording and analysis have been increasingly applied to study the pathophysiology of PD and its complications. As an oscillatory electrical activity, local field potential (LFP) reflects the comprehensive potential changes or synchrony electrical activity results from a group of neurons or synaptic structures in a local region. 4 Enhanced beta-band (13-35-Hz) oscillations and information flow in CBT circuits have been shown to have anti-kinetic functions in parkinsonian subjects, 5 and dopaminergic treatment suppresses this enhancement and destruct information flow to ameliorate bradykinesia. 6 Long-term L-dopa administration has prokinetic effects, inducing exaggerated gamma-band (>35-Hz) activity associated with abnormal involuntary movements. 7 Recent studies have examined L-dopa-induced excessive gamma oscillations in broadly distributed neural networks, such as those of the striatum 8 and motor cortex. 9 Basic research has shown that exaggerated narrowband gamma oscillations in the cortex are associated with dyskinesia and may serve as control signals for adaptive deep brain stimulation (DBS). 10 Clinical electrophysiological data suggest that broadband gamma oscillations occur in the subthalamic nucleus (STN) 11 and internal globus pallidus 12 in patients with advanced PD and dyskinesia. However, the functional consequences of enhanced gamma oscillations in the PD and LID states remain incompletely understood.
Phase-amplitude coupling (PAC) is a representation of crossfrequency coupling in which the low-frequency phase modulates the high-frequency amplitude. PAC alterations have been associated with multiple neurological disorders, 4 such as PD, Alzheimer's disease, schizophrenia, and epilepsy. The relationship between exaggerated beta-gamma PAC in the cortex and motor impairment in patients with PD has been investigated. 13 Beta-band power is known to be related to the severity of PD symptoms, but the correlation between beta and gamma power remains obscure. A clinical study demonstrated that phase-targeted DBS modulated cortical PAC, leading to motor improvement, and thus that its clinical application for the treatment of neurological disorders associated with abnormal PAC, such as PD, may be suitable. 14 However, changes in PAC in the dyskinetic state remain enigmatic, and a better appreciation of PAC may yield a promising electrophysiological target for LID therapy.
Functional connectivity measures the similarity of specific physiological signals between different regions, reflecting the synchronization of field potentials and information transfer. 15 It can also be regarded as an indicator of motor performance improvement, such as that achieved with L-dopa treatment and DBS. 16  The widely accepted potential mechanism of the pathogenesis of LID is exceptional synaptic plasticity in the corticostriatal circuit due to the non-physiological synthesis, release, and reuptake of DA from exogenous high-dose L-dopa metabolism. 17,18 However, much research has demonstrated that the pathogenesis of LID is related to not only dopaminergic receptors but also nondopaminergic receptors, such as serotonergic receptors, 19 and various nondopaminergic medications are applied to manage such complications. 20 Abnormal 5-hydroxytryptamine (5-HT) transmission is thought to be involved in various central nervous system (CNS) disorders, including anxiety, depression, schizophrenia, addiction, obsessivecompulsive disorder, PD, and Alzheimer's disease. 21 In a non-human primate study, Jiménez-Sánchez et al. 22 confirmed that the destruction of nigrostriatal dopaminergic neurons occurring in PD promotes the hyperinnervation of striatal 5-HT fibers as a compensatory mechanism. 5-HT neurons have been found to facilitate the conversion of exogenous L-dopa to DA, the storage of DA in synaptic vesicles, and the release of DA in an activity-dependent manner. As these neurons lack a negative feedback mechanism for the regulation of synaptic DA levels, these processes are highly uncontrolled, leading to persistent and prolonged abnormal DA release that significantly increases the striatal DA level and ultimately leads to LID. 23 Several 5-HT receptor agonists, such as NLX-112, 24 zonisamide, 25 and buspirone, 26 have been used to ameliorate dyskinesia in recent preclinical and clinical trials. Eltoprazine, a 5-HT1A/B autoreceptor gamma-band oscillation can be used to guide and optimize deep brain stimulation parameters. Eltoprazine has potential clinical application for dyskinesia.

K E Y W O R D S
5-HT1A receptor, abnormal involuntary movements, electrophysiology, local field potential, PAC, Parkinson's disease, serotonin agonist, inhibits DA release from 5-HT neurons and has been shown to effectively ameliorate dyskinesia in rats 27 and patients 28 with LID.
Although eltoprazine has been shown to alleviate LID efficiently in a dose-dependent manner, the changes in neural electrophysiology during its administration remain to be explored.
In this study, firstly, we investigated the accumulative effect of L-dopa on neural oscillations in the corticostriatal projection. The LFPs from M1 and DLS were simultaneously recorded in Sham + saline, Sham + LB, PD + saline, and PD + LB groups for following assessment. Furthermore, we demonstrate the efficacy of eltoprazine in the alleviation of hyperkinesia in rats with LID and neural activity alterations to clarify its underlying therapeutic mechanism at the electrophysiological level for the first time. Lastly, we also explored the impact of eltoprazine on anti-Parkinsonian efficacy of L-dopa.
Our research provided compelling evidence supporting future clinical application of eltoprazine in LID therapy.

| Animals
All experimental procedures were approved by the Institutional been completed, the rats were sacrificed and brain tissue was harvested for histological analysis ( Figure S1A).

| Experiment 2
Experiment 2 was performed with 52 rats. The rats were apportioned randomly to PD (n = 36) and sham (n = 16) groups. On day 1, were performed on days 43, 46, and LFP recording with synchronous video recording was performed on day 49. Open field test was also performed on day 48 80 min after LB, LB plus eltoprazine, or eltoprazine injection. After all electrophysiological recordings had been completed, the rats were executed for histological analysis. One rat from the Sham + saline group was eliminated for missing the target.

| Surgeries
For lesion creation, each rat's head was fixed in a stereotaxic apparatus after the induction of anesthesia with sodium pentobarbital For microelectrode implantation, two electrode arrays consisting of eight stainless-steel Teflon-insulated microwires (50μm diameter, 2 × 4 configuration with 150 μm spacing between microwires; Plexon) were implanted vertically in right primary motor cortex (M1) layers V and VI (AP +2.6 mm, ML −2.6 mm, DV −1.5 to 1.6 mm, below the dura) and the right dorsolateral striatum (DLS; AP 0 mm, ML −3.5 mm, DV -5.5 mm, below the dura), respectively. Two screws placed above the cerebellum and in contact with the dura were used as grounds, and ground wires were wrapped around them. The electrode arrays were secured to the cranium with screws and dental cement. Each rat received postoperative injections of penicillin (80,000 U/mL, 1 mL, i.p.) for 7 consecutive days after electrode implantation to prevent infection.

| Behavioral tests
All rats were allowed to habituate to the behavior cages for 5 min before test initiation. For the OFT, each rat was placed in an opaque box (100 × 100 × 50 cm). It was permitted to move freely for 5 min, and the total distance and velocity were recorded for the assessment of locomotor activity with Noldus software (Noldus).
For the cylinder test, each rat was placed in an open transparent plastic cylinder, and 20 instances in which it used its forelimb to stand against the wall were recorded. Forelimb use was defined as the placement of the entire forepaw on the cylinder wall, indicating body support. The number of contacts with the forelimb contralateral to the lesioned side was calculated as a percentage of the total 20 contacts.
The APO challenge was performed to evaluate unilateral dopaminergic neuron depletion. The rats' rotations were counted 15 min after APO injection, and lesions were considered to be sufficient when rats made >30 contralateral rotations over a 5-min period.
L-dopa-induced involuntary movement was scored using a rat dyskinesia scale. 27 The severity of motor, forelimb, and orolingual dyskinesia was scored using the AIMs on a scale that motor, forelimb, and orolingual dyskinesias were each scored ranging from 0 to 4 (0, absence of abnormal movement; 1, dyskinesia occurring during <50% of the observational period; 2, dyskinesia occurring during ≥50% of the observational period; 3, constant dyskinesia that could be interrupted artificially; 4, constant dyskinesia that could not be interrupted). After L-dopa plus benserazide (LB) injection, the rats were video-recorded and observed for 180 min, and AIMs scores were recorded for 2-min period during each of the 10-to 20-min intervals (maximum AIMS score = 120). The interval between L-dopa injection and the manifestation of abnormal involuntary movement was recorded as the response time (RT), as described previously.

| Electrophysiological recording
Local field potentials in the M1 and DLS were recorded with a 128-channel data acquisition system (Plexon). To record the baseline parkinsonian state, data were obtained 5 min before LB injection on day 29. LFPs were recorded in 11 5-min sessions [1 before and 10 (at 20-min intervals during a 180-min period) after LB injection] on days 41 and 49. The data were collected at a sampling frequency of 1 kHz, amplified (300×), and bandpass filtered (0.5-1000 Hz). A ground wire was used for reference.

| Histological analysis
Each rat was anesthetized with pentobarbital sodium (50 mg/kg, i.p.), and a 20-μA current was passed through the recording electrodes for 30 s to mark the recording sites. Then, rat was then perfused transcardially with ice-cold 0.9% w/v saline (400 mL) followed by 4% paraformaldehyde (400 mL). The brain was extracted and immersed in 4% paraformaldehyde for 24-h post-fixation, then dehydrated and paraffin embedded. Coronal sections (8 μm) through the M1, DLS, and SNc were cut on a paraffin microtome (Leica). To verify the recording sites, sections including electrode tracts were mounted on glass slides, stained with hematoxylin and eosin, and observed using a digital slide scanner (PANORAMIC; 3D Hitech Ltd).

| Immunofluorescence staining
Immunofluorescence staining of tyrosine hydroxylase (TH) in the SNc was performed to measure DA depletion. All sections were processed using the same protocol and chemical reagents. The sections were placed in a dry oven at 65°C for 60 min, then washed to remove the paraffin and rehydrate the tissue in dimethylbenzene (10 min, three times); 100%, 95%, 85%, and 75% ethanol (5 min each); and double-distilled water (5 min, three times). Then, the sections were immersed in citrate antigen retrieval solution (Beyotime) and The labeling intensity in each area was quantified as the index of light attenuation with respect to the background (neighboring corpus callosum) using the standard ImageJ (National Institutes of Health) program. The amount of TH+ cells was calculated according to the optical fractionator principle using ImageJ.

| Data preprocessing
The Fieldtrip Toolbox 29 was used to preprocess the data. Using ft_definetrial, data from the 5-min LFP sessions were divided into 300 1-s segments. Then, artifacts were identified and removed using ft_artifact_zvalue and ft_rejectartifact.

| Spectral analysis of LFPs
Power spectrum density (PSD) was estimated based on fast Fourier transform using the ft_freqanalysis function of Fieldtrip. In total, 120 1-s processed data segments were used to calculate PSDs with cfg.foilim = 1:1:150 (target frequency range, 1-150 Hz), tapsmofrq (amount of spectral smoothing achieved through multi-tapering) = 2, and a Hanning window.

| Aperiodic-adjusted power of beta and gamma oscillations
We analyzed the aperiodic-adjusted beta power in the baseline (day-29) parkinsonian state and the gamma oscillations power at 80 min after LB administration on day 41 using the Fitting Oscillations & One Over F (FOOOF) toolbox. 30 The Matlab package was utilized to separate periodic and aperiodic components from the spectral results, according to our previous research. 31 The PSD for each frequency f P(f) was expressed as: where L(f) is the aperiodic component and G n (f) is the Gaussian distribution. The periodic component was parameterized as a mixture of Gaussian dis- , where α is the height of the peak (power) above the aperiodic component, c is the center frequency of the peak (peak frequency), ω is the width of the peak (bandwidth), and f is the array of frequency values. The aperiodic component was also parameterized using a Lorentzian function as where b is the broadband offset, k is the "knee," and χ is the expo-

| Statistical analysis
All data are reported as means with standard errors of the mean.
All data were tested for normal distribution before statistical analysis. The data exhibited a normal distribution was further undergone parametric test while the data did not exhibit a normal distribution

| PD model establishment
PD was established successfully in 25 of the 29 rats ( Figure 1A). Rats with 6-OHDA lesions showed reduced total distance and velocity on the OFT (Student's t test, total distance, p < 0.0001; velocity, p < 0.0001; Figure 1B,C, Figure S1B) and impaired contralateral limb use in the cylinder test (Student's t test, p < 0.0001; Figure 1D

| L-dopa-induced exaggerated gamma oscillation in the M1 and DLS
PSDs were estimated using LFPs at 80 min, corresponding to peak dyskinesia after LB administration, on day 41. In the M1, the beta oscillations power was greater in the PD + saline group than in the Sham + saline and PD + LB groups (two-way ANOVA: LB:  Figure 2E). In the DLS, the PD + saline group showed enhanced beta activities (two-way ANOVA: LB:

| Effects on gamma oscillation power
Theta-gamma PAC in the DLS was greater in the PD + saline group than in the Sham + saline group (Tukey's test, p = 0.0226), and greater in the PD + LB group than in the Sham + LB and PD + saline groups  Figure 3A,B).
It has been proved that beta-band power was related to the severity of PD symptoms, but the correlation between beta and gamma power remains obscure. We analyzed the aperiodic-adjusted power of beta rhythm of parkinsonian state baseline on day 29 and power of gamma oscillations at 80 min after LB administration on day 41 by Fitting Oscillations & One Over f (FOOOF) toolbox ( Figure 3C). The beta and gamma power correlated positively (r = 0.6026, p = 0.0023; Figure 3D).

| DISCUSSION
To better understand the pathological mechanism underlying LID at Data are means ± SEMs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA followed by Tukey's multiple comparison test. We observed strengthened gamma-band coherence and PLVs after LB administration which indicated aberrant neural functional connectivity in rats with LID. Cortico-STN coherence with a frequency-specific topography has been detected in subjects with PD on and off of L-dopa medication. 38 Recent findings suggest that strengthened connectivity between the cerebellar dentate nucleus and putamen reduces dyskinetic symptoms, 39 and functional connectivity has been used as an indicator of DBS eligibility in patients with early PD. 40 Moreover, structural and functional connectivity predicted motor improvement independently in subjects with PD. 41 We explore the contribution of functional connectivity to the pathology of PD and LID. What's more, the aberrant alteration of functional connectivity in the LID state found by us may provide evidence for DBS candidate selection and clinical efficacy prediction.
We demonstrated that eltoprazine, which targets 5-HT1A/1B autoreceptors, affects gamma oscillations and thus could be applied in the treatment of LID; we explored its pharmacological profile which may be conducive to justify the optimal dosage of eltoprazine, it reduced AIMs scores, extended RTs in a dose-dependent manner, suppressed gamma-band activity without altering beta-band activity, ameliorated enhanced theta-gamma PAC in DLS, and reduced M1-DLS coherence and PLVs in gamma-band. We also excluded the own effect of eltoprazine on LID rats without L-dopa injection, no dyskinetic symptoms appeared in LID + E group rats. As for neural activities alteration, LID + E group rats exhibited an electrophysiological parameter resembling LID + LB + E group rats accompanied by declined gamma oscillations, theta-gamma PAC, and gamma-band functional connectivity. In addition, neural activities in beta-band were not influenced, all of which indicated no impact of eltoprazine alone. According F I G U R E 7 Effect of eltoprazine on theta-gamma PAC of rats with LID. (A) Representative images of theta-gamma PAC. (B) Comparison of theta-gamma PAC between LID + LB, LID + LB + E, LID + E groups rats. Data are means ± SEMs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA followed by Tukey's multiple comparison test.
to our observation during experiment procedure and data analysis, LB or eltoprazine injection alone showed no improvement during OFT on day 48 due to only induced pathological involuntary movements or lacking the anti-parkinsonian efficacy of L-dopa, respectively.
However, eltoprazine invention combined with LB ameliorated dyskinetic symptoms with rescued locomotor activity. Drugs targeting the 5-HT1A and/or 5-HT1B receptors have been designed to alleviate dyskinesia. It has been proved that eltoprazine suppressed sensitization of striatonigral projections (direct pathway) and modulated striatal glutamate transmission, but had no effect on striatal ectopic DA release, which may be the basis of its anti-dyskinetic effect. 42 In addition, etloprazine could improve LID symptoms by recovering the Data are means ± SEMs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA followed by Tukey's multiple comparison test. long-term potentiation and synaptic depotentiation in striatal medium spiny neurons, which was correlated to the normalization of dopamine receptor-related cAMP/PKA and ERK/mTORC signaling pathways and the restoration of NMDA receptor subunit equilibrium. 43 From the perspective of neuroinflammation, eltoprazine reduced the L-DOPA-induced upregulation of immediate-early gene zif-268 in striatum, delayed the onset of dyskinesia, and reserved the efficacy of L-dopa. 44,45 Previous study demonstrated that both gamma power and gamma burst in M1 were correlated with AIMs 9 ; thus, we infer that from view of in vivo electrophysiology, eltoprazine modulates exaggerated gamma activity accompanied with resumption of aberrant neural functional connectivity and information flow in M1-DLS projection to alleviate LID symptoms. In addition, our research provided evidence that eltoprazine exerts anti-dyskinetic effect without reducing anti-parkinsonian efficacy of L-dopa. We clarified the potential neural electrophysiological mechanism followed by eltoprazine invention in dyskinetic state for the first time and provide supporting evidence for its application in clinical field.
However, the findings of this study have to be seen in light of some limitations. Despite we observed excessive gamma oscillations induced by L-dopa administration, its parameters are incompletely investigated since we only analyzed power, more characteristics and its relationship with the severity of dyskinesia are awaiting research. As we speculated that L-dopa may affect gamma oscillations in a dose manner and a concentration gradient of L-dopa is needed to be explored for its effects on gamma oscillations. We demonstrated that enhanced striatal theta-gamma PAC in DLS is the mechanism of exaggerated broadband gamma oscillations while excessive gamma oscillations are driven by beta oscillations in M1 in LID-on state, more direct evidences with strong support are required for future. In addition, we confirmed the underlying electrophysiological mechanism of eltoprazine invention but the internal molecular mechanisms and synaptic plasticity alterations still lie on the table. Eltoprazine targets 5-HT1A/1B receptors which was relative to psychiatric disorders, since the clinical manifestation included a range of non-motor symptoms such as anxiety and depression, its effect on PD-related psychosis needs to be explored in the future. Also, the effect of eltoprazine on PD symptoms remains to be explored both on electrophysiological and behavioral perspective.
In conclusion, this study demonstrated that L-dopa induced exaggerated gamma oscillation in the M1 and DLS, accompanied by enhanced theta-gamma PAC in the DLS. Increased functional connectivity, represented by coherence and PLVs, was also detected based on neuroanatomical corticostriatal projections in the dyskinetic state. Eltoprazine modulated the abnormal gamma oscillation to ameliorate LID symptoms, providing compelling evidence supporting its future clinical application.

AUTH O R CO NTR I B UTI O N S
YB and WZ contributed to the conception and design of the study; YB, PW, JY, ZW, HY, YD, JG contributed to the acquisition and analysis of data; YB and PW contributed to drafting the text or preparing the figures.

ACK N OWLED G M ENTS
This work was supported by funds from the National Natural Science Foundation of China (No. 82071254).

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors report no competing interests. Some figures were created with Biorender.com.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.