A Direct Primary Motor Cortex-Globus Pallidus Internus Circuit Regulates Both Motor and Non-Motor Symptoms in Parkinson's Disease.

A Direct Primary Motor Cortex-Globus Pallidus Internus Circuit Regulates Both Motor and Non-Motor Symptoms in Parkinson's Disease. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A Direct Primary Motor Cortex-Globus Pallidus Internus Circuit Regulates Both Motor and Non-Motor Symptoms in Parkinson's Disease. Yong Wang, Yaqian Li, Xueping Zhang, Ruobing Zheng, Qianwen Wang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5990552/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The cortico-basal ganglia (BG) circuit is vital for motor control and Parkinson's disease (PD) symptoms. The Globus Pallidus Internus (GPi) is a principal BG output nucleus and a key target for Deep Brain Stimulation (DBS) in PD treatment. However, the structure and function of the cortico-GPi circuit have not been completely addressed. In the present studies, data demonstrate a direct Primary Motor Cortex (M1)-GPi pathway, bypassing the classical direct, indirect, and hyperdirect pathways. This direct M1-GPi pathway plays an essential role in motor regulation under normal conditions. Importantly, in PD, post-synaptic inhibition of this pathway alleviates motor deficits. Post-synaptic activation of the M1-GPi pathway ameliorates depression symptoms associated with PD but exacerbates the PD motor symptoms. Interventions targeting the pre-synaptic M1-GPi pathway do not significantly affect motor regulation or PD symptoms. It suggests that the M1-GPi pathway may play a crucial role in motor regulation, PD motor symptoms and non-motor symptoms. Aberrant activities of the post-synaptic M1-GPi pathway potentially contributing to PD deficits. And the mechanism of GPi-DBS in PD therapy may involve post-synaptic regulation of M1-GPi pathway activities, rather than the retrograde modulation of pre-synaptic M1-GPi pathways. Biological sciences/Neuroscience/Diseases of the nervous system/Parkinson's disease Biological sciences/Neuroscience/Motor control/Motor cortex Parkinson’s disease direct M1-GPi pathway motor regulation motor symptoms non-motor symptoms Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Parkinson’s disease (PD) is a common neurodegenerative disease characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNc), leading to severe motor dysfunction. [ 1 , 2 ] This deficiency manifests as severe motor dysfunctions such as tremors, rigidity, and bradykinesia, which are the hallmark symptoms of PD. In addition to the classical motor symptoms, PD patients frequently exhibit non-motor manifestations, notably depression and anxiety. [ 1 , 3 ] These PD symptoms are attributed to abnormalities in the structure and function of the basal ganglia (BG) network. [ 4 , 5 , 6 ] The Globus Pallidus Internus (GPi) in primates, and its homolog in rodents known as the entopeduncular nucleus (EP or EPN), serves as a principal output nucleus of the basal ganglia (BG). It plays a pivotal role in integrating and modulating signals originating from diverse BG nuclei. These signals are subsequently relayed to regions implicated in motor function and the manifestation of PD symptoms. [ 4 , 7 ] The primary target areas of the GPi include the ventromedial (VM) and ventroanterior lateral (VAL) nuclei of the thalamus, as well as the lateral habenula (LHb). [ 7 ] Among these, the GPi projection to the thalamus is typically implicated in motor regulation and contributes to PD motor symptoms. And in clinical practice, the GPi is a key target for deep brain stimulation (DBS) interventions aimed at alleviating PD symptoms. [ 8 ] Unfortunately, the underlying mechanism for GPi-DBS in PD therapy has not been completely elucidated. Hence, deciphering the specific contributions of the GPi and its associated circuits to PD symptoms is crucial for developing effective therapeutic interventions. According to the canonical model, it is widely accepted that cortical neural projections reach the GPi through three primary pathways: the direct, indirect, and hyperdirect pathways. These pathways are crucial for the execution of movements and the regulation of motor functions, and imbalances in these pathways are thought to contribute to the symptoms of PD. [ 4 , 9 ] In the cortico-basal ganglia-thalamus loop, information from the cerebral cortex converges in the GPi through the striatum (Str), globus pallidus externus (GPe), and subthalamic nucleus (STN), before being relayed back to the cortex via the thalamus (Tha). This complex network underscores the importance of maintaining a delicate balance within the basal ganglia circuitry for normal motor function. [ 10 ] In such traditional models, cortex information is not typically thought to reach the GPi directly. Nonetheless, some evidence suggests a direct cortical-GPi connection. For instance, studies using anterograde tracers like biotinylated dextran amine (BDA) indicate the potential existence of a direct neural pathway from the cortex to the globus pallidus. [ 11 ] Findings from human brain and rat research imply the existence of a direct cortical-globus pallidus projection. [ 12 , 13 , 14 , 15 ] However, despite substantial advancements in comprehending the basal ganglia's part in movement disorders, the architecture, functionality, and fundamental mechanisms of the direct cortico-GPi connection in motor regulation and the manifestation of PD symptoms are still not fully understood. The role of the direct cortico-GPi connection in the underlying mechanism for GPi-DBS has not been completely elucidated either. On the other hand, recent studies have highlighted the LHb's significant role in the pathophysiology of depression. [ 16 , 17 ] Considering that the LHb is one of primary nuclei projected by GPi, [ 7 ] the role of the cortico-GPi connection in PD-related depression remains incompletely understood. In the present studies, we conduct a comprehensive investigation into the direct cortical inputs to the GPi. We examine their connectivity, function, and relevance to motor and non-motor phenotypes (such as depressive-like behaviors) under normal conditions and in a PD mouse model. Our findings offer robust evidence supporting the existence of the direct cortico-GPi pathway. This pathway may play crucial roles in motor regulation and contribute significantly to both motor and non-motor symptoms of PD. Moreover, our data could help to understand the mechanisms associated with PD motor or non-motor symptoms in the context of GPi-DBS for PD treatment. 2. Results 2.1. The Primary Motor Cortex (M1) neurons directly project to the GPi, establishing the direct M1-GPi pathway. To investigate the connectivity of cortico-GPi, we utilize a strategic approach to map the neuronal projections utilizing adeno-associated virus (AAV) vectors expressing fluorescent proteins. The anterograde tracing results show that neurons in the M1 project descending fibers to the GPi (Fig. 1 A,B; Figure S1 A). Importantly, both filamentous axonal fibers originating from M1, as well as puncta labeled with EYFP, are observed in GPi (Figure S1 A). This indicates that M1 neurons might directly project to the GPi, forming synaptic connections and thereby establishing the direct M1-GPi pathway. Besides the GPi, M1 neurons also project to other basal ganglia nuclei, including the striatum (Str), the external globus pallidus (GPe), the subthalamic nucleus (STN), and the substantia nigra pars compacta (SNc) (Figure S1 B-F, Supporting Information). The retrograde tracing results show that the M1 serves as a principal input to the GPi, indicated by the abundant presence of retrogradely mCherry-labeled pyramidal neurons in the M1 (Fig. 1 C,D). The GPi receives additional inputs from various regions including the secondary motor cortex (M2), GPe, STN, centromedian nucleus (CM), and parafascicular nucleus (PF), ventral tegmental area (VTA), SNc, substantia nigra pars reticulata (SNr), deep mesencephalic nucleus (DpMe) and other nuclei (Figure S2, Supporting Information). To further validate the direct connections between cortical M1 and the GPi, we utilized a trans-monosynaptic tracing approach. This procedure entailed the injection of AAV2/1-hSyn-Cre into the M1, coupled with administration of AAV2/9-hSyn-DIO-mCherry into the GPi (Fig. 1 G). The AAV2/1-hSyn-Cre virus, a serotype 1 adeno-associated virus (AAV), is capable of trans-monosynaptic infection to express the Cre protein, allowing it to specifically label trans-monosynaptic neurons situated within downstream nuclei, thereby enabling precise mapping of direct neuronal pathways. [ 18 , 19 ] Consequently, the specific expression of mCherry, induced by trans-monosynaptic Cre in the GPi, ensures dependable labeling of the direct connectivity between the cortical M1 and GPi. The results demonstrate a robust presence of mCherry labeling in the neurons within the GPi (Fig. 1 H), providing a compelling evidence of direct connectivity between the M1 and the GPi. This indicates that the cortical M1 neurons may directly connect and innervate the GPi neurons. Additionally, the GPi neurons that are directly innervated by the M1 secondarily project to the thalamus (VAL) and the LHb (Fig. 1 I,J). These findings at the anatomical level provide substantial evidence supporting the direct connectivity between the cortical M1 and GPi, establishing a direct M1-GPi pathway. This direct M1-GPi pathway potentially functions as an alternative, shortcut circuit for projections from the cortex to the GPi, bypassing the canonical direct pathway, indirect pathway, and hyperdirect pathway. 2.2. The synaptic transmission of the direct M1-GPi pathway is potentially mediated by glutamatergic M1 neurons. To investigate the synaptic transmission of the direct M1-GPi pathway, the AAV2/9-CaMKIIα-hChR2-mCherry is injected into the M1. Results of the electrophysiological recordings show that the glutamatergic M1 neurons are effectively excited by the optical stimulation (Figure S3). And optogenetic activation of axon from glutamatergic M1 neurons results in an increased spontaneous firing frequency in GPi neurons (Fig. 1 E,F). Moreover, the electrophysiological recordings of trans-monosynaptically labeled GPi neurons exhibit a consistent response. The optogenetic activation of glutamatergic M1 neurons elicits an increased spontaneous firing frequency in the GPi neurons, which are actually innervated by glutamatergic M1 neuron axons (Fig. 1 K,L). In addition, an increased amplitude of the optogenetic-induced EPSC (oEPSC) is observed in the trans-monosynaptically labeled GPi neurons, corresponding to the optogenetic activation at the axon terminal of the glutamatergic M1 neurons (Fig. 1 M,N). These data provide electrophysiological evidence that further emphasizes the direct connectivity between the cortical M1 and the GPi, suggesting that the synaptic transmission of the M1-GPi pathway may be mediated by the glutamatergic M1 neurons. 2.3. The glutamatergic M1-GPi pathway physiologically modulates motor behavior in mice. To explore the physiological functions of the direct M1-GPi pathway, motor behaviors of mice are assessed using optogenetic manipulations under normal conditions. Results show that unilaterally optogenetic activation of the glutamatergic axon terminals of the M1-GPi pathway results in decreased motor coordination observed in the rotarod test, and an increase in rearing behavior in the cylinder test (Fig. 2 A-E), though motor activities assessed in the open field test (OFT) and the cylinder test remain unchanged (Fig. 2 E; Figure S4; S5A-E, Supporting Information). Additionally, unilaterally optogenetic inhibition of the post-synaptic neurons in the direct M1-GPi pathway also results in decreased motor coordination, as observed in the rotarod test (Fig. 2 F-H), while motor activities in OFT and cylinder test remain unchanged (Fig. 2 I,J; Figure S5F-K, Supporting Information). These data provide behavioral evidence indicating that the direct M1-GPi pathway physiologically modulates motor behavior in mice, a process that may be mediated by glutamatergic M1 neurons. 2.4 The function of the direct M1-GPi pathway in modulating motor behavior is comparable to the hyperdirect M1-STN pathway. The M1-STN pathway, known as the hyperdirect pathway, has been demonstrated to play crucial roles in motor regulation and PD symptoms. [ 5 , 20 ] . To evaluate the function of the direct M1-GPi pathway, we compare it with the M1-STN pathway under normal conditions. Results show that optogenetic activation of the glutamatergic axon terminals of the M1-STN pathway leads to a significant decrease in motor coordination, as observed in the rotarod test (Fig. 2 K-M). This outcome is similar to the effects observed from activating the direct M1-GPi pathway (Fig. 2 A-E). Different from the M1-GPi pathway, optogenetic activation of the M1-STN pathway leads to a significant increase in left forelimb preference in the cylinder test (Fig. 2 N). Motor activities assessed in OFT and cylinder test do not exhibit significant changes, similar to the observations made with the M1-GPi pathway (Fig. 2 O; Figure S5L-N). These data suggest that the motor regulatory function of the direct M1-GPi pathway exhibits similarities with the hyperdirect M1-STN pathway, further confirming the role of the direct M1-GPi pathway in motor regulation under normal condition. It is evident that both the direct M1-GPi pathway and the hyperdirect M1-STN pathway play important roles in motor regulation. At the same time, these two pathways exhibit different preferences in motor regulation. The M1-STN pathway seems to preferentially modulate forelimb movements, while the M1-GPi pathway appears to be more involved in controlling rearing behaviors. 2.5 The 6-OHDA lesions in the SNc induce typical PD motor symptoms in mice, but do not lead to typical depressive-like behaviors. To investigate the roles of the M1-GPi pathway in PD symptoms, we administer 6-OHDA to induce either bilateral or unilateral lesions in the SNc of mice. [ 1 ] Subsequently, motor behaviors and non-motor symptoms, primarily depressive-like behaviors, are assessed (Fig. 3 A). In mice subjected to a unilateral 6-OHDA lesion in the SNc, a substantial loss of dopaminergic neurons in the SNc and dopaminergic fibers in the striatum are seen (Fig. 3 B). Motor deficits are characteristically observed, as demonstrated by the APO-rotation test, rotarod test, OFT, and cylinder test (Fig. 3 C-I). Regarding depressive-like behavior, the immobility duration in the tail suspension test (TST) exhibits a significant increase (Fig. 3 J), which is indicative of the typical symptom of behavioral despair associated with depression. The anhedonia, as measured by the sucrose preference test (SPT), along with another symptom of behavioral despair observed in the forced swimming test (FST) and the novelty-suppressed feeding test (NSF), does not exhibit significant alterations in PD mice (Fig. 3 K-M; Figure S6, Supporting Information). It indicates that the 6-OHDA lesion in SNc leads to typical motor deficits in mice, but barely produces symptoms of PD-related depression (PDD). In addition, bilateral 6-OHDA lesions of the SNc also result in a substantial depletion of dopaminergic neurons in the SNc and a reduction of dopaminergic fibers in the striatum of mice (Figure S9, Supporting Information). 2.6. Unilateral ablation of the pre-synaptic M1-GPi pathway has no prominent effects on motor modulation at either normal or PD states in mice. To examine the function of the pre-synaptic M1-GPi pathway, we unilaterally inject the AAVretro-hSyn-Cre-mCherry into the GPi and the AAV2/9-EF1α-DIO-taCasp3-EGFP into the M1 (Fig. 4 A,B). The retrogradely expressed taCasp3 induce targeted ablation of pre-synaptic neurons in the M1-GPi pathway (Fig. 4 C,D). Results show that the ablation of pre-synaptic neurons in the M1-GPi pathway leads to a decreasing trend in motor coordination, as indicated by a p -value of 0.0588 in the rotarod test (Fig. 4 E) under normal conditions. No other motor behaviors exhibit significant alterations in OFT and cylinder test under normal conditions (Fig. 4 F-J; Figure S7A,B, Supporting Information). It suggests that ablation of the pre-synaptic M1-GPi pathway may have virtually no significant impact on motor behavior under normal condition. Following the ablation of the pre-synaptic M1-GPi pathway, the 6-OHDA lesion is induced in the SNc of mice to generate a PD state. This helps us understand how the pre-synaptic M1-GPi pathway contributes to the development of PD symptoms. Results demonstrate that the targeted ablation of M1 neurons in the M1-GPi pathway has virtually no influence on the motor behaviors of PD mice (Fig. 4 K-P; Figure S7C,D, Supporting Information), except the rearing behavior observed in the cylinder test (Fig. 4 N). This implies that the ablation of the pre-synaptic M1-GPi pathway may not effectively impact on PD motor symptoms except the rearing behavior in cylinder test. 2.7. The collateral projections of the M1-GPi pathway involve multiple nuclei of basal ganglia. It is confounding that the targeted ablation of the pre-synaptic M1-GPi pathway has a negligible impact on normal motor and PD symptoms in mice. To more thoroughly explore the pre-synaptic functions of the M1-GPi pathway, the collateral projections of the M1-GPi pathway are examined. The retrograde AAVretro-hSyn-Cre-mCherry is injected into the GPi, while the AAV2/9-EF1α-DIO-EGFP is concurrently injected into the M1 (Figure S8A). This approach allows the Cre to be expressed in a retrograde manner exclusively within the M1 neurons that directly innervate the GPi (Figure S8B). Consequently, the DIO-EGFP expressions mark the soma of M1 neurons and their projecting axons of the pre-synaptic M1-GPi pathway. Results show that the M1 neurons that directly innervate the GPi have extensively collateral projections to the nuclei of the basal ganglia and thalamus, primarily including the striatum, GPe, STN, and the VM/VAL of the thalamus (Figure S8C-H). These findings demonstrate that the collateral projections of the pre-synaptic M1-GPi pathway extend to a wide array of nuclei in the basal ganglia and thalamus. These neuronal nuclei have been reported to be heavily involved in motor control and the symptoms of PD. [ 4 , 5 , 6 ] It suggests that the pre-synaptic M1-GPi pathway may play a highly complex role in motor regulation and the manifestation of PD symptoms due to its extensive collateral projections. Thus, optogenetic intervention at the axon terminal of the pre-synaptic M1-GPi pathway, located just over the GPi, can significantly impact motor behavior outcomes (e.g., rotarod test in Fig. 2 ). In contrast, globally intervening at the entire pre-synaptic M1-GPi pathway may not produce significantly different effects (as shown in the rotarod tests in Fig. 4 , Fig. 5 ). 2.8. Chemogenetic manipulation of the pre-synaptic M1-GPi pathway influences neither motor nor non-motor symptoms of PD. To further investigate the function of the pre-synaptic M1-GPi pathway in PD, we bilaterally inject AAVretro-hSyn-Cre-mCherry into the GPi and simultaneously inject AAV2/9-hSyn-DIO-hM3D-EGFP or AAV2/9-hSyn-DIO-hM4D-EGFP into the M1 of mice with 6-OHDA lesioned SNc (Fig. 5 A). This strategy enables chemogenetic intervention of the pre-synaptic M1-GPi pathway activities with specific expression of hM3D or hM4D protein in M1 neurons that innervate the GPi (Fig. 5 B; Figure S10, Supporting Information). Results show that chemogenetic activation of the pre-synaptic M1-GPi pathway has no significant effect on motor behavior (the hM3D + 6OHDA group v.s. the EGFP + 6OHDA group) in 6-OHDA lesioned mice, as observed in the rotarod test and OFT (Fig. 5 C-I), though there appears to be a decreased trend of motor activities. Chemogenetic inhibition of the pre-synaptic M1-GPi pathway also leads to no significant effect on motor behavior, except a significant increase in high-speed movement during OFT (the hM4D + 6OHDA group v.s. the EGFP + 6OHDA group) in 6-OHDA lesioned (Fig. 5 G). And chemogenetic inhibition of the pre-synaptic M1-GPi pathway results in an increasing trend of motor activities, as observed in both the rotarod test and OFT (Fig. 5 C-F). Similarly, results from the depressive-like behavior test indicate that manipulating the pre-synaptic M1-GPi pathway does not significantly impact depressive-like behavior under PD conditions (Fig. 5 J-M). In addition, chemogenetic inhibition (the hM4D + 6OHDA group) of the pre-synaptic M1-GPi pathway leads to significant increased motor activities compared to chemogenetic activation (the hM3D + 6OHDA group) in 6-OHDA lesioned mice, as observed in rotarod test (Fig. 5 C), movement velocity in OFT (Fig. 5 E), immobility in OFT (Fig. 5 F) and high-speed movement in OFT (Fig. 5 G). Despite this, these findings suggest that intervening in the pre-synaptic M1-GPi pathway may neither significantly modulate motor behavior nor non-motor symptoms under PD conditions. 2.9. Chemogenetic activation of post-syntactical M1-GPi pathway alleviates depressive-like symptom of PD but exacerbates motor symptoms. Due to the collateral branch of the M1-GPi pathway (Figure S8), interventions (Fig. 5 ) and targeted ablation (Fig. 4 ) of the pre-synaptic M1-GPi pathway do not appear to significantly influence PD symptoms in mice. To further investigate the function of the M1-GPi pathway in both motor and non-motor symptoms of PD, we reapplied chemogenetic techniques to manipulate the post-synaptic M1-GPi pathway in the 6-OHDA lesioned mice (Fig. 6 A). By facilitating the trans-monosynaptic expression of the hM3D protein (Fig. 6 B), this approach enables targeted chemogenetic activation exclusively in the post-synaptic GPi neurons that are innervated by the M1, thereby offering a precise strategy to modulate this post-synaptic M1-GPi pathways. Results show a secondary projection from the M1-GPi pathway to the LHb and the thalamus (VAL), in addition to the trans-monosynaptic projection of the M1-GPi pathway (Fig. 6 B). In the absence of 6-OHDA lesioned SNc, the sole chemogenetic activation (the hM3D + Sham group) of post-syntactical M1-GPi pathway leads to no significant alteration in motor behavior, compared to the control (the mCherry + Sham group) (Fig. 6 C-J). This indicates that activation of the post-synaptic M1-GPi pathway, when isolated, may not be sufficiently potent to modulate motor behaviors under normal conditions. Nevertheless, at PD state, chemogenetic activation of the post-synaptic M1-GPi pathway results in a significant decline in motor activities of the hM3D + 6OHDA groups, compared to the mCherry + 6OHDA controls (Fig. 6 C-J). Motor coordination in rotarod test (Fig. 6 C) and local motion in OFT (Fig. 6 E-I) are significantly decreased. Intriguing, the chemogenetic activation of post-syntactical M1-GPi pathway results in anti-depressive effects in 6-OHDA lesioned mice, characterized by significantly decreased immobility of FST and increased SPT in the hM3D + 6OHDA group, compared to the mCherry + 6OHDA controls (Fig. 6 K-N). These data demonstrate that activation of post-syntactical M1-GPi pathway alleviates depressive-like symptom in PD but simultaneously exacerbates PD motor symptoms. 2.10. Chemogenetic inhibition of the post-synaptic M1-GPi pathway ameliorates motor symptoms but does not impact depressive-like symptoms in PD. To ascertain the potential rescue of PD motor symptoms, we further investigate the chemogenetic inhibition of the post-synaptic M1-GPi pathway. The trans-monosynaptic expression of the hM4D protein enables targeted chemogenetic inhibition specifically in the post-synaptic GPi neurons innervated by the M1 (Fig. 7 A,B). The results also display a secondary projection from the M1-GPi pathway to the LHb and thalamus (VAL), in addition to the trans-monosynaptic projection of the M1-GPi pathway (Fig. 7 B). Under PD conditions, chemogenetic inhibition of the post-synaptic M1-GPi pathway rescues PD motor deficits, demonstrating a significant increase in motor activities during both the rotarod test (Fig. 7 C) and OFT (Fig. 7 D-F) in mice from the hM4D + 6OHDA group, compared to the mCherry + 6OHDA group. But chemogenetic inhibitions of post-synaptic M1-GPi pathway have no significant effects on the depressive-like behavior in PD mice from the hM4D + 6OHDA group, compared to the mCherry + 6OHDA group (Fig. 7 G-I). Similar to chemogenetic activation, the chemogenetic inhibition of the post-synaptic M1-GPi pathway in the sham mice does not significantly impact motor or non-motor behaviors, comparing the hM4D + Sham group with the EGFP + Sham group (Fig. 7 C-I). These findings imply that the function of the post-synaptic M1-GPi pathway could be instrumental in the manifestation of PD symptoms. The chemogenetic inhibition of the post-synaptic M1-GPi pathway effectively ameliorates PD motor symptoms, which may elucidate the mechanism underlying DBS in clinical PD treatment. But it is intriguing that without 6-OHDA lesion, solely manipulating the post-synaptic M1-GPi pathway has no impact on sham mice. This suggests that the manipulation of the post-synaptic M1-GPi pathway may play important roles in the state of PD but not under normal conditions. 3. Discussion The cortico–basal ganglia–thalamo–cortical loop is one of the fundamental network motifs in the brain. Unraveling its structural and functional organization is vital for comprehending motor behavior, cognition, and the development of many neurological and neuropsychiatric disorders. Typically, this network involves in information channel: motor, limbic and associative. [ 21 ] In this network, the structure and function of the cortico-GPi pathway have not been addressed completely, although several studies have suggested that there might be a direct projection from cortex to GP. In the present studies, we offer substantial evidence validating a direct route from the Primary Motor Cortex to the GPi (the M1-GPi pathway), which bypasses the traditional direct, indirect, and hyperdirect pathways (Fig. 8 ). And the direct M1-GPi pathway may play an essential role in regulation of motor. Importantly, the post-synaptic inhibition of the M1-GPi pathway alleviates the motor deficits in PD. The post-synaptic activation of the M1-GPi pathway ameliorates depression-related symptoms in PD but exacerbates the motor symptoms. In addition, intervention of the pre-synaptic M1-GPi pathway has no pronounced roles in motor regulation under normal or PD conditions. These data suggest that aberrant activities of the post-synaptic M1-GPi pathway may contribute to the mechanism of PD motor and non-motor symptoms. And it also helps us understand the GPi-DBS mechanism in PD therapy, suggesting that post-synaptic regulation, rather than the retrograde modulation of pre-synaptic M1-GPi pathways, may be involved. The suppression of GPi activity may contribute to the alleviation of PD motor symptoms in GPi-DBS, whereas the activation of GPi may be linked to the amelioration of PD non-motor symptoms. The classical model of cortico-basal ganglia function has critically shaped understanding of how nuclei and circuits within contribute to motor regulation and how circuit-level changes lead to the PD motor symptoms. [ 22 , 23 ] In this classical model, cortical information flows to the GPi through three ways: direct pathway (cortex-striatum-GPi), indirect pathway (cortex-striatum-GPe-STN-GPi) and hyperdirect pathway (cortex-STN-GPi). The GPi is one of major outputs of basal ganglia, converging information of basal ganglia and then sending the integrated signal to the down streamed, for example, the VM/VAL of thalamus. The equilibrium of activities within the basal ganglia pathways is essential for motor regulation under normal condition. In PD, dopamine loss of SNc causes imbalanced activity of pathways in cortico-basal ganglia, leading to the PD symptoms. [ 22 , 23 , 24 ] And the GPi also is one of the key targets for DBS in PD therapy. Therefore, the GPi is a crucial nucleus within the basal ganglia, playing a significant role in motor regulation and the manifestation of PD. The connections and innervation patterns of the GPi have thus become particularly important scientific and clinic question. In the conventional model of cortico-basal ganglia function, it has been commonly believed that there are no direct pathways linking the cortex to the GPi. Despite this, several reports indicate the possibility of cortical projections to the GPi. [ 11 , 12 , 13 , 14 , 15 ] Unfortunately, these reports have not provided evidence sufficient enough to define the precise anatomical structure, physiological functions, and pathophysiological implications of the cortico-GPi pathway. In the present studies, we provide substantial evidence at the anatomical, electrophysiological, and behavioral levels to support the assertion that the M1 directly projects to the GPi, thereby establishing the direct M1-GPi pathway. Our findings from anterograde tracing, retrograde tracing, and anterograde trans-monosynaptic tracing indicate that the M1 directly project to the GPi and establish connections with GPi (Fig. 1 ). The electrophysiological results further substantiate that glutamatergic M1 neurons directly innervate the GPi, facilitating the direct transmission of excitation from M1 to the GPi and inducing EPSC in the GPi via a mono-synaptic synapse (Fig. 1 ). Behavioral results from optogenetic and chemogenetic manipulations suggest that the glutamatergic M1-GPi pathway may play a role in physiological motor regulation and the manifestation of PD symptoms (Figs. 2 , 5 , 6 and 7 ). Additionally, findings from the hyperdirect pathway (M1-STN pathway) indicate similar impacts on motor control, implying that the M1-GPi pathway might serve a functionally comparable role to the hyperdirect pathway in motor regulation (Fig. 2 ). These data suggest that the direct M1-GPi pathway bypasses the conventional direct, indirect, and hyperdirect pathways, substantially contributing to motor regulation and PD symptoms. Our findings support the complement to the traditional model of basal ganglia circuitry and suggest a more complex relationship between the cortex and the GPi, which could have significant implications for the study and treatment of movement disorders like PD. Neurons originating in the M1 send direct axon to the GPi, delineating a discrete M1-GPi pathway instrumental in fine-tuned motor control. Literature has suggested circuit-specific therapy for PD. [ 25 ] Based on our data, the post-synaptic intervention of the M1-GPi pathway activity bi-directionally modulates PD symptoms. Post-synaptic activation of the M1-GPi pathway exacerbates the motor symptoms of PD (Fig. 6 ). Conversely, post-synaptic inhibition of the M1-GPi pathway rescues PD motor symptoms (Fig. 7 ). These data indicate that the post-synaptic M1-GPi pathway plays a crucial role in PD symptoms, suggesting that the activities of the GPi neurons innervated by M1 could modulate the motor symptoms associated with PD. Our findings endorse the concept of circuit-specific therapy for PD and suggest that the post-synaptic M1-GPi pathway, particularly the GPi neurons innervated by the M1, could serve as a promising target for circuit-specific strategies to alleviate PD motor symptoms. On the other hand, the pre-synaptic intervention of the M1-GPi pathway does not result in a prominent effect on PD motor symptoms (Fig. 5 ). The reason might be that M1 neurons, which project to the GPi, are found to collaterally project to other nuclei of the basal ganglia, including the striatum, GPe, and STN (Figure S8). Thus, pre-synaptic intervention of the M1-GPi pathway concomitantly exerts effects on the direct, indirect, and hyperdirect pathways. In this scenario, the GPi integrates inputs from the direct, indirect, and hyperdirect pathways, together with the M1-GPi pathway, yielding a balanced or compensatory output that consequently has no impact on PD symptoms. Nonetheless, this equilibrium could be disturbed in circumstances involving unilateral intervention of the M1-GPi pathway, as evidenced by the asymmetric motor performance observed in the rotarod test during unilateral intervention in the present study (Fig. 2 ). The GPi is a key target of DBS in PD therapy. [ 8 , 26 , 27 , 28 ] However, the underlying mechanism for GPi-DBS has not been addressed completely yet. Current understanding of the GPi-DBS mechanism in PD therapy proposes that it potentially engages in inhibition, activation, or both in the GPi and basal ganglia activities, [ 29 , 30 , 31 , 32 , 33 ] and may also involve the retrograde regulation of motor cortex activity. [ 30 , 34 , 35 , 36 ] From our observations, post-synaptic inhibition of the M1-GPi pathway effectively alleviates PD motor symptoms (Fig. 7 ), while post-synaptic activation ameliorates PD non-motor symptoms such as PD-related depression (Fig. 6 ). In contrast to post-synaptic intervention, pre-synaptic intervention of the M1-GPi pathway significantly impacts neither PD motor symptoms nor non-motor symptoms (Fig. 5 ). Taking these into account, our findings clearly support the opinion that the mechanism underlying GPi-DBS in PD therapy may involve the modulation of post-synaptic M1-GPi pathway activity, rather than the retrograde modulation of pre-synaptic M1-GPi pathway activity. In addition, study has suggested that a subset of neurons in nucleus might be inhibited during DBS, while another subset might be excited. [ 34 ] Our data indicate that inhibition of post-synaptic M1-GPi pathway may be associated with mechanisms alleviating PD motor symptoms, while activation of post-synaptic GPi neurons innervated by M1 may involve mechanisms ameliorating PD non-motor symptoms. This may elucidate the observation that DBS in PD therapy exhibit variable therapeutic outcomes across motor and non-motor symptoms, wherein some cases predominantly benefit motor symptoms while others favor non-motor symptoms. [ 27 ] Depression, also referred to as PDD (Parkinson's Disease Depression) or PD-related depression, represents a typical non-motor PD symptom. [ 22 , 37 , 38 ] It is a common and often debilitating symptom that can greatly impact the quality of life for those with PD. However, the mechanism underlying the PDD has not been addressed completely yet. Our findings indicate that the LHb serves as a principal target of GPi projections and even the M1-GPi pathway (Fig. 1 I, 6 B and 7 B). Current studies have suggested a strong correlation between LHb activity and the manifestation of depressive symptoms. [ 16 , 17 ] Our data demonstrate that the post-synaptic activation of the M1-GPi pathway alleviates depressive-like symptoms associated with PD (Fig. 6 L-N). These data imply that the secondary projection from the M1-GPi pathway to the LHb or the M1-GPi-LHb pathway could be crucially implicated in the mechanisms responsible for non-motor symptoms of PD like PDD. On the other hand, our data indicate that the post-synaptic activation of the M1-GPi pathway exacerbates PD motor symptoms while alleviating PD-related depressive symptoms (Fig. 6 C-J). Fortunately, we found that post-synaptic inhibition of the M1-GPi pathway has no profound effects on depressive-like symptoms while ameliorate the motor deficits in PD (Fig. 7 ). It underscores the need for cautious deliberation when considering interventions that target GPi activity to modulate PD motor symptoms, given their potential side effects on depressive symptoms. Taken together, substantial data delineate a direct pathway from M1 to GPi (the direct M1-GPi pathway), which bypasses the traditional direct, indirect, and hyperdirect pathways. This direct glutamatergic M1-GPi pathway plays an essential role in motor regulation, as demonstrated by unilateral intervention of the pre-synaptic M1-GPi pathway under normal conditions. The M1-GPi pathway projects extensive collaterals to the basal ganglia and other brain areas, which may be responsible for the negligible effects of interventions targeting the pre-synaptic M1-GPi pathway on motor regulation and PD motor symptoms. Additionally, the secondary projection to the LHb, or even the M1-GPi-LHb pathway, might be significantly implicated in the mechanisms responsible for non-motor symptoms of PD, like PDD. Importantly, in PD, post-synaptic inhibition of the M1-GPi pathway alleviates motor deficits. Conversely, the post-synaptic activation of the M1-GPi pathway ameliorates depression symptoms associated with PD but exacerbates the motor symptoms of PD. These data suggest that the M1-GPi pathway, and potentially the extended M1-GPi-thalamus/LHb pathways, may play a crucial role in motor regulation, PD motor symptoms, and non-motor symptoms. Furthermore, our data indicate that the regulation of post-synaptic M1-GPi pathway activities may be implicated in the underlying mechanism of GPi-DBS in PD therapy, rather than the retrograde modulation of pre-synaptic M1-GPi pathways. The inhibition of post-synaptic M1-GPi pathway activities may be implicated in the underlying mechanism of GPi-DBS alleviating PD motor symptoms, while the activation of post-synaptic M1-GPi pathways may contribute to the mechanisms of GPi-DBS ameliorating PD non-motor symptoms. This direct cortico-GPi connection may allow for a more streamlined and efficient communication between the two regions, enabling rapid response and fine-tuned motor control. The existence of this pathway further emphasizes the complexity of the basal ganglia's role in motor functions and mood, suggesting underlying mechanism and potential avenues for targeted therapeutic interventions in PD motor and non-motor symptoms. Overall, these findings suggest that the M1-GPi pathway may play a complex role in regulating motor symptoms and mood behavior in PD patients. Given the association between GPi activity and depression-related symptoms, further research is important and necessary. This will enable a more comprehensive understanding of the mechanisms underlying these effects and facilitate the development of more effective interventions for PD patients, particularly those experiencing depressive-like symptoms. 4. Conclusion The M1 neurons project directly to the GPi neurons, demonstrating a direct M1-GPi pathway that bypasses the direct, indirect, and hyperdirect pathways in the classical model (Fig. 8 ). This direct M1-GPi pathway contributes to the regulation of physiological motor functions, as well as to both motor and non-motor symptoms of PD. Interventions targeting the M1-GPi pathway, particularly post-synaptic interventions, bidirectionally control PD motor symptoms. Most importantly, the post-synaptic inhibition of the M1-GPi pathway effectively alleviates PD motor deficits, and its activation ameliorates the depressive symptoms associated with PD. These could help to understand the mechanisms associated with PD motor or non-motor symptoms in the context of GPi-DBS for PD treatment. And it is the regulation of post-synaptic M1-GPi pathway activities that may underlie the mechanism of GPi-DBS in PD therapy, rather than the retrograde modulation of pre-synaptic M1-GPi pathways. 5. Experimental Section Animals : Only healthy and adult C57BL/6J mice (sourced from Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) were used. All mice were group-housed, with 3 to 5 per cage. They were housed in a temperature-controlled animal room at 22 ± 2℃ and 40–70% humidity, on a 12-hour light/dark cycle (lights on at 7:00), with food and water available ad libitum until surgery. All experiments were approved by the Animal Ethics Committee of Capital Medical University and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Viral vectors : rAAV-EF1α-EYFP-WPRE-hGH pA (Cat#: PT-0098, working titer: 2.00E + 12 vg/mL), rAAV2/1-hSyn-CRE-WPRE-hGH-pA (Cat#: PT-0136, working titer: 2.00E + 12 vg/mL), rAAV2/9-hSyn-DIO-mCherry-WPRE-hGH-pA (Cat#: PT-0115, working titer: 2.00E + 12 vg/mL), rAAV2/9-CaMKIIα-hChR2(H134R)-mCherry-WPRE-hGH-pA (Cat#: PT-0297, working titer: 2.00E + 12 vg/mL), rAAV2/9-CaMKIIα-mCherry-WPRE-hGH-pA (Cat#: PT-0108, working titer: 2.00E + 12 vg/mL), rAAV2/9-EF1α-DIO-EGFP-WPRE-hGH-pA (Cat#: PT-0795, working titer: 2.00E + 12 vg/mL), rAAV2/R-hSyn-CRE-mCherry-WPRE-hGH-pA (Cat#: PT-0407, working titer: 5.00E + 12 vg/mL) and rAAV2/9-EF1α-DIO-taCasp3-TEVp-P2A-EGFP-WPRE-hGH-pA (Cat#: PT-1230, working titer: 2.00E + 12 vg/mL) were purchased from BrainVTA. ScAAV2/1-hSyn-Cre-pA (Cat#: S0292-1, working titer: 5.00E + 12 vg/mL), AAV2/9-hEF1α-DIO-eNpHR3.0-mCherry- WPRE-pA (Cat#: S0852-9, working titer: 5.00E + 12 vg/mL), AAV2/9-hEF1α-DIO-mCherry-WPRE-pA (Cat#: S0197-9, working titer: 5.00E + 12 vg/mL), AAV2/9-hSyn-DIO-hM3D(Gq)-mCherry-WPRE-pA (Cat#: S0192-9, working titer: 5.00E + 12 vg/mL), AAV2/9-hSyn-DIO-mCherry-WPRE-pA (Cat#: S1138-9, working titer: 5.00E + 12 vg/mL), AAV2/9-hSyn-DIO-hM4D(Gi)-eGFP-WPRE-pA (Cat#: S0286-9, working titer: 5.00E + 12 vg/mL), AAV2/9-hSyn-DIO-EGFP-WPRE-pA (Cat#: S0746-9, working titer: 5.00E + 12 vg/mL), AAV2/9-hSyn-DIO-hM3D(Gq)-eGFP-WPRE-pA (Cat#: S0260-9, working titer: 5.00E + 12 vg/mL) and AAV2/2Retro Plus-hSyn-Cre-mCherry-WPRE-pA (Cat#: S0702-2RP, working titer: 5.00E + 12 vg/mL) were purchased from Taitool Bioscience. Stereotaxic viral injection : Mice were deeply anesthetized via intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg) and positioned on a stereotaxic frame (RWD, China). Virus was injected either unilaterally or bilaterally into the M1 (AP: +1.30, ML: ±1.78 mm, DV: -1.55 mm) or the GPi (AP: -1.25 mm, ML: ±1.80 mm, DV:-4.80 mm), using a pulled glass capillary connected to a pressure microinjector (GAOGE, China) and a microinjection pump (RWD, China) at a rate of 30 nL/min. The virus volumes ranged from 200 to 250 nL in the M1 and from 100 to 150 nL in the GPi per side. The injection needle was left in place for 10 min to allow diffusion of the virus and then slowly withdrawn. Following the surgical procedures, mice were returned to their home cage for 3 to 4 weeks to allow for maximal virus expression and recovery. Mouse model of PD : Mice were deeply anesthetized as previously described. The 400-500nl of 6-OHDA (5 µg/µL dissolved in 0.1% ascorbic acid; Cat#: HY-B1081A, MCE, China) or saline (as control) was unilaterally or bilaterally microinjected into each side of the SNc (AP: -3.08 mm, ML: ±1.20 mm, DV: -4.65 mm) at a rate of 100 nL/min. Animals were allowed to recover for two weeks before post-lesion behavioral testing commenced. Histology, immunohistochemistry and quantification : Mice were deeply anesthetized and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS, pH 7.4). Brains were carefully removed and post-fixed in 4% PFA for an additional 12 hours. They were sequentially transferred to 20% and 30% sucrose solutions in PBS until they sank to the bottom of the container and then cut into 40µm coronal sections on a freezing microtome (Leica, Germany). For TH immune-staining, sections were incubated with a mouse antibody against TH (1:5000; Cat#: SAB4200697, Sigma-Aldrich) overnight at 4°C. After washing three times with PBS (5 min each), sections were incubated with biotinylated horse secondary antibody against mouse (1:1000; Cat#: PK-4002, Vector Laboratories, Youngstown, OH). The antibody was visualized with a DAB solution. Sections were fixed and cover-slipped. [ 6 ] The fluorescent images were obtained with the automatic digital section scanning system (OLYMPUS, Japan) and a Zeiss LSM 880 confocal microsystem (ZEISS, Germany). Quantification of tyrosine hydroxylase (TH) staining was used as a measure of dopamine lesion in SNc and stratum as Mastro described. [ 39 ] The quantification of input-output relationships follows the method described by Lilascharoen. [ 9 ] . Acute slice preparation : Mice were anesthetized by intraperitoneal injection of 1% pentobarbital sodium and perfused with oxygenated high sucrose and ice-cold slicing solution (in mM: 213 sucrose, 10 glucose, 26 NaHCO3, 3 KCl, 1 NaH2PO4·2H2O, 5 MgCl2·6H2O, and 0.5 CaCl2·2H2O; gassed with 95% O2/5% CO2). Brains were swiftly removed, and coronal slices (300 µm thick) containing the M1 and GPi were sectioned with a vibratome (DTK-1000, Dosaka EM, Kyoto). Slices were then incubated in aCSF (in mM: 126 NaCl, 21.4 NaHCO3, 10 glucose, 2.5 KCl, 1.25 NaH2PO4·2H2O, 1.2 MgCl2·6H2O, and 2 CaCl2·2H2O; pH 7.4, bubbled with 95% O2/5% CO2) at 33°C for 1 hour before recordings. Slices were allowed to recover for at least 1 h in continuously oxygenated aCSF (95% O2/5% CO2). Ex vivo electrophysiology : Patch clamp recordings were performed on M1 and GPi neurons using an upright microscope (Nikon FN-S2N, Japan), equipped with a 4× objective and a 40× water-immersion objective. The neurons with red fluorescence were visualized using a mercury lamp through a RED filter on the microscope. During recording, coronal slices containing the M1 and GPi area were continuously perfused at a rate of 2–3 mL/min with oxygenated aCSF and maintained at 32°C. The patch pipettes, pulled with a pipette puller (O.D.: 1.5 mm, I.D.: 1.10 mm, 10 cm length, Sutter Instrument, USA) from borosilicate glass (Sutter Instrument, USA), exhibited a resistance of 3–5 MΩ. Electrophysiological recordings were conducted using a HEKA amplifier. Signals were amplified, filtered at 2 kHz, and sampled at 20 kHz using the HEKA amplifier. Patch Master was used to convert the recorded signal into a digital signal. Clampfit 10.6 (Molecular Devices, USA) was employed for offline analysis of electrophysiological data. To test the effectiveness of the ChR2 viruses, optogenetic stimulation (473 nm, 10–20 Hz, 4–10 mW, 5 ms pulses) was delivered by a LED light source. To capture the spontaneous electrical activity of neurons, a current-clamp was used to record voltage while holding the current at 0 pA, indicating that neurons were at their resting membrane potential. The internal solution consisted of (in mM): 130 K-gluconate, 20 KCl, 10 HEPES, 0.2 EGTA, 10 disodium phosphocreatine, 4 Mg-ATP, 0.3 Na2-GTP (pH 7.20–7.25; 288–295 mOsm/kg). To evoke synaptic transmission using ChR2, optogenetic stimulation (473 nm, 10–20 Hz, 4 mW, 5 ms pulses) was delivered by a LED light source, and postsynaptic recordings in voltage clamp mode (holding potential, 0 mV) were made in the GPi areas of ChR2 terminal infection using electrodes filled with a cesium-based internal solution (in mM: 120 CsMeSO3, 15 CsCl, 8 NaCl, 10 TEA, 10 HEPES, 0.2 EGTA, 2 Mg-ATP, 0.3 Na2-GTP and 5 QX-314 (Cl-salt); pH 7.20–7.25; 288–295 mOsm/kg). Optogenetic manipulations : For optogenetic stimulation of the M1-GPi pathway, following injections of ChR2, eNpHR3.0, or mCherry virus, optical fibers (200 µm O.D., 0.37 NA; Blackrock Microsystems, ASIA) were unilaterally implanted 200–300 µm above the GPi. All fibers were secured to the skull with bone screws and dental cement. [ 40 ] Mice were allowed to recover and express the virus for 3–4 weeks. They were then habituated for 1 minute after being connected to a laser source, after which the behavioral tests were performed. Different lasers (SLOC, China) were used to deliver laser light to mice expressing ChR2, eNpHR3.0, and mCherry at wavelengths of 473 nm (blue, 4–6 mW) or 589 nm (yellow, 10 mW). The Doric software (Doric, Canada) was utilized to control the illumination parameters: blue light at 473 nm (30 Hz, 5 ms pulse width) and yellow light at 589 nm (100 Hz). Chemogenetic manipulations : Clozapine N-oxide (CNO, Sigma-Aldrich, Cat#C0832; CAS: 34233-69-7) stock solution was prepared in dimethyl sulfoxide (DMSO, Beyotime Biotechnology, CAS: 67-68-5) at a concentration of 25 mg/mL and stored at -80°C. To prepare the working solution for experiments, CNO was diluted with 0.9% saline to a concentration of 0.125 mg/mL. Mice were intraperitoneally injected with either 0.9% saline (3 mg/kg) or CNO (3 mg/kg). Mice injected with CNO were allowed to rest for 40 minutes before behavioral tests. Rotarod test A rotarod apparatus was applied to assess the motor coordination of mice (UGO BASILE, Italy). This test was performed as described by Li. [ 40 ] Briefly, mice were trained twice per day over two consecutive days separately at 11 rpm on day 1 and 22 rpm on day 2. On the testing day, mice were tested at a speed ranging from 4 to 40 rpm within 120 seconds, repeated three times with a 1-hour inter-training interval. The test results were obtained by averaging the latency of each mouse to fall off the accelerating rotarod across the three trials. Open field test (OFT) The open field chamber (50 cm × 50 cm × 50 cm) was used to evaluate locomotor activity. Total distance moved (cm), motor velocity (cm/s), immobility time (s), center distance (cm), low-speed (movement speed < 5 cm/s), middle-speed (5 cm/s < movement speed 15 cm/s) were analyzed by the SuperMaze system (Xinrun, China). The center was defined as the central 25% of the arena. In PD behavior and chemogenetic behavior tests, each mouse was placed in the center of the square box and allowed to freely move for 10 min. For optogenetic behavioral tests, the test time was divided into three consecutive 30s or 120s epochs in the Pre–Light–Post periods. Cylinder test : Mice were placed in a transparent 500 mL beaker. Rearing times, net contralateral rotations, and forelimb contact times were scored blindly. Rearing was defined as the body raised and both forelimbs off the ground. [ 40 ] Rotations were defined as each 270° rotation that contained no turn of > 90° in the opposite direction. The left forelimb preference was calculated using the following equation: left forelimb touches / (left forelimb touches + right forelimb touches + simultaneous touches) * 100. In PD behavior and chemogenetic behavior tests, each mouse was videotaped for 5 minutes. For optogenetic behavioral tests, the test time was divided into three consecutive 60s or 180s epochs in the Pre–Light–Post periods. Apomorphine (APO)-Induced rotation test : Two weeks after the 6-OHDA injection, APO (0.5 mg/kg dissolved in 0.9% saline, Cat#: H116-5 MG, Sigma-Aldrich) was administered by intraperitoneal injection. Mice were placed into a 500 mL beaker and videotaped for 20 minutes. The net number of contralateral rotations (number of contralateral rotations – number of ipsilateral rotations) was recorded. PD was confirmed in mice with a net number of contralateral rotations > 5 turns/min. Tail suspension test (TST) The mice tails were wrapped with tape at approximately 2–3 cm from the end of the tail. The mice were then fixed upside down on a horizontal bar with the nose tip about 30 cm above the ground. Animal behaviors were recorded for 6 min and the immobility time was scored during the last 4 min. Forced swim test (FST) Animals were individually placed in clear 5000 ml glass beaker (17.8 cm in diameter, 26 cm in height) filled with water (21–25°C). Water depth was set to prevent mice from touching the glass bottom with their limbs or tails. The test lasted for 6 min and the immobility time was counted from the last 4 min. Mice were regarded as immobile when floating motionless or making only movements that were necessary to hold its head above the water. Sucrose preference test (SPT) Mice were single housed and habituated with 1% sucrose and water for 2 days and the bottle positions were counterbalanced every 24 h. On the testing day, mice were water-deprived for 24 h and then exposed to pre-weighed bottles (one bottle of water and one bottle of 1% sucrose) for 2 h in the dark phase. Sucrose preference was calculated by dividing the consumption of sucrose by the total liquid consumption (water and sucrose). Statistical analysis All data were presented as mean ± SEM. Graphing and statistical analysis were performed using GraphPad Prism 8.0 software (GraphPad Software, USA). Normal distribution was determined by the Shapiro-Wilk and Kolmogorov-Smirnov normality tests. Data meeting normal distribution criteria were analyzed using unpaired t-tests in two-sided manner for single factors, and data not meeting normal distribution criteria were analyzed using the Mann-Whitney tests in two-sided manner. One-way ANOVA with Tukey test and Two-way ANOVA with Bonferroni test were used for multiple comparisons. We represent p values in all figures as * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, and ns, no statistically-significant difference. Declarations Conflict of Interest The authors declare no conflict of interest. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 31971103 to Y. Wang). Y.-Q. Li, X.-P. Zhang, R.-B. Zheng contributed equally to this work. References Y. Zhang, D. S. Roy, Y. Zhu, Y. Chen, T. Aida, Y. Hou, C. Shen, N. E. Lea, M. E. Schroeder, K. M. Skaggs, H. A. Sullivan, K. B. Fischer, E. M. Callaway, I. R. Wickersham, J. Dai, X. M. Li, Z. Lu, G. Feng, Nature 2022 , 607 (7918), 321, https://doi.org/10.1038/s41586-022-04806-x. R. L. Albin, A. B. Young, J. B. Penney, Trends Neurosci 1989 , 12 (10), 366, https://doi.org/10.1016/0166-2236(89)90074-x. W. Poewe, Eur J Neurol 2008 , 15 Suppl 1 , 14, https://doi.org/10.1111/j.1468-1331.2008.02056.x. M. R. DeLong, T. Wichmann, Arch Neurol 2007 , 64 (1), 20, https://doi.org/10.1001/archneur.64.1.20. A. Nambu, H. Tokuno, M. Takada, Neurosci Res 2002 , 43 (2), 111, https://doi.org/10.1016/s0168-0102(02)00027-5. Y. Y. Wang, Y. Wang, H. F. Jiang, J. H. Liu, J. Jia, K. Wang, F. Zhao, M. H. Luo, M. M. Luo, X. M. Wang, Exp Neurol 2018 , 300 , 135, https://doi.org/10.1016/j.expneurol.2017.11.006. M. L. Wallace, A. Saunders, K. W. Huang, A. C. Philson, M. Goldman, E. Z. Macosko, S. A. McCarroll, B. L. Sabatini, Neuron 2017 , 94 (1), 138, https://doi.org/10.1016/j.neuron.2017.03.017. Y. Liu, W. Li, C. Tan, X. Liu, X. Wang, Y. Gui, L. Qin, F. Deng, C. Hu, L. Chen, J Neurosurg 2014 , 121 (3), 709, https://doi.org/10.3171/2014.4.Jns131711. V. Lilascharoen, E. H. Wang, N. Do, S. C. Pate, A. N. Tran, C. D. Yoon, J. H. Choi, X. Y. Wang, H. Pribiag, Y. G. Park, K. Chung, B. K. Lim, Nat Neurosci 2021 , 24 (4), 504, https://doi.org/10.1038/s41593-021-00810-y. J. Song, H. Lin, S. Liu, Network 2023 , 34 (1-2), 84, https://doi.org/10.1080/0954898x.2023.2173816. A. Naito, H. Kita, Brain Res 1994 , 653 (1-2), 251, https://doi.org/10.1016/0006-8993(94)90397-2. Y. Smith, T. Wichmann, Mov Disord 2015 , 30 (3), 293, https://doi.org/10.1002/mds.26095. D. Milardi, M. Gaeta, S. Marino, A. Arrigo, G. Vaccarino, E. Mormina, G. Rizzo, C. Milazzo, G. Finocchio, A. Baglieri, G. Anastasi, A. Quartarone, Mov Disord 2015 , 30 (3), 342, https://doi.org/10.1002/mds.25995. F. Karube, S. Takahashi, K. Kobayashi, F. Fujiyama, Elife 2019 , 8 , https://doi.org/10.7554/eLife.49511. A. Cacciola, A. Calamuneri, D. Milardi, E. Mormina, G. Chillemi, S. Marino, A. Naro, G. Rizzo, G. Anastasi, A. Quartarone, Front Neuroanat 2017 , 11 , 85, https://doi.org/10.3389/fnana.2017.00085. H. Hu, Y. Cui, Y. Yang, Nat Rev Neurosci 2020 , 21 (5), 277, https://doi.org/10.1038/s41583-020-0292-4. M. Chen, S. Ma, H. Liu, Y. Dong, J. Tang, Z. Ni, Y. Tan, C. Duan, H. Li, H. Huang, Y. Li, X. Cao, C. J. Lingle, Y. Yang, H. Hu, Science 2024 , 385 (6709), eado7010, https://doi.org/10.1126/science.ado7010. X. Liu, H. Li, R. Ma, X. Tong, J. Wu, X. Huang, K. F. So, Q. Tao, L. Huang, S. Lin, C. Ren, Adv Sci (Weinh) 2024 , 11 (30), e2401059, https://doi.org/10.1002/advs.202401059. H. Y. Li, M. Z. Zhu, X. R. Yuan, Z. X. Guo, Y. D. Pan, Y. Q. Li, X. H. Zhu, Cell 2023 , 186 (7), 1352, https://doi.org/10.1016/j.cell.2023.02.036. Z. Polyakova, S. Chiken, N. Hatanaka, A. Nambu, J Neurosci 2020 , 40 (39), 7451, https://doi.org/10.1523/jneurosci.0772-20.2020. N. N. Foster, J. Barry, L. Korobkova, L. Garcia, L. Gao, M. Becerra, Y. Sherafat, B. Peng, X. Li, J. H. Choi, L. Gou, B. Zingg, S. Azam, D. Lo, N. Khanjani, B. Zhang, J. Stanis, I. Bowman, K. Cotter, C. Cao, S. Yamashita, A. Tugangui, A. Li, T. Jiang, X. Jia, Z. Feng, S. Aquino, H. S. Mun, M. Zhu, A. Santarelli, N. L. Benavidez, M. Song, G. Dan, M. Fayzullina, S. Ustrell, T. Boesen, D. L. Johnson, H. Xu, M. S. Bienkowski, X. W. Yang, H. Gong, M. S. Levine, I. Wickersham, Q. Luo, J. D. Hahn, B. K. Lim, L. I. Zhang, C. Cepeda, H. Hintiryan, H. W. Dong, Nature 2021 , 598 (7879), 188, https://doi.org/10.1038/s41586-021-03993-3. M. M. McGregor, A. B. Nelson, Neuron 2019 , 101 (6), 1042, https://doi.org/10.1016/j.neuron.2019.03.004. M. R. DeLong, Trends Neurosci 1990 , 13 (7), 281, https://doi.org/10.1016/0166-2236(90)90110-v. F. Blandini, G. Nappi, C. Tassorelli, E. Martignoni, Prog Neurobiol 2000 , 62 (1), 63, https://doi.org/10.1016/s0301-0082(99)00067-2. Y. Chen, Z. Hong, J. Wang, K. Liu, J. Liu, J. Lin, S. Feng, T. Zhang, L. Shan, T. Liu, P. Guo, Y. Lin, T. Li, Q. Chen, X. Jiang, A. Li, X. Li, Y. Li, J. J. Wilde, J. Bao, J. Dai, Z. Lu, Cell 2023 , 186 (24), 5394, https://doi.org/10.1016/j.cell.2023.10.004. H. L. Combs, B. S. Folley, D. T. Berry, S. C. Segerstrom, D. Y. Han, A. J. Anderson-Mooney, B. D. Walls, C. van Horne, Neuropsychol Rev 2015 , 25 (4), 439, https://doi.org/10.1007/s11065-015-9302-0. Z. G. Tan, Q. Zhou, T. Huang, Y. Jiang, Clin Interv Aging 2016 , 11 , 777, https://doi.org/10.2147/cia.S105505. J. Zhang, J. Li, F. Chen, X. Liu, C. Jiang, X. Hu, L. Ma, Z. Xu, Clin Neurol Neurosurg 2021 , 201 , 106450, https://doi.org/10.1016/j.clineuro.2020.106450. K. W. McCairn, A. Iriki, M. Isoda, J Neurophysiol 2015 , 114 (4), 2090, https://doi.org/10.1152/jn.00223.2015. K. W. McCairn, R. S. Turner, J Neurophysiol 2015 , 113 (7), 2537, https://doi.org/10.1152/jn.00701.2014. J. O. Dostrovsky, A. M. Lozano, Mov Disord 2002 , 17 Suppl 3 , S63, https://doi.org/10.1002/mds.10143. J. L. Vitek, Mov Disord 2002 , 17 Suppl 3 , S69, https://doi.org/10.1002/mds.10144. C. C. McIntyre, M. Savasta, L. Kerkerian-Le Goff, J. L. Vitek, Clin Neurophysiol 2004 , 115 (6), 1239, https://doi.org/10.1016/j.clinph.2003.12.024. A. Muralidharan, J. Zhang, D. Ghosh, M. D. Johnson, K. B. Baker, J. L. Vitek, Brain Stimul 2017 , 10 (1), 126, https://doi.org/10.1016/j.brs.2016.10.005. Q. Li, Y. Ke, D. C. Chan, Z. M. Qian, K. K. Yung, H. Ko, G. W. Arbuthnott, W. H. Yung, Neuron 2012 , 76 (5), 1030, https://doi.org/10.1016/j.neuron.2012.09.032. L. A. Johnson, J. Wang, S. D. Nebeck, J. Zhang, M. D. Johnson, J. L. Vitek, J Neurosci 2020 , 40 (10), 2166, https://doi.org/10.1523/jneurosci.2480-19.2020. F. Faivre, A. Joshi, E. Bezard, M. Barrot, Neurosci Biobehav Rev 2019 , 96 , 335, https://doi.org/10.1016/j.neubiorev.2018.10.004. J. S. Reijnders, U. Ehrt, W. E. Weber, D. Aarsland, A. F. Leentjens, Mov Disord 2008 , 23 (2), 183, https://doi.org/10.1002/mds.21803. K. J. Mastro, K. T. Zitelli, A. M. Willard, K. H. Leblanc, A. V. Kravitz, A. H. Gittis, Nat Neurosci 2017 , 20 (6), 815, https://doi.org/10.1038/nn.4559. L. X. Li, Y. L. Li, J. T. Wu, J. Z. Song, X. M. Li, Neurosci Bull 2022 , 38 (1), 1, https://doi.org/10.1007/s12264-021-00775-9. Additional Declarations There is NO Competing Interest. Supplementary Files Supplementaryinformation.docx Supplementary information Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5990552","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":415453471,"identity":"1a90c93c-4721-4fe8-b6ce-cb04044fb4f2","order_by":0,"name":"Yong Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYBACCWYogx9KMzYQrUWygWgtMIbBAWK1SLbzHn7NU3HHbvPxw0838zDYyG44wPzsAT4t0sx8adY8Z54lbzuTZnabhyHNeMMBNnMDfFrkmHnMjHnbDieb3eBhA2o5nLjhAA+bBFFajGeAtfwnrEWamcf4MVCLnYEEWMsBwlokm3nMGOecOZwgAfTLzTkGycYzD7OZ4dUicf6M8Yc3FYft+dsPP7vxpsJOtu948zO8WoCATYqHgSGxAcwGBRUzXtVgwPzxBwODPWF1o2AUjIJRMGIBANoSRVj53fGSAAAAAElFTkSuQmCC","orcid":"","institution":"Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Capital Medical University, Beijing, 100069, China.","correspondingAuthor":true,"prefix":"","firstName":"Yong","middleName":"","lastName":"Wang","suffix":""},{"id":415453472,"identity":"af2e6d1e-3920-4a65-a0a5-dcd9f6c49205","order_by":1,"name":"Yaqian Li","email":"","orcid":"","institution":"Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Capital Medical University, Beijing, 100069, China.","correspondingAuthor":false,"prefix":"","firstName":"Yaqian","middleName":"","lastName":"Li","suffix":""},{"id":415453473,"identity":"3099da75-f78c-4bb9-9efc-4dd4e0587b5e","order_by":2,"name":"Xueping Zhang","email":"","orcid":"","institution":"Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Capital Medical University, Beijing, 100069, China.","correspondingAuthor":false,"prefix":"","firstName":"Xueping","middleName":"","lastName":"Zhang","suffix":""},{"id":415453474,"identity":"3ba34b8e-c2e9-4a9a-be5e-22b66db13055","order_by":3,"name":"Ruobing Zheng","email":"","orcid":"","institution":"Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Capital Medical University, Beijing, 100069, China.","correspondingAuthor":false,"prefix":"","firstName":"Ruobing","middleName":"","lastName":"Zheng","suffix":""},{"id":415453475,"identity":"733c8ade-7fc9-4d60-87f3-b3d2aa1f9753","order_by":4,"name":"Qianwen Wang","email":"","orcid":"","institution":"Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Capital Medical University, Beijing, 100069, China.","correspondingAuthor":false,"prefix":"","firstName":"Qianwen","middleName":"","lastName":"Wang","suffix":""},{"id":415453476,"identity":"6309cd62-9a52-4d89-a882-730ffbc90cd7","order_by":5,"name":"Zikang Liu","email":"","orcid":"","institution":"Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Capital Medical University, Beijing, 100069, China.","correspondingAuthor":false,"prefix":"","firstName":"Zikang","middleName":"","lastName":"Liu","suffix":""},{"id":415453477,"identity":"3a970690-09a2-4a1a-8732-c1d90ae44b4e","order_by":6,"name":"Derong Li","email":"","orcid":"","institution":"Departments of Basic Medical Sciences, School of Basic Medical Sciences, Capital Medical University, Beijing, 100069, China.","correspondingAuthor":false,"prefix":"","firstName":"Derong","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-02-09 05:00:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5990552/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5990552/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":76312757,"identity":"de6c0cc5-a6d5-4f27-a5a0-102eaacac2fd","added_by":"auto","created_at":"2025-02-14 16:03:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1168636,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDirect M1-GPi projection with mono-synaptic connection and secondary projections to LHb and thalamus.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Schematic of antegrade tracing strategy for the direct M1-GPi pathway. The M1 is injected with AAV2/9-EF1α-EYFP.\u003c/p\u003e\n\u003cp\u003eB) Representative images showing direct M1 inputs projecting to the GPi, as demonstrated by anterograde tracing. Neurons in the motor cortex M1 are robustly labeled by expressed EYFP. The filamentous axonal fibers in the GPi are visible in green and originate from M1.\u003c/p\u003e\n\u003cp\u003eC) Schematic of the retrograde tracing strategy for the direct M1-GPi pathway. The GPi is injected with AAVretro-Cre-mCherry.\u003c/p\u003e\n\u003cp\u003eD) Representative images showing direct M1 inputs projecting to the GPi, as demonstrated by retrograde tracing. Neurons in the motor cortex M1 are robustly labeled by the retrograde expression of mCherry.\u003c/p\u003e\n\u003cp\u003eE) Schematic of the electrophysiological experiment using whole-cell patch-clamp technique. An optic fiber is implanted over the GPi to optogenetic-activate the axon terminal of M1 glutamatergic neurons that directly project to the GPi.\u003c/p\u003e\n\u003cp\u003eF) Representative tracing of neuronal firing demonstrates that optogenetic activation of glutamatergic terminals from M1 neurons leads to an increased firing frequency of action potentials in GPi neurons.\u003c/p\u003e\n\u003cp\u003eG) Schematic of anterograde trans-monosynaptic tracing. The AAV2/1-hsyn-Cre injected into M1 trans-monosynapticly infects GPi and express the Cre protein. A subsequent AAV2/9-hSyn-DIO-mCherry injection labels GPi neurons innervated by M1 inputs via expressed Cre.\u003c/p\u003e\n\u003cp\u003eH) Representative images of trans-monosynaptic projection of M1 to GPi. The GPi neurons directly innervated by M1 are labeled with red fluorescence.\u003c/p\u003e\n\u003cp\u003eI-J) Representative images show the M1-innervated GPi neurons secondarily project to the LHb (I) and the VAL (J).\u003c/p\u003e\n\u003cp\u003eK) Schematic of oEPSC experiment. The AAV2/1-hSyn-Cre and AAV2/9-CaMKIIα-hChR2(H134R)-mCherry are co-injected into M1, while AAV-hSyn-DIO-mCherry is injected into the GPi. Axonal terminals from M1 are optogenetic-activated and electrophysiological responses are recorded in GPi neurons innervated by M1. This setup exclusively observes and records synaptic transmission of the direct M1-GPi pathway.\u003c/p\u003e\n\u003cp\u003eL) Representative tracing depicts increased action potential firing in GPi neurons with optogenetic activation of M1 neuron terminals.\u003c/p\u003e\n\u003cp\u003eM) Representative tracing illustrates oEPSC in GPi neurons. Optogenetic activation of M1 axonal terminals enhances the oEPSC amplitude in GPi neurons innervated by M1.\u003c/p\u003e\n\u003cp\u003eN) Histogram of oEPSC amplitudes in GPi neurons. Optogenetic activation of M1 neuron terminals significantly elevates the oEPSC amplitude (n = 4 neurons, 2 mice. Unpaired t-test in two-sided manner, **p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5990552/v1/db47653f8f85b9730f8c66f7.png"},{"id":76312756,"identity":"a6df9191-3486-4a3d-ae91-3b089495d7cc","added_by":"auto","created_at":"2025-02-14 16:03:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1016078,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe glutamatergic M1-GPi pathway modulates motor behavior in mice, similarly to optogenetic activation of the unilateral M1-STN hyperdirect pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Schematic of optogenetic activation experiments and motor behavior tests. Unilateral injection of AAV2/9-CaMKII-ChR2-mCherry makes the glutamatergic neurons in the M1 expressing ChR2. Optic fibers are implanted over GPi to activate the axon terminal from the M1 glutamatergic neurons at 473 nm.\u003c/p\u003e\n\u003cp\u003eB) Representative images illustrate that ChR2 is expressed in glutamatergic M1 neurons which project to the GPi.\u003c/p\u003e\n\u003cp\u003eC) Rotarod Test. Unilateral activation of glutamatergic M1 neurons in the ChR2-expressed mice results in a significant decrease in motor coordination compared to the mCherry mice. (n = 6 mice for the mCherry group, n = 6 mice for the ChR2 group. Two-way ANOVA with Bonferroni’s multiple comparisons test, *p \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003eD) Rearing in cylinder test is significantly increased with optogenetic activation of the unilateral axon terminal of the glutamatergic M1-GPi pathway. (n = 6-7 mice for the mCherry group, n = 6 mice for the ChR2 group. Two-way ANOVA with Bonferroni’s multiple comparisons test, **p \u0026lt; 0.01).\u003c/p\u003e\n\u003cp\u003eE) Total distance of OFT. No significant changes found. (n = 6 mice for the mCherry group, n = 6 mice for the ChR2 group. Two-way ANOVA with Bonferroni’s multiple comparisons test, n.s., no statistically-significant difference).\u003c/p\u003e\n\u003cp\u003eF) Schematic of optogenetic inhibition. Injecting AAV2/1-hsyn-Cre into M1 and AAV2/9-hEFLa-DIO-eNpHR3.0-mCherry into GPi leads to specific expression of eNpHR3.0 in GPi neurons innervated by M1. This configuration allows for the trans-monosynaptic inhibition of the M1-GPi pathway at 589 nm.\u003c/p\u003e\n\u003cp\u003eG) Representative images illustrate post-synaptic expression of eNpHR3.0 in the GPi.\u003c/p\u003e\n\u003cp\u003eH) Rotarod tests show that post-synaptic inhibition of the M1-GPi pathway significantly decreases motor coordination. (n = 9-10 mice for the mCherry group, n = 14-15 mice for the eNpHR group. Two-way ANOVA with Bonferroni’s multiple comparisons test, **p \u0026lt; 0.01).\u003c/p\u003e\n\u003cp\u003eI) Rearing in cylinder test is unaffected by optogenetic inhibition of the post-synaptic M1-GPi pathway in the eNpHR-expressed mice. (n = 10 mice for the mCherry group, n = 13-15 mice for the eNpHR group. Two-way ANOVA with Bonferroni’s multiple comparisons test, n.s., no statistically-significant difference).\u003c/p\u003e\n\u003cp\u003eJ) OFT. No significant changes found in the total distance by optogenetic inhibition. (n = 10 mice for the mCherry group, n = 15 mice for the eNpHR group. Two-way ANOVA with Bonferroni’s multiple comparisons test, n.s., no statistically-significant difference).\u003c/p\u003e\n\u003cp\u003eK) Schematic of the optogenetic experiment and motor behavior test. The ChR2 is expressed in the M1 glutamatergic neurons through unilateral injection of AAV2/9-CaMKII-ChR2-mCherry. An optic fiber is implanted over the STN to activate the axon terminal of the glutamatergic neurons from the M1.\u003c/p\u003e\n\u003cp\u003eL) Representative images depict the expression of ChR2 in M1 glutamatergic neurons that projection of M1 to STN.\u003c/p\u003e\n\u003cp\u003eM) Rotarod test. Optogenetic activation of the M1-STN glutamatergic axon terminal significantly decreases motor coordination in mice expressing ChR2, compared to those expressing mCherry. (n = 6 mice for the mCherry group, n = 6 mice for the ChR2 group. Two-way ANOVA with Bonferroni’s multiple comparisons test, n.s., no statistically-significant difference, *p \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003eN) Cylinder test. Optogenetic activation of glutamatergic M1-STN pathway leads to significantly increased left forelimb preference. (n = 5 mice for the mCherry group, n = 7 mice for the ChR2 group. Two-way ANOVA with Bonferroni’s multiple comparisons test, n.s., no statistically-significant difference, *p \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003eO) OFT. No significant changes in total distance. (n = 6 mice for the mCherry group, n = 7 mice for the ChR2 group. Two-way ANOVA with Bonferroni’s multiple comparisons test, n.s., no statistically-significant difference).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5990552/v1/1d5d82aca6687579f1407500.png"},{"id":76313544,"identity":"df6d3bb5-95b2-45dc-bde0-bf957b74e283","added_by":"auto","created_at":"2025-02-14 16:11:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":324643,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMotor behavior and depressive-like behavior in the mice with unilateral 6-OHDA lesioned SNc.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Schematic of the experiment. Unilateral injection of 6-OHDA into the SNc of mice is conducted. The motor behavior assessments encompass APO-induced rotation test (APO-rotation), rotarod test (Rotarod), OFT, and cylinder test (Cylinder). The assessment of depressive-like behaviors includes the tail suspension test (TST), forced swimming test (FST), sucrose preference test (SPT), and novelty suppressed food test (NSF).\u003c/p\u003e\n\u003cp\u003eB) Representative images and quantitation reveal the loss of both dopaminergic neurons in SNc and dopaminergic fibers in stratum of mice with unilateral 6-OHDA lesioned SNc. (n = 6 mice for the Sham group, n = 6 mice for the 6OHDA group. Unpaired t-test in two-sided manner, ****p \u0026lt; 0.0001).\u003c/p\u003e\n\u003cp\u003eC-I) Motor behavior test. Unilateral 6-OHDA lesions lead to significantly increased APO-induced contralateral rotations (C), right forelimb preference in cylinder test (F), and immobility in OFT (I). They also cause significantly decreased latency to fall in rotarod test (D), rearing in cylinder test (E), as well as total distance (G) and velocity in OFT (H). (n = 6 mice for the Sham group, n = 7-9 mice for the 6OHDA group. Unpaired t-test in two-sided manner for E, G, and I, Mann-Whitney test in two-sided manner for C, D, F, and H, *p \u0026lt; 0.05, ***p \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003eJ-M) Depressive-like behavior tests. Unilateral 6-OHDA lesion leads to a significantly increased immobility in the tail suspension test (J) but no significant changes in the forced swim test (K), sucrose preference test (L), or novelty-suppressed feeding test (M). (n = 6 mice for the Sham group, n = 8-9 mice for the 6OHDA group. Unpaired t-test in two-sided manner for J, K, and L, Mann-Whitney test in two-sided manner for M, n.s., no statistically-significant difference, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5990552/v1/ec2b3516b643d6f7e9e9dfbc.png"},{"id":76313546,"identity":"74fe618f-4f61-4049-b0dc-d8317bb03bde","added_by":"auto","created_at":"2025-02-14 16:11:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1206555,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of unilateral ablation of pre-synaptic M1-GPi pathway on motor behaviors in mice under normal and PD conditions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Schematic of experiments. AAVretro-Cre-mCherry is injected into GPi and AAV-DIO-taCasp3-EGFP is injected into M1. SNc is lesioned with 6-OHDA in mice.\u003c/p\u003e\n\u003cp\u003eB) Representative images display expression of mCherry and EGFP in GPi and M1. In M1, pyramidal neurons labeled in green or yellow demonstrate expression of DIO-EGFP induced by retrogradely expressed Cre from GPi. Green EGFP labeling in the GPi represents the axon terminal from M1.\u003c/p\u003e\n\u003cp\u003eC-D) Ablation of M1 neurons induced by taCasp3 expression. (n = 8 mice for the EGFP group, n = 12 mice for the taCasp3 group. Mann-Whitney test in two-sided manner, ****p \u0026lt; 0.0001).\u003c/p\u003e\n\u003cp\u003eE-J) Motor behaviors test. Unilateral ablation of the pre-synaptic M1-GPi pathway results in a decreased trend in motor coordination in rotarod test (E) with a p value of 0.0588. No significant changes in OFT (F-G) or cylinder test (H-J). (n = 8 mice for the EGFP group, n = 12 mice for the taCasp3 group. Unpaired t-test in two-sided manner for E-F and H-J, Mann-Whitney test in two-sided manner for G, n.s., no statistically-significant difference).\u003c/p\u003e\n\u003cp\u003eK-P) Motor behaviors with both 6-OHDA lesions and ablation of the pre-synaptic M1-GPi pathway. A significant decrease seen in rearing of cylinder test (N), while no significant changes are observed in the rotarod test (K), OFT (L-M), and other aspects of the cylinder test (O-P). (n = 7-8 mice for the EGFP+6OHDA group, n = 11-12 mice for the taCasp3+6OHDA group. Unpaired t-test in two-sided manner for K-O, Mann-Whitney test in two-sided manner for P, n.s., no statistically-significant difference, *p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5990552/v1/5dee3c53140f2593c147ef4b.png"},{"id":76312760,"identity":"b220e2a3-5972-4c89-9b91-ee2ec9949b49","added_by":"auto","created_at":"2025-02-14 16:03:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":804577,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBilaterally chemogenetic manipulation of the pre-synaptic M1-GPi pathway does not have a significant impact on PD symptoms.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Schematics of experiments. AAVretro-Cre-mCherry is injected into GPi. Injection of AAV-DIO-hM3D-EGFP or AAV-DIO-hM4D-EGFP into M1, in the presence of Cre, ensures the specific expression of hM3D or hM4D proteins in the M1 neurons that project to the GPi. The SNc is lesioned using 6-OHDA, and motor behaviors are assessed.\u003c/p\u003e\n\u003cp\u003eB) Representative images display that in M1, neurons labeled in green or yellow (arrow) demonstrate EGFP expression induced by retrograde Cre from GPi. In GPi, axons from M1 neurons expressing EGFP are represented by green fibers.\u003c/p\u003e\n\u003cp\u003eC-I) Motor behavior test. Inhibition with hM4D results in a significant increase in the percentage of high speed movement in OFT (G), compared to the EGFP group in the 6-OHDA lesioned mice. But neither the activation of hM3D nor the inhibition of hM4D leads to significant changes in performance on rotarod (C), total distance (D), velocity (E), immobility (F), middle speed movement percentage (H), and low speed movement percentage (I) in OFT, compared to the EGFP group in the 6-OHDA lesioned mice. Intriguingly, in the 6-OHDA lesioned mice, the hM4D inhibition results in a significant increase in rotarod (C), velocity (E), and the percentage of high speed movements (G), while significant decrease in immobility (F) of OFT, compared to the hM3D activation. (n = 14-16 mice for the EGFP+6OHDA group, n = 10-15 mice for the hM3D+6OHDA group, n = 12-15 mice for the hM4D+6OHDA group. One-way ANOVA with Tukey’s multiple comparisons test, n.s., no statistically-significant difference, *p \u0026lt; 0.05, **p \u0026lt; 0.01).\u003c/p\u003e\n\u003cp\u003eJ-M) Depressive-like behavior tests. Neither the activation of hM3D nor the inhibition of hM4D in the 6-OHDA lesioned mice leads to significant changes in performance on TST (J), FST (K), SPT (L) and center time in OFT (M), compared to the EGFP group. (n = 8-16 mice for the EGFP+6OHDA group, n = 9-13 mice for the hM3D+6OHDA group, n = 7-15 mice for the hM4D+6OHDA group. One-way ANOVA with Tukey’s multiple comparisons test, n.s., no statistically-significant difference, *p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5990552/v1/e6b8d0c0e408759af730f872.png"},{"id":76313549,"identity":"283b8b88-3bc6-4710-87e7-32995a21b129","added_by":"auto","created_at":"2025-02-14 16:11:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":748208,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBilateral chemogenetic activation of the post-synaptic M1-GPi pathway alleviates depressive-like symptoms in PD mice while exacerbating motor symptoms.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Schematics of experiment. The injected AAV2/1-hsyn-Cre in M1 trans-monosynaptic infects GPi and induced post-synaptic expression of the hM3D-mCherry in GPi with AAV2/9-hsyn-DIO-hM3D-mCherry injected. The SNc is lesioned by injection of 6-OHDA. The motor behavior and depressive-like behaviors are assessed after 45 minutes following intraperitoneal injections of CNO.\u003c/p\u003e\n\u003cp\u003eB) Representative image shows the post-synaptic expression of hM3D-mCherry in the GPi. And GPi neurons innervated by M1 neurons secondarily project to both the LHb and the thalamus (VAL).\u003c/p\u003e\n\u003cp\u003eC) Rotarod test. Chemogenetic activation of post-synapticM1-GPi pathway leads to significant decreased motor coordination in 6-OHDA lesioned mice (the hM3D+6OHDA), compared to the mCherry mice (the mCherry+6OHDA). (n = 7 mice for the mCherry+Sham group, n = 8 mice for the hM3D+Sham group, n = 6 mice for the mCherry+6OHDA group, n = 7 mice for the hM3D+6OHDA group. One-way ANOVA with Tukey’s multiple comparisons test, n.s., no statistically-significant difference, *p \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003eD) Rearing of the cylinder test. No significant changes found in 6-OHDA lesioned mice. (n = 7 mice for the mCherry+Sham group, n = 8 mice for the hM3D+Sham group, n = 10 mice for the mCherry+6OHDA group, n = 8 mice for the hM3D+6OHDA group. One-way ANOVA with Tukey’s multiple comparisons test, n.s., no statistically-significant difference).\u003c/p\u003e\n\u003cp\u003eE-J) OFT. Chemogenetic activation of post-synaptic M1-GPi pathway leads to significantly decreased motor activities in the 6-OHDA lesioned mice, including decreased total distance (E), decreased motor velocity (F), increased immobility (G), decreased high speed movement percentage (H) and middle speed movement percentage (I) (the mCherry+6OHDA group v.s. the hM3D+6OHDA group). (n = 7 mice for the mCherry+Sham group, n = 8 mice for the hM3D+Sham group, n = 8 mice for the mCherry+6OHDA group, n = 8-9 mice for the hM3D+6OHDA group. One-way ANOVA with Tukey’s multiple comparisons test, n.s., no statistically-significant difference, *p \u0026lt; 0.05, **p \u0026lt; 0.01).\u003c/p\u003e\n\u003cp\u003eK-N) Depressive-like behavior. Chemogenetic activation of post-synaptic M1-GPi pathway leads to significantly decreased immobility in FST (L) and increased SPT (M) in the 6-OHDA lesioned mice (the mCherry+6OHDA group v.s. the hM3D+6OHDA group). (n = 7-9 mice for the mCherry+6OHDA group, n = 6-9 mice for the hM3D+6OHDA group. Unpaired t-test in two-sided manner for K, L, and M, Mann-Whitney test in two-sided manner for N, n.s., no statistically-significant difference, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5990552/v1/a6ba3beabd6884567462d6fd.png"},{"id":76312769,"identity":"4a7b4d24-f18f-49b1-abec-fe2cb492eb62","added_by":"auto","created_at":"2025-02-14 16:03:12","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":522496,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBilaterally chemogenetic Inhibition of post-synaptic M1-GPi pathway rescues motor symptoms but has no effects on the depressive-like symptoms of PD mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Schematics of experiments. The AAV2/1-hsyn-Cre is injected into M1, trans-monosynapticly expressing Cre protein in GPi. In GPi, neurons innervated by M1 inputs specifically express hM4D protein with EGFP tag through injection of AAV2/9-hsyn-DIO-hM4D-EGFP. The SNc is lesioned by 6-OHDA.\u003c/p\u003e\n\u003cp\u003eB) Representative images illustrate the post-synaptic expression of the hM4D-EGFP protein in GPi. And GPi neurons innervated by M1 neurons secondary project to LHb and VAL.\u003c/p\u003e\n\u003cp\u003eC-F) Motor behavior test. Chemogenetic inhibition of the post-synaptic M1-GPi pathway rescues the PD motor symptoms of 6-OHDA lesioned mice. This is illustrated by the significantly increased motor coordination in rotarod test (C) and significantly increased motor activities in total distance (D), velocity (E), and decreased immobility (F) of OFT (the EGFP+6OHDA group v.s. the hM4D+6OHDA group). (n = 10 mice for the EGFP+Sham group, n = 8-9 mice for the hM4D+Sham group, n = 10-12 mice for the EGFP+6OHDA group, n = 12-13 mice for the hM4D+6OHDA group. One-way ANOVA with Tukey’s multiple comparisons test, n.s., no statistically-significant difference, *p \u0026lt; 0.05, **p \u0026lt; 0.01).\u003c/p\u003e\n\u003cp\u003eG-I) Chemogenetic inhibition of post-synaptic M1-GPi pathway has no significant effects on the PD related depressive-like behavior in 6-OHDA lesioned mice, illustrated by no significant changes in TST (G), SPT (H) and center time of OFT (I). (n = 8-10 mice for the EGFP+Sham group, n = 8-9 mice for the hM4D+Sham group, n = 9-11 mice for the EGFP+6OHDA group, n = 12 mice for the hM4D+6OHDA group. One-way ANOVA with Tukey’s multiple comparisons test, n.s., no statistically-significant difference).\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-5990552/v1/ada138fd569a93aa68045733.png"},{"id":76312764,"identity":"a8767422-b76c-4592-8cf6-d3856036a394","added_by":"auto","created_at":"2025-02-14 16:03:12","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":843196,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic summary of the direct M1-GPi pathway and its function.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe direct M1-GPi pathway, bypassing classical direct, indirect and hyperdirect pathways, plays an essential role in motor regulation under normal conditions. In PD, post-synaptic inhibition of M1-GPi pathway alleviates motor deficits. Post-synaptic activation of this pathway ameliorates depression symptoms associated with PD but exacerbates the PD motor symptoms.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-5990552/v1/6ed4ed29f0c4bfd869b2675c.png"},{"id":79381865,"identity":"a7079fad-2407-40ca-9f4e-3792db13d800","added_by":"auto","created_at":"2025-03-27 16:44:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8738428,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5990552/v1/2fc8d36f-91e3-4ee1-b881-46372dd974e4.pdf"},{"id":76312768,"identity":"957ee08f-592b-4209-be0c-5410f2ed6006","added_by":"auto","created_at":"2025-02-14 16:03:12","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":30587515,"visible":true,"origin":"","legend":"Supplementary information","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5990552/v1/ac93f405e0652a3e573a6c18.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"A Direct Primary Motor Cortex-Globus Pallidus Internus Circuit Regulates Both Motor and Non-Motor Symptoms in Parkinson's Disease.","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eParkinson\u0026rsquo;s disease (PD) is a common neurodegenerative disease characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNc), leading to severe motor dysfunction.\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e This deficiency manifests as severe motor dysfunctions such as tremors, rigidity, and bradykinesia, which are the hallmark symptoms of PD. In addition to the classical motor symptoms, PD patients frequently exhibit non-motor manifestations, notably depression and anxiety.\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e These PD symptoms are attributed to abnormalities in the structure and function of the basal ganglia (BG) network.\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe Globus Pallidus Internus (GPi) in primates, and its homolog in rodents known as the entopeduncular nucleus (EP or EPN), serves as a principal output nucleus of the basal ganglia (BG). It plays a pivotal role in integrating and modulating signals originating from diverse BG nuclei. These signals are subsequently relayed to regions implicated in motor function and the manifestation of PD symptoms.\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e The primary target areas of the GPi include the ventromedial (VM) and ventroanterior lateral (VAL) nuclei of the thalamus, as well as the lateral habenula (LHb).\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e Among these, the GPi projection to the thalamus is typically implicated in motor regulation and contributes to PD motor symptoms. And in clinical practice, the GPi is a key target for deep brain stimulation (DBS) interventions aimed at alleviating PD symptoms.\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e Unfortunately, the underlying mechanism for GPi-DBS in PD therapy has not been completely elucidated. Hence, deciphering the specific contributions of the GPi and its associated circuits to PD symptoms is crucial for developing effective therapeutic interventions.\u003c/p\u003e \u003cp\u003eAccording to the canonical model, it is widely accepted that cortical neural projections reach the GPi through three primary pathways: the direct, indirect, and hyperdirect pathways. These pathways are crucial for the execution of movements and the regulation of motor functions, and imbalances in these pathways are thought to contribute to the symptoms of PD.\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e In the cortico-basal ganglia-thalamus loop, information from the cerebral cortex converges in the GPi through the striatum (Str), globus pallidus externus (GPe), and subthalamic nucleus (STN), before being relayed back to the cortex via the thalamus (Tha). This complex network underscores the importance of maintaining a delicate balance within the basal ganglia circuitry for normal motor function.\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e In such traditional models, cortex information is not typically thought to reach the GPi directly.\u003c/p\u003e \u003cp\u003eNonetheless, some evidence suggests a direct cortical-GPi connection. For instance, studies using anterograde tracers like biotinylated dextran amine (BDA) indicate the potential existence of a direct neural pathway from the cortex to the globus pallidus.\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e Findings from human brain and rat research imply the existence of a direct cortical-globus pallidus projection.\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e However, despite substantial advancements in comprehending the basal ganglia's part in movement disorders, the architecture, functionality, and fundamental mechanisms of the direct cortico-GPi connection in motor regulation and the manifestation of PD symptoms are still not fully understood. The role of the direct cortico-GPi connection in the underlying mechanism for GPi-DBS has not been completely elucidated either. On the other hand, recent studies have highlighted the LHb's significant role in the pathophysiology of depression.\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e Considering that the LHb is one of primary nuclei projected by GPi,\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e the role of the cortico-GPi connection in PD-related depression remains incompletely understood.\u003c/p\u003e \u003cp\u003eIn the present studies, we conduct a comprehensive investigation into the direct cortical inputs to the GPi. We examine their connectivity, function, and relevance to motor and non-motor phenotypes (such as depressive-like behaviors) under normal conditions and in a PD mouse model. Our findings offer robust evidence supporting the existence of the direct cortico-GPi pathway. This pathway may play crucial roles in motor regulation and contribute significantly to both motor and non-motor symptoms of PD. Moreover, our data could help to understand the mechanisms associated with PD motor or non-motor symptoms in the context of GPi-DBS for PD treatment.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003e \u003cb\u003e2.1. The Primary Motor Cortex (M1) neurons directly project to the GPi, establishing the direct M1-GPi pathway.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the connectivity of cortico-GPi, we utilize a strategic approach to map the neuronal projections utilizing adeno-associated virus (AAV) vectors expressing fluorescent proteins. The anterograde tracing results show that neurons in the M1 project descending fibers to the GPi (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA,B; Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Importantly, both filamentous axonal fibers originating from M1, as well as puncta labeled with EYFP, are observed in GPi (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). This indicates that M1 neurons might directly project to the GPi, forming synaptic connections and thereby establishing the direct M1-GPi pathway. Besides the GPi, M1 neurons also project to other basal ganglia nuclei, including the striatum (Str), the external globus pallidus (GPe), the subthalamic nucleus (STN), and the substantia nigra pars compacta (SNc) (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB-F, Supporting Information). The retrograde tracing results show that the M1 serves as a principal input to the GPi, indicated by the abundant presence of retrogradely mCherry-labeled pyramidal neurons in the M1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC,D). The GPi receives additional inputs from various regions including the secondary motor cortex (M2), GPe, STN, centromedian nucleus (CM), and parafascicular nucleus (PF), ventral tegmental area (VTA), SNc, substantia nigra pars reticulata (SNr), deep mesencephalic nucleus (DpMe) and other nuclei (Figure S2, Supporting Information).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further validate the direct connections between cortical M1 and the GPi, we utilized a trans-monosynaptic tracing approach. This procedure entailed the injection of AAV2/1-hSyn-Cre into the M1, coupled with administration of AAV2/9-hSyn-DIO-mCherry into the GPi (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). The AAV2/1-hSyn-Cre virus, a serotype 1 adeno-associated virus (AAV), is capable of trans-monosynaptic infection to express the Cre protein, allowing it to specifically label trans-monosynaptic neurons situated within downstream nuclei, thereby enabling precise mapping of direct neuronal pathways.\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e Consequently, the specific expression of mCherry, induced by trans-monosynaptic Cre in the GPi, ensures dependable labeling of the direct connectivity between the cortical M1 and GPi. The results demonstrate a robust presence of mCherry labeling in the neurons within the GPi (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH), providing a compelling evidence of direct connectivity between the M1 and the GPi. This indicates that the cortical M1 neurons may directly connect and innervate the GPi neurons. Additionally, the GPi neurons that are directly innervated by the M1 secondarily project to the thalamus (VAL) and the LHb (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI,J). These findings at the anatomical level provide substantial evidence supporting the direct connectivity between the cortical M1 and GPi, establishing a direct M1-GPi pathway. This direct M1-GPi pathway potentially functions as an alternative, shortcut circuit for projections from the cortex to the GPi, bypassing the canonical direct pathway, indirect pathway, and hyperdirect pathway.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.2. The synaptic transmission of the direct M1-GPi pathway is potentially mediated by glutamatergic M1 neurons.\u003c/h2\u003e \u003cp\u003eTo investigate the synaptic transmission of the direct M1-GPi pathway, the AAV2/9-CaMKIIα-hChR2-mCherry is injected into the M1. Results of the electrophysiological recordings show that the glutamatergic M1 neurons are effectively excited by the optical stimulation (Figure S3). And optogenetic activation of axon from glutamatergic M1 neurons results in an increased spontaneous firing frequency in GPi neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE,F). Moreover, the electrophysiological recordings of trans-monosynaptically labeled GPi neurons exhibit a consistent response. The optogenetic activation of glutamatergic M1 neurons elicits an increased spontaneous firing frequency in the GPi neurons, which are actually innervated by glutamatergic M1 neuron axons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK,L). In addition, an increased amplitude of the optogenetic-induced EPSC (oEPSC) is observed in the trans-monosynaptically labeled GPi neurons, corresponding to the optogenetic activation at the axon terminal of the glutamatergic M1 neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM,N). These data provide electrophysiological evidence that further emphasizes the direct connectivity between the cortical M1 and the GPi, suggesting that the synaptic transmission of the M1-GPi pathway may be mediated by the glutamatergic M1 neurons.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.3. The glutamatergic M1-GPi pathway physiologically modulates motor behavior in mice.\u003c/h2\u003e \u003cp\u003eTo explore the physiological functions of the direct M1-GPi pathway, motor behaviors of mice are assessed using optogenetic manipulations under normal conditions. Results show that unilaterally optogenetic activation of the glutamatergic axon terminals of the M1-GPi pathway results in decreased motor coordination observed in the rotarod test, and an increase in rearing behavior in the cylinder test (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-E), though motor activities assessed in the open field test (OFT) and the cylinder test remain unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE; Figure S4; S5A-E, Supporting Information). Additionally, unilaterally optogenetic inhibition of the post-synaptic neurons in the direct M1-GPi pathway also results in decreased motor coordination, as observed in the rotarod test (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-H), while motor activities in OFT and cylinder test remain unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI,J; Figure S5F-K, Supporting Information). These data provide behavioral evidence indicating that the direct M1-GPi pathway physiologically modulates motor behavior in mice, a process that may be mediated by glutamatergic M1 neurons.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.4 The function of the direct M1-GPi pathway in modulating motor behavior is comparable to the hyperdirect M1-STN pathway.\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe M1-STN pathway, known as the hyperdirect pathway, has been demonstrated to play crucial roles in motor regulation and PD symptoms.\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. To evaluate the function of the direct M1-GPi pathway, we compare it with the M1-STN pathway under normal conditions. Results show that optogenetic activation of the glutamatergic axon terminals of the M1-STN pathway leads to a significant decrease in motor coordination, as observed in the rotarod test (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK-M). This outcome is similar to the effects observed from activating the direct M1-GPi pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-E). Different from the M1-GPi pathway, optogenetic activation of the M1-STN pathway leads to a significant increase in left forelimb preference in the cylinder test (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eN). Motor activities assessed in OFT and cylinder test do not exhibit significant changes, similar to the observations made with the M1-GPi pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eO; Figure S5L-N). These data suggest that the motor regulatory function of the direct M1-GPi pathway exhibits similarities with the hyperdirect M1-STN pathway, further confirming the role of the direct M1-GPi pathway in motor regulation under normal condition. It is evident that both the direct M1-GPi pathway and the hyperdirect M1-STN pathway play important roles in motor regulation. At the same time, these two pathways exhibit different preferences in motor regulation. The M1-STN pathway seems to preferentially modulate forelimb movements, while the M1-GPi pathway appears to be more involved in controlling rearing behaviors.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.5 The 6-OHDA lesions in the SNc induce typical PD motor symptoms in mice, but do not lead to typical depressive-like behaviors.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the roles of the M1-GPi pathway in PD symptoms, we administer 6-OHDA to induce either bilateral or unilateral lesions in the SNc of mice.\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e Subsequently, motor behaviors and non-motor symptoms, primarily depressive-like behaviors, are assessed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In mice subjected to a unilateral 6-OHDA lesion in the SNc, a substantial loss of dopaminergic neurons in the SNc and dopaminergic fibers in the striatum are seen (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Motor deficits are characteristically observed, as demonstrated by the APO-rotation test, rotarod test, OFT, and cylinder test (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-I). Regarding depressive-like behavior, the immobility duration in the tail suspension test (TST) exhibits a significant increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ), which is indicative of the typical symptom of behavioral despair associated with depression. The anhedonia, as measured by the sucrose preference test (SPT), along with another symptom of behavioral despair observed in the forced swimming test (FST) and the novelty-suppressed feeding test (NSF), does not exhibit significant alterations in PD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK-M; Figure S6, Supporting Information). It indicates that the 6-OHDA lesion in SNc leads to typical motor deficits in mice, but barely produces symptoms of PD-related depression (PDD). In addition, bilateral 6-OHDA lesions of the SNc also result in a substantial depletion of dopaminergic neurons in the SNc and a reduction of dopaminergic fibers in the striatum of mice (Figure S9, Supporting Information).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e2.6. Unilateral ablation of the pre-synaptic M1-GPi pathway has no prominent effects on motor modulation at either normal or PD states in mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo examine the function of the pre-synaptic M1-GPi pathway, we unilaterally inject the AAVretro-hSyn-Cre-mCherry into the GPi and the AAV2/9-EF1α-DIO-taCasp3-EGFP into the M1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA,B). The retrogradely expressed taCasp3 induce targeted ablation of pre-synaptic neurons in the M1-GPi pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC,D). Results show that the ablation of pre-synaptic neurons in the M1-GPi pathway leads to a decreasing trend in motor coordination, as indicated by a \u003cem\u003ep\u003c/em\u003e-value of 0.0588 in the rotarod test (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) under normal conditions. No other motor behaviors exhibit significant alterations in OFT and cylinder test under normal conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-J; Figure S7A,B, Supporting Information). It suggests that ablation of the pre-synaptic M1-GPi pathway may have virtually no significant impact on motor behavior under normal condition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFollowing the ablation of the pre-synaptic M1-GPi pathway, the 6-OHDA lesion is induced in the SNc of mice to generate a PD state. This helps us understand how the pre-synaptic M1-GPi pathway contributes to the development of PD symptoms. Results demonstrate that the targeted ablation of M1 neurons in the M1-GPi pathway has virtually no influence on the motor behaviors of PD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK-P; Figure S7C,D, Supporting Information), except the rearing behavior observed in the cylinder test (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eN). This implies that the ablation of the pre-synaptic M1-GPi pathway may not effectively impact on PD motor symptoms except the rearing behavior in cylinder test.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.7. The collateral projections of the M1-GPi pathway involve multiple nuclei of basal ganglia.\u003c/h2\u003e \u003cp\u003eIt is confounding that the targeted ablation of the pre-synaptic M1-GPi pathway has a negligible impact on normal motor and PD symptoms in mice. To more thoroughly explore the pre-synaptic functions of the M1-GPi pathway, the collateral projections of the M1-GPi pathway are examined. The retrograde AAVretro-hSyn-Cre-mCherry is injected into the GPi, while the AAV2/9-EF1α-DIO-EGFP is concurrently injected into the M1 (Figure S8A). This approach allows the Cre to be expressed in a retrograde manner exclusively within the M1 neurons that directly innervate the GPi (Figure S8B). Consequently, the DIO-EGFP expressions mark the soma of M1 neurons and their projecting axons of the pre-synaptic M1-GPi pathway. Results show that the M1 neurons that directly innervate the GPi have extensively collateral projections to the nuclei of the basal ganglia and thalamus, primarily including the striatum, GPe, STN, and the VM/VAL of the thalamus (Figure S8C-H). These findings demonstrate that the collateral projections of the pre-synaptic M1-GPi pathway extend to a wide array of nuclei in the basal ganglia and thalamus. These neuronal nuclei have been reported to be heavily involved in motor control and the symptoms of PD.\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e It suggests that the pre-synaptic M1-GPi pathway may play a highly complex role in motor regulation and the manifestation of PD symptoms due to its extensive collateral projections. Thus, optogenetic intervention at the axon terminal of the pre-synaptic M1-GPi pathway, located just over the GPi, can significantly impact motor behavior outcomes (e.g., rotarod test in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In contrast, globally intervening at the entire pre-synaptic M1-GPi pathway may not produce significantly different effects (as shown in the rotarod tests in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Chemogenetic manipulation of the pre-synaptic M1-GPi pathway influences neither motor nor non-motor symptoms of PD.\u003c/h2\u003e \u003cp\u003eTo further investigate the function of the pre-synaptic M1-GPi pathway in PD, we bilaterally inject AAVretro-hSyn-Cre-mCherry into the GPi and simultaneously inject AAV2/9-hSyn-DIO-hM3D-EGFP or AAV2/9-hSyn-DIO-hM4D-EGFP into the M1 of mice with 6-OHDA lesioned SNc (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). This strategy enables chemogenetic intervention of the pre-synaptic M1-GPi pathway activities with specific expression of hM3D or hM4D protein in M1 neurons that innervate the GPi (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB; Figure S10, Supporting Information). Results show that chemogenetic activation of the pre-synaptic M1-GPi pathway has no significant effect on motor behavior (the hM3D\u0026thinsp;+\u0026thinsp;6OHDA group \u003cem\u003ev.s.\u003c/em\u003e the EGFP\u0026thinsp;+\u0026thinsp;6OHDA group) in 6-OHDA lesioned mice, as observed in the rotarod test and OFT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-I), though there appears to be a decreased trend of motor activities. Chemogenetic inhibition of the pre-synaptic M1-GPi pathway also leads to no significant effect on motor behavior, except a significant increase in high-speed movement during OFT (the hM4D\u0026thinsp;+\u0026thinsp;6OHDA group \u003cem\u003ev.s.\u003c/em\u003e the EGFP\u0026thinsp;+\u0026thinsp;6OHDA group) in 6-OHDA lesioned (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). And chemogenetic inhibition of the pre-synaptic M1-GPi pathway results in an increasing trend of motor activities, as observed in both the rotarod test and OFT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-F). Similarly, results from the depressive-like behavior test indicate that manipulating the pre-synaptic M1-GPi pathway does not significantly impact depressive-like behavior under PD conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ-M). In addition, chemogenetic inhibition (the hM4D\u0026thinsp;+\u0026thinsp;6OHDA group) of the pre-synaptic M1-GPi pathway leads to significant increased motor activities compared to chemogenetic activation (the hM3D\u0026thinsp;+\u0026thinsp;6OHDA group) in 6-OHDA lesioned mice, as observed in rotarod test (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), movement velocity in OFT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), immobility in OFT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF) and high-speed movement in OFT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Despite this, these findings suggest that intervening in the pre-synaptic M1-GPi pathway may neither significantly modulate motor behavior nor non-motor symptoms under PD conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Chemogenetic activation of post-syntactical M1-GPi pathway alleviates depressive-like symptom of PD but exacerbates motor symptoms.\u003c/h2\u003e \u003cp\u003eDue to the collateral branch of the M1-GPi pathway (Figure S8), interventions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) and targeted ablation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) of the pre-synaptic M1-GPi pathway do not appear to significantly influence PD symptoms in mice. To further investigate the function of the M1-GPi pathway in both motor and non-motor symptoms of PD, we reapplied chemogenetic techniques to manipulate the post-synaptic M1-GPi pathway in the 6-OHDA lesioned mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). By facilitating the trans-monosynaptic expression of the hM3D protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), this approach enables targeted chemogenetic activation exclusively in the post-synaptic GPi neurons that are innervated by the M1, thereby offering a precise strategy to modulate this post-synaptic M1-GPi pathways. Results show a secondary projection from the M1-GPi pathway to the LHb and the thalamus (VAL), in addition to the trans-monosynaptic projection of the M1-GPi pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). In the absence of 6-OHDA lesioned SNc, the sole chemogenetic activation (the hM3D\u0026thinsp;+\u0026thinsp;Sham group) of post-syntactical M1-GPi pathway leads to no significant alteration in motor behavior, compared to the control (the mCherry\u0026thinsp;+\u0026thinsp;Sham group) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-J). This indicates that activation of the post-synaptic M1-GPi pathway, when isolated, may not be sufficiently potent to modulate motor behaviors under normal conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNevertheless, at PD state, chemogenetic activation of the post-synaptic M1-GPi pathway results in a significant decline in motor activities of the hM3D\u0026thinsp;+\u0026thinsp;6OHDA groups, compared to the mCherry\u0026thinsp;+\u0026thinsp;6OHDA controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-J). Motor coordination in rotarod test (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) and local motion in OFT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-I) are significantly decreased. Intriguing, the chemogenetic activation of post-syntactical M1-GPi pathway results in anti-depressive effects in 6-OHDA lesioned mice, characterized by significantly decreased immobility of FST and increased SPT in the hM3D\u0026thinsp;+\u0026thinsp;6OHDA group, compared to the mCherry\u0026thinsp;+\u0026thinsp;6OHDA controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK-N). These data demonstrate that activation of post-syntactical M1-GPi pathway alleviates depressive-like symptom in PD but simultaneously exacerbates PD motor symptoms.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.10. Chemogenetic inhibition of the post-synaptic M1-GPi pathway ameliorates motor symptoms but does not impact depressive-like symptoms in PD.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo ascertain the potential rescue of PD motor symptoms, we further investigate the chemogenetic inhibition of the post-synaptic M1-GPi pathway. The trans-monosynaptic expression of the hM4D protein enables targeted chemogenetic inhibition specifically in the post-synaptic GPi neurons innervated by the M1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA,B). The results also display a secondary projection from the M1-GPi pathway to the LHb and thalamus (VAL), in addition to the trans-monosynaptic projection of the M1-GPi pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Under PD conditions, chemogenetic inhibition of the post-synaptic M1-GPi pathway rescues PD motor deficits, demonstrating a significant increase in motor activities during both the rotarod test (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC) and OFT (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD-F) in mice from the hM4D\u0026thinsp;+\u0026thinsp;6OHDA group, compared to the mCherry\u0026thinsp;+\u0026thinsp;6OHDA group. But chemogenetic inhibitions of post-synaptic M1-GPi pathway have no significant effects on the depressive-like behavior in PD mice from the hM4D\u0026thinsp;+\u0026thinsp;6OHDA group, compared to the mCherry\u0026thinsp;+\u0026thinsp;6OHDA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG-I).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilar to chemogenetic activation, the chemogenetic inhibition of the post-synaptic M1-GPi pathway in the sham mice does not significantly impact motor or non-motor behaviors, comparing the hM4D\u0026thinsp;+\u0026thinsp;Sham group with the EGFP\u0026thinsp;+\u0026thinsp;Sham group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-I). These findings imply that the function of the post-synaptic M1-GPi pathway could be instrumental in the manifestation of PD symptoms. The chemogenetic inhibition of the post-synaptic M1-GPi pathway effectively ameliorates PD motor symptoms, which may elucidate the mechanism underlying DBS in clinical PD treatment. But it is intriguing that without 6-OHDA lesion, solely manipulating the post-synaptic M1-GPi pathway has no impact on sham mice. This suggests that the manipulation of the post-synaptic M1-GPi pathway may play important roles in the state of PD but not under normal conditions.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eThe cortico\u0026ndash;basal ganglia\u0026ndash;thalamo\u0026ndash;cortical loop is one of the fundamental network motifs in the brain. Unraveling its structural and functional organization is vital for comprehending motor behavior, cognition, and the development of many neurological and neuropsychiatric disorders. Typically, this network involves in information channel: motor, limbic and associative.\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e In this network, the structure and function of the cortico-GPi pathway have not been addressed completely, although several studies have suggested that there might be a direct projection from cortex to GP. In the present studies, we offer substantial evidence validating a direct route from the Primary Motor Cortex to the GPi (the M1-GPi pathway), which bypasses the traditional direct, indirect, and hyperdirect pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). And the direct M1-GPi pathway may play an essential role in regulation of motor. Importantly, the post-synaptic inhibition of the M1-GPi pathway alleviates the motor deficits in PD. The post-synaptic activation of the M1-GPi pathway ameliorates depression-related symptoms in PD but exacerbates the motor symptoms. In addition, intervention of the pre-synaptic M1-GPi pathway has no pronounced roles in motor regulation under normal or PD conditions. These data suggest that aberrant activities of the post-synaptic M1-GPi pathway may contribute to the mechanism of PD motor and non-motor symptoms. And it also helps us understand the GPi-DBS mechanism in PD therapy, suggesting that post-synaptic regulation, rather than the retrograde modulation of pre-synaptic M1-GPi pathways, may be involved. The suppression of GPi activity may contribute to the alleviation of PD motor symptoms in GPi-DBS, whereas the activation of GPi may be linked to the amelioration of PD non-motor symptoms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe classical model of cortico-basal ganglia function has critically shaped understanding of how nuclei and circuits within contribute to motor regulation and how circuit-level changes lead to the PD motor symptoms.\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e In this classical model, cortical information flows to the GPi through three ways: direct pathway (cortex-striatum-GPi), indirect pathway (cortex-striatum-GPe-STN-GPi) and hyperdirect pathway (cortex-STN-GPi). The GPi is one of major outputs of basal ganglia, converging information of basal ganglia and then sending the integrated signal to the down streamed, for example, the VM/VAL of thalamus. The equilibrium of activities within the basal ganglia pathways is essential for motor regulation under normal condition. In PD, dopamine loss of SNc causes imbalanced activity of pathways in cortico-basal ganglia, leading to the PD symptoms.\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e And the GPi also is one of the key targets for DBS in PD therapy. Therefore, the GPi is a crucial nucleus within the basal ganglia, playing a significant role in motor regulation and the manifestation of PD. The connections and innervation patterns of the GPi have thus become particularly important scientific and clinic question. In the conventional model of cortico-basal ganglia function, it has been commonly believed that there are no direct pathways linking the cortex to the GPi. Despite this, several reports indicate the possibility of cortical projections to the GPi.\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e Unfortunately, these reports have not provided evidence sufficient enough to define the precise anatomical structure, physiological functions, and pathophysiological implications of the cortico-GPi pathway. In the present studies, we provide substantial evidence at the anatomical, electrophysiological, and behavioral levels to support the assertion that the M1 directly projects to the GPi, thereby establishing the direct M1-GPi pathway. Our findings from anterograde tracing, retrograde tracing, and anterograde trans-monosynaptic tracing indicate that the M1 directly project to the GPi and establish connections with GPi (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The electrophysiological results further substantiate that glutamatergic M1 neurons directly innervate the GPi, facilitating the direct transmission of excitation from M1 to the GPi and inducing EPSC in the GPi via a mono-synaptic synapse (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Behavioral results from optogenetic and chemogenetic manipulations suggest that the glutamatergic M1-GPi pathway may play a role in physiological motor regulation and the manifestation of PD symptoms (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Additionally, findings from the hyperdirect pathway (M1-STN pathway) indicate similar impacts on motor control, implying that the M1-GPi pathway might serve a functionally comparable role to the hyperdirect pathway in motor regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These data suggest that the direct M1-GPi pathway bypasses the conventional direct, indirect, and hyperdirect pathways, substantially contributing to motor regulation and PD symptoms. Our findings support the complement to the traditional model of basal ganglia circuitry and suggest a more complex relationship between the cortex and the GPi, which could have significant implications for the study and treatment of movement disorders like PD. Neurons originating in the M1 send direct axon to the GPi, delineating a discrete M1-GPi pathway instrumental in fine-tuned motor control.\u003c/p\u003e \u003cp\u003eLiterature has suggested circuit-specific therapy for PD.\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e Based on our data, the post-synaptic intervention of the M1-GPi pathway activity bi-directionally modulates PD symptoms. Post-synaptic activation of the M1-GPi pathway exacerbates the motor symptoms of PD (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Conversely, post-synaptic inhibition of the M1-GPi pathway rescues PD motor symptoms (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These data indicate that the post-synaptic M1-GPi pathway plays a crucial role in PD symptoms, suggesting that the activities of the GPi neurons innervated by M1 could modulate the motor symptoms associated with PD. Our findings endorse the concept of circuit-specific therapy for PD and suggest that the post-synaptic M1-GPi pathway, particularly the GPi neurons innervated by the M1, could serve as a promising target for circuit-specific strategies to alleviate PD motor symptoms. On the other hand, the pre-synaptic intervention of the M1-GPi pathway does not result in a prominent effect on PD motor symptoms (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The reason might be that M1 neurons, which project to the GPi, are found to collaterally project to other nuclei of the basal ganglia, including the striatum, GPe, and STN (Figure S8). Thus, pre-synaptic intervention of the M1-GPi pathway concomitantly exerts effects on the direct, indirect, and hyperdirect pathways. In this scenario, the GPi integrates inputs from the direct, indirect, and hyperdirect pathways, together with the M1-GPi pathway, yielding a balanced or compensatory output that consequently has no impact on PD symptoms. Nonetheless, this equilibrium could be disturbed in circumstances involving unilateral intervention of the M1-GPi pathway, as evidenced by the asymmetric motor performance observed in the rotarod test during unilateral intervention in the present study (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe GPi is a key target of DBS in PD therapy.\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e However, the underlying mechanism for GPi-DBS has not been addressed completely yet. Current understanding of the GPi-DBS mechanism in PD therapy proposes that it potentially engages in inhibition, activation, or both in the GPi and basal ganglia activities,\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e and may also involve the retrograde regulation of motor cortex activity.\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e From our observations, post-synaptic inhibition of the M1-GPi pathway effectively alleviates PD motor symptoms (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), while post-synaptic activation ameliorates PD non-motor symptoms such as PD-related depression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In contrast to post-synaptic intervention, pre-synaptic intervention of the M1-GPi pathway significantly impacts neither PD motor symptoms nor non-motor symptoms (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Taking these into account, our findings clearly support the opinion that the mechanism underlying GPi-DBS in PD therapy may involve the modulation of post-synaptic M1-GPi pathway activity, rather than the retrograde modulation of pre-synaptic M1-GPi pathway activity.\u003c/p\u003e \u003cp\u003eIn addition, study has suggested that a subset of neurons in nucleus might be inhibited during DBS, while another subset might be excited.\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e Our data indicate that inhibition of post-synaptic M1-GPi pathway may be associated with mechanisms alleviating PD motor symptoms, while activation of post-synaptic GPi neurons innervated by M1 may involve mechanisms ameliorating PD non-motor symptoms. This may elucidate the observation that DBS in PD therapy exhibit variable therapeutic outcomes across motor and non-motor symptoms, wherein some cases predominantly benefit motor symptoms while others favor non-motor symptoms.\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eDepression, also referred to as PDD (Parkinson's Disease Depression) or PD-related depression, represents a typical non-motor PD symptom.\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e It is a common and often debilitating symptom that can greatly impact the quality of life for those with PD. However, the mechanism underlying the PDD has not been addressed completely yet. Our findings indicate that the LHb serves as a principal target of GPi projections and even the M1-GPi pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Current studies have suggested a strong correlation between LHb activity and the manifestation of depressive symptoms.\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e Our data demonstrate that the post-synaptic activation of the M1-GPi pathway alleviates depressive-like symptoms associated with PD (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL-N). These data imply that the secondary projection from the M1-GPi pathway to the LHb or the M1-GPi-LHb pathway could be crucially implicated in the mechanisms responsible for non-motor symptoms of PD like PDD. On the other hand, our data indicate that the post-synaptic activation of the M1-GPi pathway exacerbates PD motor symptoms while alleviating PD-related depressive symptoms (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-J). Fortunately, we found that post-synaptic inhibition of the M1-GPi pathway has no profound effects on depressive-like symptoms while ameliorate the motor deficits in PD (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). It underscores the need for cautious deliberation when considering interventions that target GPi activity to modulate PD motor symptoms, given their potential side effects on depressive symptoms.\u003c/p\u003e \u003cp\u003eTaken together, substantial data delineate a direct pathway from M1 to GPi (the direct M1-GPi pathway), which bypasses the traditional direct, indirect, and hyperdirect pathways. This direct glutamatergic M1-GPi pathway plays an essential role in motor regulation, as demonstrated by unilateral intervention of the pre-synaptic M1-GPi pathway under normal conditions. The M1-GPi pathway projects extensive collaterals to the basal ganglia and other brain areas, which may be responsible for the negligible effects of interventions targeting the pre-synaptic M1-GPi pathway on motor regulation and PD motor symptoms. Additionally, the secondary projection to the LHb, or even the M1-GPi-LHb pathway, might be significantly implicated in the mechanisms responsible for non-motor symptoms of PD, like PDD. Importantly, in PD, post-synaptic inhibition of the M1-GPi pathway alleviates motor deficits. Conversely, the post-synaptic activation of the M1-GPi pathway ameliorates depression symptoms associated with PD but exacerbates the motor symptoms of PD. These data suggest that the M1-GPi pathway, and potentially the extended M1-GPi-thalamus/LHb pathways, may play a crucial role in motor regulation, PD motor symptoms, and non-motor symptoms. Furthermore, our data indicate that the regulation of post-synaptic M1-GPi pathway activities may be implicated in the underlying mechanism of GPi-DBS in PD therapy, rather than the retrograde modulation of pre-synaptic M1-GPi pathways. The inhibition of post-synaptic M1-GPi pathway activities may be implicated in the underlying mechanism of GPi-DBS alleviating PD motor symptoms, while the activation of post-synaptic M1-GPi pathways may contribute to the mechanisms of GPi-DBS ameliorating PD non-motor symptoms.\u003c/p\u003e \u003cp\u003eThis direct cortico-GPi connection may allow for a more streamlined and efficient communication between the two regions, enabling rapid response and fine-tuned motor control. The existence of this pathway further emphasizes the complexity of the basal ganglia's role in motor functions and mood, suggesting underlying mechanism and potential avenues for targeted therapeutic interventions in PD motor and non-motor symptoms. Overall, these findings suggest that the M1-GPi pathway may play a complex role in regulating motor symptoms and mood behavior in PD patients. Given the association between GPi activity and depression-related symptoms, further research is important and necessary. This will enable a more comprehensive understanding of the mechanisms underlying these effects and facilitate the development of more effective interventions for PD patients, particularly those experiencing depressive-like symptoms.\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThe M1 neurons project directly to the GPi neurons, demonstrating a direct M1-GPi pathway that bypasses the direct, indirect, and hyperdirect pathways in the classical model (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). This direct M1-GPi pathway contributes to the regulation of physiological motor functions, as well as to both motor and non-motor symptoms of PD. Interventions targeting the M1-GPi pathway, particularly post-synaptic interventions, bidirectionally control PD motor symptoms. Most importantly, the post-synaptic inhibition of the M1-GPi pathway effectively alleviates PD motor deficits, and its activation ameliorates the depressive symptoms associated with PD. These could help to understand the mechanisms associated with PD motor or non-motor symptoms in the context of GPi-DBS for PD treatment. And it is the regulation of post-synaptic M1-GPi pathway activities that may underlie the mechanism of GPi-DBS in PD therapy, rather than the retrograde modulation of pre-synaptic M1-GPi pathways.\u003c/p\u003e"},{"header":"5. Experimental Section","content":"\u003cp\u003e\u003cem\u003eAnimals\u003c/em\u003e: Only healthy and adult C57BL/6J mice (sourced from Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) were used. All mice were group-housed, with 3 to 5 per cage. They were housed in a temperature-controlled animal room at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2℃ and 40\u0026ndash;70% humidity, on a 12-hour light/dark cycle (lights on at 7:00), with food and water available ad libitum until surgery. All experiments were approved by the Animal Ethics Committee of Capital Medical University and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e \u003cp\u003e \u003cem\u003eViral vectors\u003c/em\u003e: rAAV-EF1α-EYFP-WPRE-hGH pA (Cat#: PT-0098, working titer: 2.00E\u0026thinsp;+\u0026thinsp;12 vg/mL), rAAV2/1-hSyn-CRE-WPRE-hGH-pA (Cat#: PT-0136, working titer: 2.00E\u0026thinsp;+\u0026thinsp;12 vg/mL), rAAV2/9-hSyn-DIO-mCherry-WPRE-hGH-pA (Cat#: PT-0115, working titer: 2.00E\u0026thinsp;+\u0026thinsp;12 vg/mL), rAAV2/9-CaMKIIα-hChR2(H134R)-mCherry-WPRE-hGH-pA (Cat#: PT-0297, working titer: 2.00E\u0026thinsp;+\u0026thinsp;12 vg/mL), rAAV2/9-CaMKIIα-mCherry-WPRE-hGH-pA (Cat#: PT-0108, working titer: 2.00E\u0026thinsp;+\u0026thinsp;12 vg/mL), rAAV2/9-EF1α-DIO-EGFP-WPRE-hGH-pA (Cat#: PT-0795, working titer: 2.00E\u0026thinsp;+\u0026thinsp;12 vg/mL), rAAV2/R-hSyn-CRE-mCherry-WPRE-hGH-pA (Cat#: PT-0407, working titer: 5.00E\u0026thinsp;+\u0026thinsp;12 vg/mL) and rAAV2/9-EF1α-DIO-taCasp3-TEVp-P2A-EGFP-WPRE-hGH-pA (Cat#: PT-1230, working titer: 2.00E\u0026thinsp;+\u0026thinsp;12 vg/mL) were purchased from BrainVTA. ScAAV2/1-hSyn-Cre-pA (Cat#: S0292-1, working titer: 5.00E\u0026thinsp;+\u0026thinsp;12 vg/mL), AAV2/9-hEF1α-DIO-eNpHR3.0-mCherry- WPRE-pA (Cat#: S0852-9, working titer: 5.00E\u0026thinsp;+\u0026thinsp;12 vg/mL), AAV2/9-hEF1α-DIO-mCherry-WPRE-pA (Cat#: S0197-9, working titer: 5.00E\u0026thinsp;+\u0026thinsp;12 vg/mL), AAV2/9-hSyn-DIO-hM3D(Gq)-mCherry-WPRE-pA (Cat#: S0192-9, working titer: 5.00E\u0026thinsp;+\u0026thinsp;12 vg/mL), AAV2/9-hSyn-DIO-mCherry-WPRE-pA (Cat#: S1138-9, working titer: 5.00E\u0026thinsp;+\u0026thinsp;12 vg/mL), AAV2/9-hSyn-DIO-hM4D(Gi)-eGFP-WPRE-pA (Cat#: S0286-9, working titer: 5.00E\u0026thinsp;+\u0026thinsp;12 vg/mL), AAV2/9-hSyn-DIO-EGFP-WPRE-pA (Cat#: S0746-9, working titer: 5.00E\u0026thinsp;+\u0026thinsp;12 vg/mL), AAV2/9-hSyn-DIO-hM3D(Gq)-eGFP-WPRE-pA (Cat#: S0260-9, working titer: 5.00E\u0026thinsp;+\u0026thinsp;12 vg/mL) and AAV2/2Retro Plus-hSyn-Cre-mCherry-WPRE-pA (Cat#: S0702-2RP, working titer: 5.00E\u0026thinsp;+\u0026thinsp;12 vg/mL) were purchased from Taitool Bioscience.\u003c/p\u003e \u003cp\u003e \u003cem\u003eStereotaxic viral injection\u003c/em\u003e: Mice were deeply anesthetized via intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg) and positioned on a stereotaxic frame (RWD, China). Virus was injected either unilaterally or bilaterally into the M1 (AP: +1.30, ML: \u0026plusmn;1.78 mm, DV: -1.55 mm) or the GPi (AP: -1.25 mm, ML: \u0026plusmn;1.80 mm, DV:-4.80 mm), using a pulled glass capillary connected to a pressure microinjector (GAOGE, China) and a microinjection pump (RWD, China) at a rate of 30 nL/min. The virus volumes ranged from 200 to 250 nL in the M1 and from 100 to 150 nL in the GPi per side. The injection needle was left in place for 10 min to allow diffusion of the virus and then slowly withdrawn. Following the surgical procedures, mice were returned to their home cage for 3 to 4 weeks to allow for maximal virus expression and recovery.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMouse model of PD\u003c/em\u003e: Mice were deeply anesthetized as previously described. The 400-500nl of 6-OHDA (5 \u0026micro;g/\u0026micro;L dissolved in 0.1% ascorbic acid; Cat#: HY-B1081A, MCE, China) or saline (as control) was unilaterally or bilaterally microinjected into each side of the SNc (AP: -3.08 mm, ML: \u0026plusmn;1.20 mm, DV: -4.65 mm) at a rate of 100 nL/min. Animals were allowed to recover for two weeks before post-lesion behavioral testing commenced.\u003c/p\u003e \u003cp\u003e \u003cem\u003eHistology, immunohistochemistry and quantification\u003c/em\u003e: Mice were deeply anesthetized and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS, pH 7.4). Brains were carefully removed and post-fixed in 4% PFA for an additional 12 hours. They were sequentially transferred to 20% and 30% sucrose solutions in PBS until they sank to the bottom of the container and then cut into 40\u0026micro;m coronal sections on a freezing microtome (Leica, Germany). For TH immune-staining, sections were incubated with a mouse antibody against TH (1:5000; Cat#: SAB4200697, Sigma-Aldrich) overnight at 4\u0026deg;C. After washing three times with PBS (5 min each), sections were incubated with biotinylated horse secondary antibody against mouse (1:1000; Cat#: PK-4002, Vector Laboratories, Youngstown, OH). The antibody was visualized with a DAB solution. Sections were fixed and cover-slipped.\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e The fluorescent images were obtained with the automatic digital section scanning system (OLYMPUS, Japan) and a Zeiss LSM 880 confocal microsystem (ZEISS, Germany). Quantification of tyrosine hydroxylase (TH) staining was used as a measure of dopamine lesion in SNc and stratum as Mastro described.\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e The quantification of input-output relationships follows the method described by Lilascharoen.\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAcute slice preparation\u003c/em\u003e: Mice were anesthetized by intraperitoneal injection of 1% pentobarbital sodium and perfused with oxygenated high sucrose and ice-cold slicing solution (in mM: 213 sucrose, 10 glucose, 26 NaHCO3, 3 KCl, 1 NaH2PO4\u0026middot;2H2O, 5 MgCl2\u0026middot;6H2O, and 0.5 CaCl2\u0026middot;2H2O; gassed with 95% O2/5% CO2). Brains were swiftly removed, and coronal slices (300 \u0026micro;m thick) containing the M1 and GPi were sectioned with a vibratome (DTK-1000, Dosaka EM, Kyoto). Slices were then incubated in aCSF (in mM: 126 NaCl, 21.4 NaHCO3, 10 glucose, 2.5 KCl, 1.25 NaH2PO4\u0026middot;2H2O, 1.2 MgCl2\u0026middot;6H2O, and 2 CaCl2\u0026middot;2H2O; pH 7.4, bubbled with 95% O2/5% CO2) at 33\u0026deg;C for 1 hour before recordings. Slices were allowed to recover for at least 1 h in continuously oxygenated aCSF (95% O2/5% CO2).\u003c/p\u003e \u003cp\u003e \u003cem\u003eEx vivo electrophysiology\u003c/em\u003e: Patch clamp recordings were performed on M1 and GPi neurons using an upright microscope (Nikon FN-S2N, Japan), equipped with a 4\u0026times; objective and a 40\u0026times; water-immersion objective. The neurons with red fluorescence were visualized using a mercury lamp through a RED filter on the microscope. During recording, coronal slices containing the M1 and GPi area were continuously perfused at a rate of 2\u0026ndash;3 mL/min with oxygenated aCSF and maintained at 32\u0026deg;C. The patch pipettes, pulled with a pipette puller (O.D.: 1.5 mm, I.D.: 1.10 mm, 10 cm length, Sutter Instrument, USA) from borosilicate glass (Sutter Instrument, USA), exhibited a resistance of 3\u0026ndash;5 MΩ. Electrophysiological recordings were conducted using a HEKA amplifier. Signals were amplified, filtered at 2 kHz, and sampled at 20 kHz using the HEKA amplifier. Patch Master was used to convert the recorded signal into a digital signal. Clampfit 10.6 (Molecular Devices, USA) was employed for offline analysis of electrophysiological data. To test the effectiveness of the ChR2 viruses, optogenetic stimulation (473 nm, 10\u0026ndash;20 Hz, 4\u0026ndash;10 mW, 5 ms pulses) was delivered by a LED light source. To capture the spontaneous electrical activity of neurons, a current-clamp was used to record voltage while holding the current at 0 pA, indicating that neurons were at their resting membrane potential. The internal solution consisted of (in mM): 130 K-gluconate, 20 KCl, 10 HEPES, 0.2 EGTA, 10 disodium phosphocreatine, 4 Mg-ATP, 0.3 Na2-GTP (pH 7.20\u0026ndash;7.25; 288\u0026ndash;295 mOsm/kg). To evoke synaptic transmission using ChR2, optogenetic stimulation (473 nm, 10\u0026ndash;20 Hz, 4 mW, 5 ms pulses) was delivered by a LED light source, and postsynaptic recordings in voltage clamp mode (holding potential, 0 mV) were made in the GPi areas of ChR2 terminal infection using electrodes filled with a cesium-based internal solution (in mM: 120 CsMeSO3, 15 CsCl, 8 NaCl, 10 TEA, 10 HEPES, 0.2 EGTA, 2 Mg-ATP, 0.3 Na2-GTP and 5 QX-314 (Cl-salt); pH 7.20\u0026ndash;7.25; 288\u0026ndash;295 mOsm/kg).\u003c/p\u003e \u003cp\u003e \u003cem\u003eOptogenetic manipulations\u003c/em\u003e: For optogenetic stimulation of the M1-GPi pathway, following injections of ChR2, eNpHR3.0, or mCherry virus, optical fibers (200 \u0026micro;m O.D., 0.37 NA; Blackrock Microsystems, ASIA) were unilaterally implanted 200\u0026ndash;300 \u0026micro;m above the GPi. All fibers were secured to the skull with bone screws and dental cement.\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e Mice were allowed to recover and express the virus for 3\u0026ndash;4 weeks. They were then habituated for 1 minute after being connected to a laser source, after which the behavioral tests were performed. Different lasers (SLOC, China) were used to deliver laser light to mice expressing ChR2, eNpHR3.0, and mCherry at wavelengths of 473 nm (blue, 4\u0026ndash;6 mW) or 589 nm (yellow, 10 mW). The Doric software (Doric, Canada) was utilized to control the illumination parameters: blue light at 473 nm (30 Hz, 5 ms pulse width) and yellow light at 589 nm (100 Hz).\u003c/p\u003e \u003cp\u003e \u003cem\u003eChemogenetic manipulations\u003c/em\u003e: Clozapine N-oxide (CNO, Sigma-Aldrich, Cat#C0832; CAS: 34233-69-7) stock solution was prepared in dimethyl sulfoxide (DMSO, Beyotime Biotechnology, CAS: 67-68-5) at a concentration of 25 mg/mL and stored at -80\u0026deg;C. To prepare the working solution for experiments, CNO was diluted with 0.9% saline to a concentration of 0.125 mg/mL. Mice were intraperitoneally injected with either 0.9% saline (3 mg/kg) or CNO (3 mg/kg). Mice injected with CNO were allowed to rest for 40 minutes before behavioral tests.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eRotarod test\u003c/strong\u003e \u003cp\u003eA rotarod apparatus was applied to assess the motor coordination of mice (UGO BASILE, Italy). This test was performed as described by Li.\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e Briefly, mice were trained twice per day over two consecutive days separately at 11 rpm on day 1 and 22 rpm on day 2. On the testing day, mice were tested at a speed ranging from 4 to 40 rpm within 120 seconds, repeated three times with a 1-hour inter-training interval. The test results were obtained by averaging the latency of each mouse to fall off the accelerating rotarod across the three trials.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eOpen field test (OFT)\u003c/strong\u003e \u003cp\u003eThe open field chamber (50 cm \u0026times; 50 cm \u0026times; 50 cm) was used to evaluate locomotor activity. Total distance moved (cm), motor velocity (cm/s), immobility time (s), center distance (cm), low-speed (movement speed\u0026thinsp;\u0026lt;\u0026thinsp;5 cm/s), middle-speed (5 cm/s\u0026thinsp;\u0026lt;\u0026thinsp;movement speed\u0026thinsp;\u0026lt;\u0026thinsp;15 cm/s) and high-speed (movement speed\u0026thinsp;\u0026gt;\u0026thinsp;15 cm/s) were analyzed by the SuperMaze system (Xinrun, China). The center was defined as the central 25% of the arena. In PD behavior and chemogenetic behavior tests, each mouse was placed in the center of the square box and allowed to freely move for 10 min. For optogenetic behavioral tests, the test time was divided into three consecutive 30s or 120s epochs in the Pre\u0026ndash;Light\u0026ndash;Post periods.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eCylinder test\u003c/em\u003e: Mice were placed in a transparent 500 mL beaker. Rearing times, net contralateral rotations, and forelimb contact times were scored blindly. Rearing was defined as the body raised and both forelimbs off the ground.\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e Rotations were defined as each 270\u0026deg; rotation that contained no turn of \u0026gt;\u0026thinsp;90\u0026deg; in the opposite direction. The left forelimb preference was calculated using the following equation: left forelimb touches / (left forelimb touches\u0026thinsp;+\u0026thinsp;right forelimb touches\u0026thinsp;+\u0026thinsp;simultaneous touches) * 100. In PD behavior and chemogenetic behavior tests, each mouse was videotaped for 5 minutes. For optogenetic behavioral tests, the test time was divided into three consecutive 60s or 180s epochs in the Pre\u0026ndash;Light\u0026ndash;Post periods.\u003c/p\u003e \u003cp\u003e \u003cem\u003eApomorphine (APO)-Induced rotation test\u003c/em\u003e: Two weeks after the 6-OHDA injection, APO (0.5 mg/kg dissolved in 0.9% saline, Cat#: H116-5 MG, Sigma-Aldrich) was administered by intraperitoneal injection. Mice were placed into a 500 mL beaker and videotaped for 20 minutes. The net number of contralateral rotations (number of contralateral rotations \u0026ndash; number of ipsilateral rotations) was recorded. PD was confirmed in mice with a net number of contralateral rotations\u0026thinsp;\u0026gt;\u0026thinsp;5 turns/min.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eTail suspension test (TST)\u003c/strong\u003e \u003cp\u003eThe mice tails were wrapped with tape at approximately 2\u0026ndash;3 cm from the end of the tail. The mice were then fixed upside down on a horizontal bar with the nose tip about 30 cm above the ground. Animal behaviors were recorded for 6 min and the immobility time was scored during the last 4 min.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eForced swim test (FST)\u003c/strong\u003e \u003cp\u003eAnimals were individually placed in clear 5000 ml glass beaker (17.8 cm in diameter, 26 cm in height) filled with water (21\u0026ndash;25\u0026deg;C). Water depth was set to prevent mice from touching the glass bottom with their limbs or tails. The test lasted for 6 min and the immobility time was counted from the last 4 min. Mice were regarded as immobile when floating motionless or making only movements that were necessary to hold its head above the water.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eSucrose preference test (SPT)\u003c/strong\u003e \u003cp\u003eMice were single housed and habituated with 1% sucrose and water for 2 days and the bottle positions were counterbalanced every 24 h. On the testing day, mice were water-deprived for 24 h and then exposed to pre-weighed bottles (one bottle of water and one bottle of 1% sucrose) for 2 h in the dark phase. Sucrose preference was calculated by dividing the consumption of sucrose by the total liquid consumption (water and sucrose).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eStatistical analysis\u003c/strong\u003e \u003cp\u003eAll data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Graphing and statistical analysis were performed using GraphPad Prism 8.0 software (GraphPad Software, USA). Normal distribution was determined by the Shapiro-Wilk and Kolmogorov-Smirnov normality tests. Data meeting normal distribution criteria were analyzed using unpaired t-tests in two-sided manner for single factors, and data not meeting normal distribution criteria were analyzed using the Mann-Whitney tests in two-sided manner. One-way ANOVA with Tukey test and Two-way ANOVA with Bonferroni test were used for multiple comparisons. We represent \u003cem\u003ep\u003c/em\u003e values in all figures as *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.001, ****\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.0001, and ns, no statistically-significant difference.\u003c/p\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Grant No. 31971103 to Y. Wang). Y.-Q. Li, X.-P. Zhang, R.-B. Zheng contributed equally to this work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eY. Zhang, D. S. Roy, Y. Zhu, Y. Chen, T. Aida, Y. Hou, C. Shen, N. E. Lea, M. E. Schroeder, K. M. Skaggs, H. A. Sullivan, K. B. Fischer, E. M. Callaway, I. R. Wickersham, J. Dai, X. M. Li, Z. Lu, G. Feng, \u003cem\u003eNature \u003c/em\u003e\u003cstrong\u003e2022\u003c/strong\u003e,\u003cem\u003e607\u003c/em\u003e (7918), 321, https://doi.org/10.1038/s41586-022-04806-x.\u003c/li\u003e\n\u003cli\u003eR. L. Albin, A. B. Young, J. B. Penney, \u003cem\u003eTrends Neurosci \u003c/em\u003e\u003cstrong\u003e1989\u003c/strong\u003e,\u003cem\u003e12\u003c/em\u003e (10), 366, https://doi.org/10.1016/0166-2236(89)90074-x.\u003c/li\u003e\n\u003cli\u003eW. Poewe, \u003cem\u003eEur J Neurol \u003c/em\u003e\u003cstrong\u003e2008\u003c/strong\u003e,\u003cem\u003e15 Suppl 1\u003c/em\u003e, 14, https://doi.org/10.1111/j.1468-1331.2008.02056.x.\u003c/li\u003e\n\u003cli\u003eM. R. DeLong, T. Wichmann, \u003cem\u003eArch Neurol \u003c/em\u003e\u003cstrong\u003e2007\u003c/strong\u003e,\u003cem\u003e64\u003c/em\u003e (1), 20, https://doi.org/10.1001/archneur.64.1.20.\u003c/li\u003e\n\u003cli\u003eA. Nambu, H. Tokuno, M. Takada, \u003cem\u003eNeurosci Res \u003c/em\u003e\u003cstrong\u003e2002\u003c/strong\u003e,\u003cem\u003e43\u003c/em\u003e (2), 111, https://doi.org/10.1016/s0168-0102(02)00027-5.\u003c/li\u003e\n\u003cli\u003eY. Y. Wang, Y. Wang, H. F. Jiang, J. H. Liu, J. Jia, K. Wang, F. Zhao, M. H. Luo, M. M. Luo, X. M. Wang, \u003cem\u003eExp Neurol \u003c/em\u003e\u003cstrong\u003e2018\u003c/strong\u003e,\u003cem\u003e300\u003c/em\u003e, 135, https://doi.org/10.1016/j.expneurol.2017.11.006.\u003c/li\u003e\n\u003cli\u003eM. L. Wallace, A. Saunders, K. W. Huang, A. C. Philson, M. Goldman, E. Z. Macosko, S. A. McCarroll, B. L. Sabatini, \u003cem\u003eNeuron \u003c/em\u003e\u003cstrong\u003e2017\u003c/strong\u003e,\u003cem\u003e94\u003c/em\u003e (1), 138, https://doi.org/10.1016/j.neuron.2017.03.017.\u003c/li\u003e\n\u003cli\u003eY. Liu, W. Li, C. Tan, X. Liu, X. Wang, Y. Gui, L. Qin, F. Deng, C. Hu, L. Chen, \u003cem\u003eJ Neurosurg \u003c/em\u003e\u003cstrong\u003e2014\u003c/strong\u003e,\u003cem\u003e121\u003c/em\u003e (3), 709, https://doi.org/10.3171/2014.4.Jns131711.\u003c/li\u003e\n\u003cli\u003eV. Lilascharoen, E. H. Wang, N. Do, S. C. Pate, A. N. Tran, C. D. Yoon, J. H. Choi, X. Y. Wang, H. Pribiag, Y. G. Park, K. Chung, B. K. Lim, \u003cem\u003eNat Neurosci \u003c/em\u003e\u003cstrong\u003e2021\u003c/strong\u003e,\u003cem\u003e24\u003c/em\u003e (4), 504, https://doi.org/10.1038/s41593-021-00810-y.\u003c/li\u003e\n\u003cli\u003eJ. Song, H. Lin, S. Liu, \u003cem\u003eNetwork \u003c/em\u003e\u003cstrong\u003e2023\u003c/strong\u003e,\u003cem\u003e34\u003c/em\u003e (1-2), 84, https://doi.org/10.1080/0954898x.2023.2173816.\u003c/li\u003e\n\u003cli\u003eA. Naito, H. Kita, \u003cem\u003eBrain Res \u003c/em\u003e\u003cstrong\u003e1994\u003c/strong\u003e,\u003cem\u003e653\u003c/em\u003e (1-2), 251, https://doi.org/10.1016/0006-8993(94)90397-2.\u003c/li\u003e\n\u003cli\u003eY. Smith, T. Wichmann, \u003cem\u003eMov Disord \u003c/em\u003e\u003cstrong\u003e2015\u003c/strong\u003e,\u003cem\u003e30\u003c/em\u003e (3), 293, https://doi.org/10.1002/mds.26095.\u003c/li\u003e\n\u003cli\u003eD. Milardi, M. Gaeta, S. Marino, A. Arrigo, G. Vaccarino, E. Mormina, G. Rizzo, C. Milazzo, G. Finocchio, A. Baglieri, G. Anastasi, A. Quartarone, \u003cem\u003eMov Disord \u003c/em\u003e\u003cstrong\u003e2015\u003c/strong\u003e,\u003cem\u003e30\u003c/em\u003e (3), 342, https://doi.org/10.1002/mds.25995.\u003c/li\u003e\n\u003cli\u003eF. Karube, S. Takahashi, K. Kobayashi, F. Fujiyama, \u003cem\u003eElife \u003c/em\u003e\u003cstrong\u003e2019\u003c/strong\u003e,\u003cem\u003e8\u003c/em\u003e, https://doi.org/10.7554/eLife.49511.\u003c/li\u003e\n\u003cli\u003eA. Cacciola, A. Calamuneri, D. Milardi, E. Mormina, G. Chillemi, S. Marino, A. Naro, G. Rizzo, G. Anastasi, A. Quartarone, \u003cem\u003eFront Neuroanat \u003c/em\u003e\u003cstrong\u003e2017\u003c/strong\u003e,\u003cem\u003e11\u003c/em\u003e, 85, https://doi.org/10.3389/fnana.2017.00085.\u003c/li\u003e\n\u003cli\u003eH. Hu, Y. Cui, Y. Yang, \u003cem\u003eNat Rev Neurosci \u003c/em\u003e\u003cstrong\u003e2020\u003c/strong\u003e,\u003cem\u003e21\u003c/em\u003e (5), 277, https://doi.org/10.1038/s41583-020-0292-4.\u003c/li\u003e\n\u003cli\u003eM. Chen, S. Ma, H. Liu, Y. Dong, J. Tang, Z. Ni, Y. Tan, C. Duan, H. Li, H. Huang, Y. Li, X. Cao, C. J. Lingle, Y. Yang, H. Hu, \u003cem\u003eScience \u003c/em\u003e\u003cstrong\u003e2024\u003c/strong\u003e,\u003cem\u003e385\u003c/em\u003e (6709), eado7010, https://doi.org/10.1126/science.ado7010.\u003c/li\u003e\n\u003cli\u003eX. Liu, H. Li, R. Ma, X. Tong, J. Wu, X. Huang, K. F. So, Q. Tao, L. Huang, S. Lin, C. Ren, \u003cem\u003eAdv Sci (Weinh) \u003c/em\u003e\u003cstrong\u003e2024\u003c/strong\u003e,\u003cem\u003e11\u003c/em\u003e (30), e2401059, https://doi.org/10.1002/advs.202401059.\u003c/li\u003e\n\u003cli\u003eH. Y. Li, M. Z. Zhu, X. R. Yuan, Z. X. Guo, Y. D. Pan, Y. Q. Li, X. H. Zhu, \u003cem\u003eCell \u003c/em\u003e\u003cstrong\u003e2023\u003c/strong\u003e,\u003cem\u003e186\u003c/em\u003e (7), 1352, https://doi.org/10.1016/j.cell.2023.02.036.\u003c/li\u003e\n\u003cli\u003eZ. Polyakova, S. Chiken, N. Hatanaka, A. Nambu, \u003cem\u003eJ Neurosci \u003c/em\u003e\u003cstrong\u003e2020\u003c/strong\u003e,\u003cem\u003e40\u003c/em\u003e (39), 7451, https://doi.org/10.1523/jneurosci.0772-20.2020.\u003c/li\u003e\n\u003cli\u003eN. N. Foster, J. Barry, L. Korobkova, L. Garcia, L. Gao, M. Becerra, Y. Sherafat, B. Peng, X. Li, J. H. Choi, L. Gou, B. Zingg, S. Azam, D. Lo, N. Khanjani, B. Zhang, J. Stanis, I. Bowman, K. Cotter, C. Cao, S. Yamashita, A. Tugangui, A. Li, T. Jiang, X. Jia, Z. Feng, S. Aquino, H. S. Mun, M. Zhu, A. Santarelli, N. L. Benavidez, M. Song, G. Dan, M. Fayzullina, S. Ustrell, T. Boesen, D. L. Johnson, H. Xu, M. S. Bienkowski, X. W. Yang, H. Gong, M. S. Levine, I. Wickersham, Q. Luo, J. D. Hahn, B. K. Lim, L. I. Zhang, C. Cepeda, H. Hintiryan, H. W. Dong, \u003cem\u003eNature \u003c/em\u003e\u003cstrong\u003e2021\u003c/strong\u003e,\u003cem\u003e598\u003c/em\u003e (7879), 188, https://doi.org/10.1038/s41586-021-03993-3.\u003c/li\u003e\n\u003cli\u003eM. M. McGregor, A. B. Nelson, \u003cem\u003eNeuron \u003c/em\u003e\u003cstrong\u003e2019\u003c/strong\u003e,\u003cem\u003e101\u003c/em\u003e (6), 1042, https://doi.org/10.1016/j.neuron.2019.03.004.\u003c/li\u003e\n\u003cli\u003eM. R. DeLong, \u003cem\u003eTrends Neurosci \u003c/em\u003e\u003cstrong\u003e1990\u003c/strong\u003e,\u003cem\u003e13\u003c/em\u003e (7), 281, https://doi.org/10.1016/0166-2236(90)90110-v.\u003c/li\u003e\n\u003cli\u003eF. Blandini, G. Nappi, C. Tassorelli, E. Martignoni, \u003cem\u003eProg Neurobiol \u003c/em\u003e\u003cstrong\u003e2000\u003c/strong\u003e,\u003cem\u003e62\u003c/em\u003e (1), 63, https://doi.org/10.1016/s0301-0082(99)00067-2.\u003c/li\u003e\n\u003cli\u003eY. Chen, Z. Hong, J. Wang, K. Liu, J. Liu, J. Lin, S. Feng, T. Zhang, L. Shan, T. Liu, P. Guo, Y. Lin, T. Li, Q. Chen, X. Jiang, A. Li, X. Li, Y. Li, J. J. Wilde, J. Bao, J. Dai, Z. Lu, \u003cem\u003eCell \u003c/em\u003e\u003cstrong\u003e2023\u003c/strong\u003e,\u003cem\u003e186\u003c/em\u003e (24), 5394, https://doi.org/10.1016/j.cell.2023.10.004.\u003c/li\u003e\n\u003cli\u003eH. L. Combs, B. S. Folley, D. T. Berry, S. C. Segerstrom, D. Y. Han, A. J. Anderson-Mooney, B. D. Walls, C. van Horne, \u003cem\u003eNeuropsychol Rev \u003c/em\u003e\u003cstrong\u003e2015\u003c/strong\u003e,\u003cem\u003e25\u003c/em\u003e (4), 439, https://doi.org/10.1007/s11065-015-9302-0.\u003c/li\u003e\n\u003cli\u003eZ. G. Tan, Q. Zhou, T. Huang, Y. Jiang, \u003cem\u003eClin Interv Aging \u003c/em\u003e\u003cstrong\u003e2016\u003c/strong\u003e,\u003cem\u003e11\u003c/em\u003e, 777, https://doi.org/10.2147/cia.S105505.\u003c/li\u003e\n\u003cli\u003eJ. Zhang, J. Li, F. Chen, X. Liu, C. Jiang, X. Hu, L. Ma, Z. Xu, \u003cem\u003eClin Neurol Neurosurg \u003c/em\u003e\u003cstrong\u003e2021\u003c/strong\u003e,\u003cem\u003e201\u003c/em\u003e, 106450, https://doi.org/10.1016/j.clineuro.2020.106450.\u003c/li\u003e\n\u003cli\u003eK. W. McCairn, A. Iriki, M. Isoda, \u003cem\u003eJ Neurophysiol \u003c/em\u003e\u003cstrong\u003e2015\u003c/strong\u003e,\u003cem\u003e114\u003c/em\u003e (4), 2090, https://doi.org/10.1152/jn.00223.2015.\u003c/li\u003e\n\u003cli\u003eK. W. McCairn, R. S. Turner, \u003cem\u003eJ Neurophysiol \u003c/em\u003e\u003cstrong\u003e2015\u003c/strong\u003e,\u003cem\u003e113\u003c/em\u003e (7), 2537, https://doi.org/10.1152/jn.00701.2014.\u003c/li\u003e\n\u003cli\u003eJ. O. Dostrovsky, A. M. Lozano, \u003cem\u003eMov Disord \u003c/em\u003e\u003cstrong\u003e2002\u003c/strong\u003e,\u003cem\u003e17 Suppl 3\u003c/em\u003e, S63, https://doi.org/10.1002/mds.10143.\u003c/li\u003e\n\u003cli\u003eJ. L. Vitek, \u003cem\u003eMov Disord \u003c/em\u003e\u003cstrong\u003e2002\u003c/strong\u003e,\u003cem\u003e17 Suppl 3\u003c/em\u003e, S69, https://doi.org/10.1002/mds.10144.\u003c/li\u003e\n\u003cli\u003eC. C. McIntyre, M. Savasta, L. Kerkerian-Le Goff, J. L. Vitek, \u003cem\u003eClin Neurophysiol \u003c/em\u003e\u003cstrong\u003e2004\u003c/strong\u003e,\u003cem\u003e115\u003c/em\u003e (6), 1239, https://doi.org/10.1016/j.clinph.2003.12.024.\u003c/li\u003e\n\u003cli\u003eA. Muralidharan, J. Zhang, D. Ghosh, M. D. Johnson, K. B. Baker, J. L. Vitek, \u003cem\u003eBrain Stimul \u003c/em\u003e\u003cstrong\u003e2017\u003c/strong\u003e,\u003cem\u003e10\u003c/em\u003e (1), 126, https://doi.org/10.1016/j.brs.2016.10.005.\u003c/li\u003e\n\u003cli\u003eQ. Li, Y. Ke, D. C. Chan, Z. M. Qian, K. K. Yung, H. Ko, G. W. Arbuthnott, W. H. Yung, \u003cem\u003eNeuron \u003c/em\u003e\u003cstrong\u003e2012\u003c/strong\u003e,\u003cem\u003e76\u003c/em\u003e (5), 1030, https://doi.org/10.1016/j.neuron.2012.09.032.\u003c/li\u003e\n\u003cli\u003eL. A. Johnson, J. Wang, S. D. Nebeck, J. Zhang, M. D. Johnson, J. L. Vitek, \u003cem\u003eJ Neurosci \u003c/em\u003e\u003cstrong\u003e2020\u003c/strong\u003e,\u003cem\u003e40\u003c/em\u003e (10), 2166, https://doi.org/10.1523/jneurosci.2480-19.2020.\u003c/li\u003e\n\u003cli\u003eF. Faivre, A. Joshi, E. Bezard, M. Barrot, \u003cem\u003eNeurosci Biobehav Rev \u003c/em\u003e\u003cstrong\u003e2019\u003c/strong\u003e,\u003cem\u003e96\u003c/em\u003e, 335, https://doi.org/10.1016/j.neubiorev.2018.10.004.\u003c/li\u003e\n\u003cli\u003eJ. S. Reijnders, U. Ehrt, W. E. Weber, D. Aarsland, A. F. Leentjens, \u003cem\u003eMov Disord \u003c/em\u003e\u003cstrong\u003e2008\u003c/strong\u003e,\u003cem\u003e23\u003c/em\u003e (2), 183, https://doi.org/10.1002/mds.21803.\u003c/li\u003e\n\u003cli\u003eK. J. Mastro, K. T. Zitelli, A. M. Willard, K. H. Leblanc, A. V. Kravitz, A. H. Gittis, \u003cem\u003eNat Neurosci \u003c/em\u003e\u003cstrong\u003e2017\u003c/strong\u003e,\u003cem\u003e20\u003c/em\u003e (6), 815, https://doi.org/10.1038/nn.4559.\u003c/li\u003e\n\u003cli\u003eL. X. Li, Y. L. Li, J. T. Wu, J. Z. Song, X. M. Li, \u003cem\u003eNeurosci Bull \u003c/em\u003e\u003cstrong\u003e2022\u003c/strong\u003e,\u003cem\u003e38\u003c/em\u003e (1), 1, https://doi.org/10.1007/s12264-021-00775-9.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Parkinson’s disease, direct M1-GPi pathway, motor regulation, motor symptoms, non-motor symptoms","lastPublishedDoi":"10.21203/rs.3.rs-5990552/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5990552/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe cortico-basal ganglia (BG) circuit is vital for motor control and Parkinson's disease (PD) symptoms. The Globus Pallidus Internus (GPi) is a principal BG output nucleus and a key target for Deep Brain Stimulation (DBS) in PD treatment. However, the structure and function of the cortico-GPi circuit have not been completely addressed. In the present studies, data demonstrate a direct Primary Motor Cortex (M1)-GPi pathway, bypassing the classical direct, indirect, and hyperdirect pathways. This direct M1-GPi pathway plays an essential role in motor regulation under normal conditions. Importantly, in PD, post-synaptic inhibition of this pathway alleviates motor deficits. Post-synaptic activation of the M1-GPi pathway ameliorates depression symptoms associated with PD but exacerbates the PD motor symptoms. Interventions targeting the pre-synaptic M1-GPi pathway do not significantly affect motor regulation or PD symptoms. It suggests that the M1-GPi pathway may play a crucial role in motor regulation, PD motor symptoms and non-motor symptoms. Aberrant activities of the post-synaptic M1-GPi pathway potentially contributing to PD deficits. And the mechanism of GPi-DBS in PD therapy may involve post-synaptic regulation of M1-GPi pathway activities, rather than the retrograde modulation of pre-synaptic M1-GPi pathways.\u003c/p\u003e","manuscriptTitle":"A Direct Primary Motor Cortex-Globus Pallidus Internus Circuit Regulates Both Motor and Non-Motor Symptoms in Parkinson's Disease.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-14 16:03:07","doi":"10.21203/rs.3.rs-5990552/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8e934ed2-eb92-42fb-ae4c-51cfbcfc0fe2","owner":[],"postedDate":"February 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":44303225,"name":"Biological sciences/Neuroscience/Diseases of the nervous system/Parkinson's disease"},{"id":44303226,"name":"Biological sciences/Neuroscience/Motor control/Motor cortex"}],"tags":[],"updatedAt":"2025-03-27T16:36:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-14 16:03:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5990552","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5990552","identity":"rs-5990552","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}