Electroacupuncture Suppresses Premature Ventricular Complexes Occurring Post-myocardial Infarction through corticothalamic circuit

Electroacupuncture Suppresses Premature Ventricular Complexes Occurring Post-myocardial Infarction through corticothalamic circuit | 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 Biological Sciences - Article Electroacupuncture Suppresses Premature Ventricular Complexes Occurring Post-myocardial Infarction through corticothalamic circuit ronglin CAI, Fan ZHANG, Qian-yi WANG, Xia Zhu, Li-bin WU, Qi SHU, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4473024/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Electroacupuncture (EA) has been shown to suppress premature ventricular complexes (PVCs) following myocardial infarction (MI) in humans. However, the specific neural circuitry and causal mechanisms underlying this effect remain unclear. Here, we reveal a previously unrecognized connection from the primary motor cortex (M1) to the nucleus rostral ventrolateral medulla (RVLM) circuitry via the layer 5 of the primary motor cortex (M1L5)-zona incerta (ZI) pathway, which selectively suppresses PVCs in post-MI mice. Utilizing viral tracing, fiber photometry recordings, and optogenetic stimulation, we demonstrate that EA inhibits glutamatergic projections from M1L5 to ZI, leading to the activation of local GABAergic neurons and subsequent inhibition of RVLM (M1L5-ZI-RVLM). Furthermore, optogenetic or chemogenetic inhibition of the M1L5-ZI-RVLM circuit replicates the anti-PVC effects observed with EA in MI mice. Artificial activation of M1L5-projecting ZI neurons reverses the suppressive effects of EA on PVCs in MI mice. Overall, our findings highlight the M1L5-ZI-RVLM circuit as a crucial mediator of EA-induced suppression of PVCs following myocardial infarction. Additionally, this newly identified corticothalamic circuit may represent a promising target for mitigating PVCs post-myocardial infarction. Biological sciences/Neuroscience/Neural circuits Health sciences/Neurology Electroacupuncture myocardial ischemia Primary motor cortex premature ventricular complex CNS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Highlights EA suppresses PVCs occurring post-MI HT7 (Shenmen) acupoint afferent nerves drive a brain-to-heart circuit through M1L5 EA reduces hyperactivity of M1L5 neurons in MI model mice EA-induced suppression of PVCs occurring post-MI via M1L5-ZI-RVLM circuit INTRODUCTION Premature ventricular complexes (PVCs) following myocardial infarction (MI) pose a significant risk factor for subsequent sudden cardiac death (SCD) 1 – 3 . While treatments over the past decade have encompassed antiarrhythmic drug therapy, defibrillation, or ablation, early-stage use of antiarrhythmic drugs such as encainide, flecainide, or moricizine has been associated with increased rates of SCD and overall mortality 4 . Consequently, effectively mitigating PVCs post-MI remains a formidable scientific challenge. Bioelectronic medicine therapy, which includes nerve electrical stimulation, has emerged as a promising alternative. Acupuncture, a practice with roots dating back over 2000 years in China and now globally recognized, is increasingly utilized as a form of bioelectronic medicine therapy. Electroacupuncture (EA), in particular, has demonstrated efficacy in alleviating PVCs post-MI, ameliorating disorders of the sympathetic system, and reducing the risk of SCD 5 – 8 . The tachycardias precipitating cardiac arrest are primarily driven by abnormal sympathetic excitation 9 , 10 . Human studies have shown that electroacupuncture can regulate abnormal states of autonomic nerves 11 , 12 . Furthermore, expert consensus recommends acupuncture as an effective adjunctive therapy for regulating heartbeat rhythm in arrhythmia patients 13 . Leveraging the somatosensory autonomic nervous system, acupuncture's effects can extend to distant areas 14 , 15 . The inherent characteristics of EA effects have led to the hypothesis that they improve PVCs post-MI via modulation of the autonomic nervous system. However, the precise mechanisms underlying these effects remain to be elucidated. Shenmen (HT7), an acupoint situated in the anterior carpal region at the ulnar end of the distal metacarpal crease and along the radial margin of the flexor carpi ulnaris tendon (Fig. S1A) , has been specifically targeted for the treatment of myocardial ischemia, particularly post-myocardial infarction PVCs 16 , 17 . Alterations in activities in various brain regions involved in sensation and visceral motor processing have been revealed in functional magnetic resonance imaging (fMRI) studies with individuals receiving EA treatment at HT7 18 . The thalamus serves as a relay for multimodal sensory information and reciprocally connects with the sympathetic nervous system 19 . Within the thalamus, the zona incerta (ZI) receives diverse inputs from cortical and subcortical regions 20 , suggesting a potential role for the ZI as a conduit for somatosensory processing during EA. Nevertheless, the specific organization of cell types and the functions of corticothalamic circuits underlying EA-induced suppression of post-myocardial infarction PVCs remain largely unexplored. In the present study, we investigated the functional organization of the M1L5-ZI-RVLM circuit and elucidated its role in suppressing post-MI PVCs in response to EA stimulation. Artificial manipulation of this circuitry can replicate or abolish the effects of EA-induced suppression of post-MI PVCs. Our findings indicate that inhibition of the M1L5-ZI-RVLM circuit is essential for the manifestation of EA's suppressive effects on post-MI PVCs. RESULTS EA suppresses PVCs depends on balance sympathetic nervous system in mice We investigated whether stimulation of the HT7 acupoint by EA suppresses post-myocardial infarction PVCs in mice. Mouse models of premature ventricular complexes were induced by ligation of the left anterior descending coronary artery (MI) (Fig. 1A) . Male mice were the focus of our study unless otherwise specified. At 3 hours post-ligation, mice exhibited a significant increase in PVCs and ST-segment elevation compared to sham-operated mice (Fig. S1B, S1C) . To assess the effect of different EA intensities on post-MI PVC suppression in mice, we evaluated the impact of EA treatment at two intensities (0.5mA or 3mA). We found that only an EA intensity of 0.5mA elicited PVC suppression in MI mice (Fig. 1B-D) . Additionally, the 0.5mA EA intensity reduced the QRS width in MI mice (Fig. 1E) . It is noteworthy that QRS width reflects ventricular electrical activity and exhibits abnormal prolongation during ventricular arrhythmias 21 . We investigated whether EA suppresses PVCs in MI mice by potentially reducing sympathetic nerve activation. The autonomic nervous system significantly contributes to the development of PVCs 22,23 . In human studies, inhibiting abnormal sympathetic excitation after myocardial ischemia effectively reduces PVC occurrence 24 . We conducted heart rate variability (HRV) analysis and detected cardiac norepinephrine (NE) content to assess the potential effects of EA (0.5mA, 2Hz, pulse width of 50 μs) on the cardiac sympathetic component of PVCs occurring post-MI in mice. Our findings indicate that compared to the MI group, EA treatment significantly attenuated cardiac sympathetic excitability (Fig. 1F, 1G) ( p = 0.0045, Sham: n = 7; MI: n = 7; EA: n = 6), reduced cardiac NE content (Fig. 1H) ( p = 0.0053, n = 6), improved survival rate in MI mice (Fig. 1I) ( p = 0.006, Sham: n = 8; MI: n = 11; EA: n = 13), and decreased myocardial damage in MI mice (Fig. 1J) (AAR/LV: p = 0.0027; n = 13). We also observed that EA 0.5mA reduced QRS width ( p = 0.026; n = 6) and ST deviation ( p = 0.0319; n = 6) in MI mice during electrocardiogram (ECG) analysis. However, it did not affect RR interval, heart rate, PR interval, QTc interval, and R amplitude (Fig . S1H, S1I) . Increased cardiac sympathetic activity can induce severe PVCs during MI 25,26 , which worsens damage to myocardial cells and fibrosis. Furthermore, activated myocardial fibrosis perpetuates PVC occurrence 27,28 . Several studies have shown that reducing excessive cardiac sympathetic nerve activation is crucial for improving cardiac function and reducing PVCs 9,29 . We then examined ventricular infarction and fibrosis levels through histological analysis in MI mice following EA treatment. We observed reductions in cell atrophy and nuclear shrinkage proportions, cross-section of cardiomyocytes, interstitial congestion, and inflammatory cell infiltration (Fig . S1D) , and a decrease in cell fibrosis in the MI+EA 0.5mA group compared to the MI group (Fig . S1E, S1F) ( p = 0.0076, n = 6). Furthermore, considering the unique structure and restricted expression of cardiac troponin T (cTnT) and creatine kinase (CK)-MB by heart myocytes, they are utilized as biomarkers for acute myocardial injury. Hence, we assessed the expression of cTnT and CK-MB in myocardial tissue. Our results indicated significantly lower expression of cTnT (Fig. 1K) ( p = 0.0088, n = 6) and CK-MB (Fig . S1G) ( p = 0.0083, n = 6) in the MI+EA 0.5mA group compared to the MI group. These findings suggest that EA at 0.5mA effectively reduces PVCs and substantially mitigates risk of sudden cardiac death in MI mice. HT7 (Shenmen) acupoint afferent nerves drive a brain-to-heart circuit through the Layer 5 neurons in motor cortex (M1L5) We then investigated the neural circuitry underlying the observed suppression of PVCs following post-MI induced by EA. Given the pivotal role of the primary cortex in processing and integrating afferent somatosensory inputs 30,31 , we examined the neurons receiving somatosensory input from EA (HT7 acupoint) sites to the primary cortex. We employed herpes simplex virus (HSV) as a tracer, as it selectively targets somatosensory neurons and can traverse the somatosensory circuit. Considering the widespread distribution of somatosensory neurons at the HT7 acupoint in the dorsal root ganglion (DRG) of the T3 spinal cord segment (Fig . S2C, S2D, S2E, S2F) , we performed microinjections to directly administer the virus into the T3 DRG, ensuring a high concentration around the soma (Fig. 2A, 2B) . Additionally, to mitigate immune elimination by the host, we combined DRG microinjection with bortezomib to enhance HSV infection in T3 DRG neurons 32 (Fig . S3A) . Our observations revealed that HSV requires a minimum of 48 hours to transport from the soma of DRG neurons to the central terminals (Fig. S3B) . Secondary spinal cord neurons can be labeled approximately 72 hours after viral injection (Fig . S3C ), after which the virus spreads along the ascending pathway and gradually labels the downstream neurons (Fig . S3D) . We found that S1 and M1 neurons of the cortical area were labeled by HSV (green) (Fig. 2C, 2D) ( p = 0.9383, n = 6). The M1 serves as a crucial command center in somatosensory signal processing. Recent studies have suggested the possibility that dorsal root ganglion neurons directly transmit sensory signals to the motor cortex through ascending transmission of spinal cord projection neurons 33,34 . To verify whether afferent somatosensory nerves from the HT7 acupoint project to the M1L5 via a direct spino-cortical circuit, we injected the EnvA-pseudotyped glycoprotein (G)-deleted rabies virus (EnvA-RV-ΔG-eGFP)21 after expressing AAV2/9-DIO-RVG-TVA-mCherry for 3 weeks in the M1 of mice, with fluorogold (FG) injected at the HT7 acupoint site (Fig. 2E) . We observed a cluster of eGFP-labeled neurons located in the IV to VI layers of the T3 thoracic spinal cord. Remarkably, in the same animals, FG-positive fibers were also identified in the IV to VI layers of the T3 thoracic spinal cord (Fig. 2F, 2G) . Conversely, no labeled neurons were observed in the dorsal horn of the T3 spinal cord after injecting AAV2/9-DIO-TVA-mCherry alone into the layer 5 neurons in M1 (Fig. 2H, 2I) . These findings suggest that afferent somatosensory nerves from the HT7 acupoint relay information to the M1L5. Furthermore, the direct spinal cortical pathway may be implicated in the transmission of HT7 acupoint afferent somatosensory nerves. Although the HT7 acupoint's afferent somatosensory nerves sending projections to the M1L5 in mice are well established, it remains unknown whether the M1L5 can modulate sympathetic outflow to the heart. To identify cortical neurons involved in heart control and sympathetic nervous regulation, we initiated experiments by injecting pseudorabies virus (PRV) 531 encoding EGFP into the left ventricular wall of C57BL/6J mice to label upstream neurons retrogradely and trans-synaptically (Fig. 2J) . Validating this method, we began observations 80-90 hours post PRV injection, revealing the emergence of viral labeling in subcortical areas previously implicated in cardiac function (Fig. S2A) . Critically, at 110-120 hours post PRV injection, A small cluster of EGFP-positive neurons was consistently observed in the bilateral primary motor cortex (M1; bregma: 0.20 to –0.85 mm, lateral from midline: ±0.78 to ±1.36 mm) (Fig. 2K) . Cell counting indicated that the labeled cortical region was situated in M1 rather than the primary sensory cortex (S1) (Fig. 2L, 2M) (p < 0.0001, n = 8). In contrast, control animals receiving the same PRV injection into the chest or other viscera showed no labeled neurons in these regions (Fig. 2N, 2O, and Fig. S2B) . Moreover, the overwhelming majority of EGFP+ neurons in M1 (~95%) were localized in layer 5 and exhibited the characteristic morphology of projecting pyramidal neurons. Through immunofluorescence identification, we found that the upstream M1L5 pyramidal neurons of the heart primarily co-labeled with glutamatergic antibodies rather than GABAergic antibodies (Fig. 2P, 2Q) (p < 0.0001, n = 6). Based on these findings, the M1L5 glutamatergic neuron (M1L5 Glu ) links HT7 acupoint afferent somatosensory nerves to the mice heart sympathetic outflow center. The necessity of cardiac sympathetic regulation by M1L5 Glu For a long time, the ventrolateral region of the rostral medulla (RVLM), housing cardiac sympathetic premotor neurons, has been considered pivotal in cardiovascular disease 35 . Subsequently, we investigated c-Fos expression in the RVLM, observing an increase in c-Fos levels in the RVLM of post-MI mice compared to sham mice, with this phenomenon reversed by EA treatment (Fig. 3A, 3B) (p = 0.0439, n = 6). To further analyze whether M1L5 activation can induce RVLM activity and augment sympathetic outflow, we chemogenetically activated glutamatergic (Glu) neurons in M1L5, significantly increasing c-Fos expression in RVLM neurons (Fig. 3C, 3D) (p = 0.0019, n = 4). Next, we investigated whether stimulation of M1L5 Glu input to the heart alters cardiac sympathetic activity. Specifically, we conducted optogenetic manipulations that were temporally restricted using the CaMKIIα promoter-driven expression of Channelrhodopsin (ChR2). Optogenetic stimulation of Glu neurons in M1L5 in WT mice injected with AAV1-CaMKIIα-ChR2-mCherry (20Hz, 5ms, 3-5mw/mm 3 ) significantly increased. We also verified ChR2 cells co-expression and anti-glutamate positive cells (Fig. S4A, S4B) , heart rate (Fig. 3E, 3F) , and the HRV LF/HF ratio (an indication of cardiac sympathetic nerve excitation) (Fig. 3G, S4C) in male mice. Additionally, the score of cardiac sympathetic nerve activity (Fig. S4E) and the content of NE in cardiac tissue increased after optogenetic stimulation (Fig. 3H) (p = 0.0034, n = 7). Conversely, there was no change in heart NE levels in mice injected with the control virus at the end of 20 minutes of optogenetic stimulation. These data suggest that M1L5 Glu represents a potential target for EA to suppress ventricular extrasystole after myocardial infarction. The effect of electroacupuncture on PVCs occurring post-MI mice depends on the M1L5 Glu neurons Based on our aforementioned findings, we sought to examine changes in M1L5 Glu activity in MI mice induced by EA stimulation. We conducted c-Fos staining, a marker for neuronal activation, on M1L5 neurons upstream of the heart (Fig. 4A) . We observed increased c-Fos expression in M1L5 pyramidal neurons from MI mice. Interestingly, c-Fos expression was reduced in MI+EA mice compared with MI mice (Fig. 4B, 4C) (p = 0.0010, n = 18). To further investigate M1L5 neuron activity in MI+EA mouse models, we performed in vivo electrophysiological recordings (Fig. 4D) . Recorded M1L5 neurons were classified as putative excitatory pyramidal neurons (Eps, trough to peak duration 406.4 ± 3.312 μs, n = 154) and putative inhibitory interneurons (INs, trough to peak duration 593.3 ± 9.399 μs, n = 113) using unsupervised clustering techniques (Fig. 4E) and cross-correlogram analyses (Fig. S5A, S5B) based on the area under peak, trough to peak duration, and firing rate 36 . Interestingly, we observed an increase in the spike action potential firing rate of M1L5 Eps neurons in MI mice compared to sham mice, and this effect was reversed by EA treatment (Fig. 4F) (p = 0.0044, Sham: n = 53; MI: n = 27; MI+EA: n = 37). To delve deeper into the in vivo neuronal activity of M1L5 glutamatergic neurons following EA treatment, we conducted fiber photometry recordings in MI+EA mice by infusing an adeno-associated virus (AAV) expressing the fluorescent Ca 2+ indicator GCaMP6m (AAV-CaMKIIα-GCaMP6m) into M1L5 (Fig. 4G, 4H) . We observed that under EA treatment (0.5mA, 2Hz, pulse width of 50 μs), the fluorescence intensity of GCaMP6m-expressing neurons in MI mice was lower than before treatment (mean ΔF/F(%), p = 0.0107; calcium events, p = 0.0062, n = 7) ( Fig. 4I, 4J, 4K) , but this effect was not observed under 3mA EA intensity (Fig. S5D, S5E, S5F) . Additionally, to mitigate the effects of isoflurane, we conducted fiber photometry recordings in freely moving MI mice (Fig. S5G) . We found that the average ΔF/F(%) and calcium events of M1L5 neurons in MI+EA mice were significantly lower than those in MI model mice (mean ΔF/F(%), p = 0.0021; calcium events, p = 0.0024, n = 7) (Fig . S5H, S5I, S5J, S5K) . Microendoscopic calcium imaging (Fig. 4L, 4M) revealed that the fluorescence intensity (p = 0.0221, Sham: n = 47; MI: n = 122; EA+MI: n = 95) (Fig. 4N, 4O) and spontaneous calcium event rates (p = 0.0434) (Fig. 4P) from M1L5 neurons were significantly enhanced in MI mice compared with those in sham mice, and this enhancement was reversed by EA treatment. The aforementioned studies consistently demonstrate a heightened firing rate in M1L5 Glu neurons during PVCs, which was effectively reversed by EA treatment. To ascertain the necessity of M1L5 neurons in EA-mediated suppression of PVCs in MI mice, we employed a pharmacogenetic approach to selectively activate bilateral M1L5 Glu neurons (Fig. 4Q) . Specifically, we utilized an AAV vector to deliver hM3Dq, a designer receptor exclusively activated by the inert agonist clozapine-n-oxide (CNO) into the M1L5 region of mice (AAV-CaMKIIα-hM3Dq-mCherry) 37 . Subsequently, we assessed the therapeutic efficacy of EA in MI mice following the pharmacogenetic activation of M1L5 Glu neurons by analyzing PVC counts (refer to Fig. 4R) , LF/HF ratio (Fig. 4S) , QRS width (Fig. 4T ), and NE levels (Fig. 4U) . Intriguingly, our findings indicate that the EA-induced reduction in PVCs and attenuation of cardiac sympathetic excitability in MI mice were significantly impeded upon activation of M1L5 Glu excitatory neurons. The ZI receives direct inputs from M1L5 Glu neurons The primary motor cortex (M1) has been extensively studied for its role in regulating the autonomic nervous system and its involvement in motor initiation processes 38,39 . Notably, the cerebral cortex appears to be crucial in activating somatic-visceral reflexes induced by electroacupuncture. Studies utilizing intracortical recordings and transcranial magnetic stimulation have revealed that the perception of cardiac activity triggers responses in the human primary motor cortex 40 . One of the most distinctive features of the cerebral cortex is its reciprocal connections with the thalamus and midbrain 41,42 . In order to elucidate the acupoint (HT7)-heart pathway between these regions, we investigated the functional connections of various cortical circuits. Initially, an AAV expressing channelrhodopsin-2 (AAV-CAMKIIα–ChR2–mCherry) was administered into the M1L5 of wild-type mice (Fig. 5A) . Subsequently, three weeks later, we observed mCherry + fibers in the ZI (Fig. 5B) and in the ventrolateral periaqueductal gray (vlPAG) of the midbrain (Fig. S6A) . Notably, anterograde viral tracing experiments revealed that the RVLM does not receive direct fiber projections from M1L5 Glu (Fig. S6B) , indicating indirect downstream modulation. To further elucidate this pathway, we initially expressed ChR2 in all Glu neurons of the M1L5 and optogenetically stimulated M1 projection fibers to the ZI (Fig. S6C) or the vlPAG (Fig. S6D) . Remarkably, we observed that optogenetic stimulation of M1L5 Glu -ZI, but not M1L5 Glu -vlPAG, increased the expression of c-Fos (a neuronal activity marker) in the RVLM (Fig. S6E) . Additionally, optogenetic stimulation of M1 projection fibers to the ZI (Fig. S6F) elevated the HRV LF/HF ratio in male mice (Fig. S6G, S6H ), and increased the content of NE in cardiac tissue (Fig. S6I, S6J) . To elucidate the organization of the M1L5-ZI connection, we utilized a retrograde trans-monosynaptic tracing system employing a modified rabies virus (EnvA-pseudotyped RV-△G-eGFP) and Cre-dependent helper viruses (AAV-Ef1a-DIO-TVA-GFP and AAV-Ef1a-DIO-RVG) in mice (Fig.5C) . Incorporation of these helper viruses facilitated the monosynaptic retrograde spread of the RV. Neurons intensely labeled with eGFP were identified in several brain regions, including the anterior cingulate cortex (ACC), primary somatosensory cortex (S1L4), primary visual cortex (V1L5), and bilateral primary motor cortex (M1L5) (Fig.S7A-C) . Notably, the eGFP+ signal was predominantly observed in layer 5 of M1, co-localizing with the glutamate-specific antibody signal, while being absent in layers 1, 2, 3, 4, and 6 (Fig.5D,E) . Despite these observations, there exists a paucity of knowledge regarding the cellular circuitry underlying the modulation of the ZI by M1, a field which remains unexplored. The ZI, a subthalamic structure conserved across mammals, primarily comprises GABAergic neurons exerting widespread inhibitory influence throughout the brain 43 . ZI neurons display functional heterogeneity, exhibiting specificity in modulating various behaviors such as defensive behaviors 44,45 , binge eating 46 , hunting 47 , and sleep 48,49 , contingent upon their neurochemical properties and subdivisions across different anatomical domains. Considering potential tracer biases and limitations associated with viral tropism, we conducted retrograde tracing using retro-AAV-YFP virus injected into the ZI. Subsequently, we observed labeled neurons in layer 5 of the M1 (Fig. 5N) , while layer 6 of the M1 remained devoid of labeled neurons. Several observations collectively suggest that M1 activation primarily enhances output from the ZI to non-GABAergic neurons. Firstly, employing a viral strategy for fluorescent labeling of synaptic projection targets and GABAergic neurons, we identified that GABA neurons in the ZI predominantly reside in ZIv. Most ZI neurons receiving monosynaptic inputs from the M1 mainly consist of non-GABAergic neurons from ZId and are not co-labeled with GABA neurons in ZIv (Fig. 5F, 5G) . Notably, employing an anterograde trans-monosynaptic tracing system to track downstream neurons of M1L5 Glu -ZId non-GABA circuit revealed that ZIv GABA neurons received fiber projections from M1L5 Glu -ZId non-GABA . Secondly, optogenetic stimulation of the M1 in mice with MI led to increased c-Fos expression in Zid but not in Ziv, which was non-GABAergic neuron-specific (Fig. 5H, 5I) ( Fig. S7D, S7E) . In summary, GABAergic neurons exhibited a decrease in c-Fos expression following M1 stimulation, suggesting modulation within the local ZI microcircuit. To elucidate the reciprocal modulation of neuronal microcircuits in ZI (Fig. 5J) , we administered AAV carrying eNpHR4.0 (AAV-CAMKIIα-eNpHR-mCherry) into the M1L5 of MI mice, enabling optical inhibition of M1L5 Glu terminals in ZI (Fig. 5K) . A significant presence of mCherry nerve fibers in ZI was observed. Immunostaining revealed that c-Fos primarily colocalized with the GABA antibody (Fig. 5L) . Furthermore, we selectively monitored the responses of ZI GABA neurons in freely behaving mice following optogenetic activation or inhibition of the M1L5 Glu -ZI circuit. To achieve this, we delivered Retro-AAV1-CAMKIIα-NpHR4.0-mCherry/Retro-AAV1-CAMKIIα-ChR2-mCherry and AAV-GAD2-GCaMP6m virus into the ZI of mice. Fiber photometry recordings demonstrated a notable decrease in fluorescence intensity observed in GCaMP6m-expressing ZI GABA neurons after optogenetic stimulation of ChR2-expressing M1L5 Glu neurons (Fig. 5M) . Concurrently, the activity of ZI GABA neurons increased after optogenetic inhibition of M1L5 Glu -ZI (Fig. 5O) . These findings unveil modulation within local ZId-ZIv microcircuits (Fig. 5P) . M1L5-ZI-RVLM circuit regulates cardiac sympathetic and mediates EA to reduce PVCs occurring post-MI in mice We investigated the impact of EA on ZI GABA neurons in mice with MI (Fig. 6A) . AAV-GAD2-GCaMP6m virus was introduced into the ZI, and optic fibers were implanted above these regions in MI mice (Fig. 6B) . By examining the fluorescence intensity of GCaMP6m-expressing ZI GABA neurons, we established a direct correlation between their activity and EA stimulation. Notably, EA significantly increased the fluorescence intensity of these neurons (mean △F/F (%), p = 0.0002; calcium events, p = 0.0048; n = 6) (Fig. 6C, 6D, 6E) . Subsequently, we bilaterally administered AAV2/9-CAMKIIα-hM3dq-mCherry into the M1L5 region, activating M1L5 Glu neurons chemogenetically with CNO via intraperitoneal injection prior to EA stimulation (Fig. S9A, S9B) . Interestingly, we observed that the EA-induced increase in ZI GABA neuron activity in MI mice was inhibited (mean △F/F (%), p = 0.0004; calcium events, p = 0.0028; n = 7) ( Fig. S9C, S9D, S9E, S9F) . Concurrently, the EA-induced reduction in c-Fos expression in RVLM neurons was abolished (Fig. S9G) . Given previous findings suggesting the presence of non-GABA neurons in the ZI, we deemed it crucial to verify that ZIGABA neuron activation resulted from EA stimulation rather than technical artifacts or isoflurane effects. To address this, we utilized Fos-targeted recombination in active populations (Fos-TRAP) labeling technology to selectively identify EA-activated neurons in a time-dependent manner. Recombinant adeno-associated viruses rAAV-TRE-tight-mCherry and rAAV-c-Fos-tTA were injected into the ZI (Fig. 6F) . To validate Fos-TRAP labeling system reliability, we compared mCherry expression in neurons across different conditions (Fig. 6G) . Following a 30-day period of drinking non-Dox water, approximately 57.27±4.09% of ZI neurons exhibited mCherry expression. Conversely, Dox treatment resulted in only about 1.87±0.15% of ZI neurons expressing mCherry, confirming effective TRE promoter suppression by Dox. Furthermore, 12.72±2.09% of ZI neurons expressed mCherry three days after EA stimulation at the HT7 acupoint in MI mice (Fig. 6I, 6J) . Notably, there was no significant difference in water consumption between Dox water-drinking mice and non-Dox water-drinking mice (Fig. 6H) . Subsequent immunofluorescence staining revealed that approximately 60.27±9.64% of the mCherry signal colocalized with a specific GABA antibody (Fig. 6K ) , confirming the activation of ZI GABA neurons following EA. We then proceeded to examine the intricate circuits connecting the ZI to the medulla. Employing sparse neuronal type-specific labeling, we introduced rAAV-EF1a-DIO-Ypet-2A-mGFP-WPRE-pA and AAV2/9-Vgat-Cre virus into the ZI, facilitating selective labeling of ZI GABA dendritic spines. Subsequently, mGFP + fibers were observed in various brain regions (Fig. 6M and Fig. S8A) . Given our previous investigation demonstrating the reversal of cardiac sympathetic nerve hyperexcitability in MI mice through EA, we correlated this with the well-documented projections of the M1, particularly emphasizing the RVLM (Fig. 6L) . This discovery is intriguing, given the ZI's role as an integrative hub for modulating sensory integration, behavioral control, and visceral activity regulation 43,50,51 . To verify the ZI-RVLM projection, we infused AAV-DIO–eGFP virus into the ZI and AAV-hsyn-Cre virus into the RVLM of C57BL/6J mice, leading to the identification of eGFP-expressing neurons in the RVLM (Fig. 6N) . However, the specific neuronal types in the ZI projecting to the RVLM remained unknown. Therefore, we conducted anterograde trans-monosynaptic tracing by administering AAV1-Vgat-cre/AAV-DIO-TK-mRFP helper virus and HSV-△TK-eGFP into the ZI of mice. Remarkably, we found that RVLM primarily receives fiber projections from ZI GABA neurons (Fig. 6O and fig. S8B) . Subsequently, we employed a retrograde trans-monosynaptic tracing system, introducing modified rabies virus (EnvA-pseudotyped RV-△G-eGFP) into the RVLM along with Cre-dependent helper viruses (AAV-Ef1a-DIO-TVA-mCherry and AAV-Ef1a-DIO-RVG) in mice. Our results revealed relatively abundant eGFP signals in the ventral part of the Zona Incerta (ZIv) but scarcity in the dorsal part (ZId) ( Fig. 6P) . Notably, no eGFP signal was detected in M1L5. To explore the functional connections within the M1L5 Glu -ZI GABA -RVLM circuit, we administered Retro-AAV-CaMKIIα-ChR2 virus and AAV-Vgat-hM3dq-mCherry virus into the ZI of C57 mice (Fig. 7A) . Using fiber photometry recordings, we stimulated ChR2-containing upstream ZI cells in M1L5 and observed an increase in △F/F and calcium events in the RVLM. Notably, these responses were attenuated by chemogenetic activation of ZI GABA neurons (Fig. 7B-D) . These observations elucidate the microcircuitry wherein ZI GABA neurons receive innervation from local ZI non -GABA interneurons, both of which directly receive inputs from M1L5 Glu neurons. Given that premotor neurons for cardiac sympathetic activity are located in the RVLM, and M1L5 mediates the somatosensory effects of acupuncture at the HT7 point, we investigated whether the functional connection of the M1L5 Glu -ZI GABA -RVLM circuit contributes to the anti-PVCs effects induced by 0.5mA EA in MI mice. To this end, we infused AAV2/9-Vgat-hM3Dq-mCherry & Retro-AAV-CaMKIIα-ChR2 virus into the bilateral ZI and implanted optic fibers above the ipsilateral M1L5 regions in MI mice (Fig. 7E) . Upon optogenetic activation of the M1L5Glu-ZI pathway, we observed a blockade in the EA-induced anti-PVCs effect in MI mice (Fig . 7F) , along with a blockage in the reduction of cardiac sympathetic excitability induced by EA. However, simultaneous activation of both M1L5Glu and ZIGABA neurons reduced PVCs in MI mice subjected to EA (Fig . 7G-I) . Subsequently, we investigated the potential physiological characteristics of the M1L5 Glu -ZI GABA -RVLM pathway in anti-PVCs in MI mice (Fig . S9H, S9I) . The 0.5mA EA-induced reduction in PVC counts in MI mice was replicated upon optogenetic inhibition of the M1L5Glu-ZI pathway and was blocked upon neural activation of the RVLM (Fig . S9J-S9O) . DISCUSSION The neural pathway underlying the regulation of cardiac function via acupuncture at the HT7 point has long been enigmatic, with an intricate interplay between somatosensory systems and internal organs. Our investigation has delineated a complex polysynaptic circuit comprising M1L5-ZI-RVLM neurons, pivotal in conveying HT7 afferent signals to modulate sympathetic outflow and, consequently, heart function. Specifically, this M1L5-ZI-RVLM pathway we've elucidated suppresses post-myocardial infarction PVCs in response to EA stimulation, underscoring its role in the cardioprotective effects of HT7 activation in MI-afflicted mice. Employing HSV for polysynaptic tracing of HT7 acupoint somatosensory neural networks has proven highly effective. HSV's broad host range, particularly its robust amplification and propagation efficiency in mice 52 , establishes it as the optimal tracer for such investigations. Nonetheless, variations in host immunity necessitated the use of the immunosuppressant bortezomib 53 to enhance HSV infection rates consistently. While our focus primarily centered on HSV-labeled cases within the S1 and M1 regions, intriguingly, our observations revealed the involvement of not just the S1 but also the M1 in the reception and processing of sensory input from the HT7 acupoint. For tracking autonomic motor networks, PRV emerged as the preferred tracer. Our investigation identified a distinct cluster of M1L5 pyramidal neurons, primarily located upstream of the heart in the cortex, emphasizing the sensory-cardiac autonomic motor connection specifically within the M1L5 region. Notably, recent human imaging studies have corroborated a parallel pattern of sensory-motor integration within the M1L5 cortex, further substantiating our findings. In essence, the intricate intertwining 54 of sensory and motor pathways, forged through evolutionary processes, emerges as a fundamental mechanism underpinning life's development. Recent research has shed light on the influence of transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS) on the modulation of sympathetic outflow to the heart through motor cortex activity 55 . While the corticothalamic circuit appears to be a key player in PVCs 56 , the specific organization of corticothalamic connections by cell types and their roles in PVCs remain largely unexplored. This study aims to investigate the involvement of M1 neurons in PVCs using a mouse model of myocardial ischemia-induced left ventricular PVCs. Our findings reveal heightened activity in the RVLM and M1L5 Glu neurons, along with reduced activity in ZIGABA neurons in MI mice. Notably, inhibition of either M1L5 or ZI GABA neuronal activity leads to the suppression of PVCs induced by MI, underscoring their respective roles. Our data provide compelling evidence for the modulation of PVCs by the M1L5-ZI circuit via the RVLM. Support for this hypothesis comes from human imaging studies suggesting distinct roles for M1 and ZI neurons in regulating sympathetic outflow to the heart 57 , 58 . Compared to extensively studied autonomic nervous pathways activated by acupoints 59 – 61 , the specific central pathway transmitting afferent nerve signals from HT7 acupoints to sympathetic outflow towards the heart remains uncharted. Employing virus tracing techniques, we identified neurons that were innervated by spinal afferents from HT7 acupoints, predominantly congregating in M1L5. These glutamatergic neurons within M1L5 project to the ZI, and subsequently to the RVLM, serving as a pivotal conduit for regulating sympathetic outflow to the heart. While the somatosensory cortex is conventionally recognized as the primary pathway for receiving somatosensory signals, relying on thalamocortical relay pathways in the thalamus 62 , 63 , our findings reveal a novel route. We discovered that layer 5 of the somatic motor cortex (M1L5) directly receives somatosensory signals, bypassing the thalamus, thus providing morphological evidence for the regulation of visceral function, particularly the heart, through bioelectrical stimulation at HT7. It is noteworthy that the neural mechanism underlying the regulation of cardiac function by human acupuncture at HT7 is undoubtedly more intricate than observed in mice. Human studies have indicated the involvement of multiple brain regions in autonomic regulation, including the hypothalamus, cerebellum, prefrontal cortex, and amygdala, in response to acupuncture 64 – 66 . Consequently, further exploration is necessary to clarify how other downstream brain regions receiving projections from M1L5 (excluding ZI and PAG), or transmitting afferent nerve signals from HT7 acupoints, may elucidate the acupuncture treatment for MI. While our findings underscore the significant role of the brain in processing acupuncture signals, we acknowledge the potential for acupuncture to directly modulate the autonomic nervous system of the heart through sensory transmission in the spinal cord. Overall, our study has identified a neural pathway linking the brain and the heart, implicating the role of HT7 in the processing of heart function, which could catalyze research into the connection between the body surface and internal organs. In the future, these discoveries hold promise for inspiring the development of alternative interventions for myocardial diseases. Limitations of the study In this investigation, we primarily observed cortical involvement in the inhibitory effect of EA at HT7 on PVCs. However, our findings also indicate widespread distribution of somatosensory neurons at HT7 within the dorsal root ganglion of the T3 spinal cord. Given that the cardiac sympathetic nerve is distributed in the T3-T4 spinal cord, further investigation is warranted to ascertain whether a direct connection exists between HT7 and the cardiac sympathetic nerve at the spinal cord level, thereby contributing to the effect of EA at HT7. Simultaneously, somatosensory neural projections from the HT7 acupoint extend to multiple brain regions, including the periaqueductal gray, lateral hypothalamic area, and parvocellular medial vestibular nucleus. However, whether these projections are implicated in the suppression of PVCs in MI mice by EA remains unknown. Declarations ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (82074536) awarded to RLC, Natural Science Foundation of Anhui Province (2108085Y30) awarded to RLC, Distinguished Young Youth Scientific Research Project in Universities of Anhui Province (2022AH020043) awarded to RLC, Research Funds of Center for Xin'an Medicine and Modernization of Traditional Chinese Medicine of IHM (2023CXMMTCM019) awarded to RLC, The Plans for Major Provincial Science & Technology Projects (202303a07020002) to ZZ, National Natural Science Foundation of China (82104999) awarded to QY, Natural Science Foundation of Anhui Province (2108085QH364) awarded to QY, Excellent Young Youth Scientific Research Project in Universities of Anhui Province (2022AH030062) awarded to QY, Higher Education Teaching Quality and Teaching Reform Project of Anhui Provincial (2023xscx092) awarded to FZ, National Natural Science Foundation of China (81973757) awarded to LH, College Natural Science Project of Anhui Provincial(2022AH050514). AUTHOR CONTRIBUTIONS Conceptualization: Fan ZHANG, Rong-lin CAI, Qing YU, Zhi ZHANG, Xia ZHU. Data curation: Fan ZHANG, Qian-yi WANG, Li-bin WU, Jie ZHOU, Liu YANG, Wen-xiu DUAN. Formal analysis: Fan ZHANG, Qian-yi WANG, Xiang ZHOU, Bin ZHANG. Funding acquisition: Ling HU, Qing YU, Rong-lin CAI, Zhi ZHANG. Investigation: Fan ZHANG, Li-bin WU, Qi SHU. Methodology: Fan ZHANG, Xia WEI, Qing YU, Hui-min CHANG. Project administration: Zhi ZHANG, Ling HU, Rong-lin CAI, Qing YU. Resources: Rong-lin CAI, Qing YU, Xia ZHU. Software: Fan ZHANG, Yan WU, Qian-yi WANG, Wen-jing SHAO, Zheng-jie LUO. Supervision: Fan ZHANG, Ling HU, Qing YU, Rong-lin CAI, Zhi ZHANG. Validation: Qing YU, Rong-lin CAI. Visualization: Fan ZHANG, Rong-lin CAI. Writing–original draft: Fan ZHANG, Qian-yi WANG, Xia WEI. Writing – review & editing: Fan ZHANG, Rong-lin CAI. All authors have read and agreed to the published version of the manuscript. DECLARATION OF INTERESTS The authors declare that they have no conflicts of interest. Data availability statement The author will unreservedly provide the raw data to support the conclusions of this article. All data generated in this study can be found at: https://data.mendeley.com/datasets/vs34h73gr3/1, or through contact of the lead author, Rong-lin CAI ( [email protected] ). Ethics statement We obtained six-week-old male C57BL/6J mice from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd., for inclusion in this study. The production license for experimental animals is SCXK (ZHE) 2019-0004. The mice were housed in cages with ad libitum access to food and water, and the ambient temperature was maintained at a relative range of 23–25 °C. 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Medicine","correspondingAuthor":false,"prefix":"","firstName":"Li-bin","middleName":"","lastName":"WU","suffix":""},{"id":311081412,"identity":"022f15a2-3d21-4371-8e4d-0ec1a94c418c","order_by":5,"name":"Qi SHU","email":"","orcid":"","institution":"Anhui University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"SHU","suffix":""},{"id":311081413,"identity":"1a83ea10-5745-419e-9bf7-e36393cd06c3","order_by":6,"name":"Hui-min CHANG","email":"","orcid":"","institution":"Anhui University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hui-min","middleName":"","lastName":"CHANG","suffix":""},{"id":311081414,"identity":"24e5a61c-bb92-4aa7-b2ab-c0427ad8c8b1","order_by":7,"name":"Yan WU","email":"","orcid":"","institution":"Anhui University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"WU","suffix":""},{"id":311081415,"identity":"32d480a8-a606-432f-b50d-13a8e834ab9d","order_by":8,"name":"Wen-jing SHAO","email":"","orcid":"","institution":"Anhui University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Wen-jing","middleName":"","lastName":"SHAO","suffix":""},{"id":311081416,"identity":"f6026d04-08b4-4f9b-a018-03f7bf0b6954","order_by":9,"name":"Xia WEI","email":"","orcid":"","institution":"Anhui University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xia","middleName":"","lastName":"WEI","suffix":""},{"id":311081417,"identity":"87ff8e4d-88f0-4ca6-b9f0-c24b567a6c52","order_by":10,"name":"Xiang ZHOU","email":"","orcid":"","institution":"Anhui University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"ZHOU","suffix":""},{"id":311081418,"identity":"c1a304c1-5ba4-4bc2-8ee3-8433149ac9b7","order_by":11,"name":"Jie ZHOU","email":"","orcid":"","institution":"Anhui University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"ZHOU","suffix":""},{"id":311081419,"identity":"4bfe28f6-cdd9-4603-8101-47a2b4346451","order_by":12,"name":"Zheng-jie LUO","email":"","orcid":"","institution":"Anhui University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zheng-jie","middleName":"","lastName":"LUO","suffix":""},{"id":311081420,"identity":"3064522e-5045-43ae-ba26-ec8087393ca4","order_by":13,"name":"Liu YANG","email":"","orcid":"","institution":"Anhui University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Liu","middleName":"","lastName":"YANG","suffix":""},{"id":311081421,"identity":"a735b7b8-06a0-4d95-a61a-9cf77ec4f94b","order_by":14,"name":"Wen-xiu DUAN","email":"","orcid":"","institution":"Anhui University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Wen-xiu","middleName":"","lastName":"DUAN","suffix":""},{"id":311081422,"identity":"2e1edd9f-3d9b-4193-9c27-77158ffc664e","order_by":15,"name":"Bin ZHANG","email":"","orcid":"","institution":"Anhui University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"ZHANG","suffix":""},{"id":311081423,"identity":"70ec6d06-7a95-4313-bbb8-f5e56ff50688","order_by":16,"name":"Ling HU","email":"","orcid":"","institution":"Anhui University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ling","middleName":"","lastName":"HU","suffix":""},{"id":311081424,"identity":"8548f6fa-4dbc-4890-a224-36c12cf7f5b1","order_by":17,"name":"Qing YU","email":"","orcid":"","institution":"Anhui University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Qing","middleName":"","lastName":"YU","suffix":""},{"id":311081425,"identity":"7a321aee-a373-4824-b6a1-4e7cbcf29fdc","order_by":18,"name":"Zhi Zhang","email":"","orcid":"https://orcid.org/0000-0002-4205-3181","institution":"University of Science and Technology of China","correspondingAuthor":false,"prefix":"","firstName":"Zhi","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-05-24 14:21:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4473024/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4473024/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62599846,"identity":"2299734a-db47-4ea8-ae7d-6ad51e9cd9f1","added_by":"auto","created_at":"2024-08-16 09:42:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":236798,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEA suppresses PVCs occurring post-myocardial infarction.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Schematic for myocardial ischemia(MI) and the electrocardiographic monitoring to assess the PVCs. Experimental paradigms for electroacupuncture(EA) treatment. \u003cstrong\u003e(B) \u003c/strong\u003eTypical ECG tracks of PVCs in mice. \u003cstrong\u003e(C, D) \u003c/strong\u003eThe PVCs events in MI mice treated with or without EA stimulation within 5 min. Red indicates that at least one PVCs event occurs within 30 s. (n=10 mice per group, Ordinary one-way ANOVA with Tukey’s post-test). \u003cstrong\u003e(E)\u003c/strong\u003e The QRS width(ms) in MI mice treated with or without EA stimulation within 5 min. (n=6 mice per group, Ordinary one-way ANOVA with Tukey’s post-test). \u003cstrong\u003e(F, G)\u003c/strong\u003e The autonomic nervous tone assessed by the HRV frequency domain analysis in MI mice treated to EA stimulation at different time periods. (SH: n=7 mice; MI: n=7 mice; EA: n=6 mice, Ordinary one-way ANOVA with Tukey’s post-test). \u003cstrong\u003e(H) \u003c/strong\u003eHeart tissue expression of NE in each group. (n=6 mice per group, Ordinary one-way ANOVA with Tukey’s post-test). \u003cstrong\u003e(I)\u003c/strong\u003e EA (0.5 mA) at the HT7 site increased survival rates in MI mice. (SH: n=8 mice; MI: n=11 mice; EA: n=13 mice, two-sided log-rank test). \u003cstrong\u003e(J)\u003c/strong\u003e The representative images of TTC staining of heart sections in each group of mice. Non-ischemic areas are shown in blue, dangerous areas (AAR) in red, and the infarct area (INF) in white, Scale bar, 2.5 mm. Myocardial AAR per left ventricle (LV) and infarct (INF) per AAR for each group of mice after myocardial ischemia. (n = 6 mice per group, One-way ANOVA with Tukey’s post-test). \u003cstrong\u003e(K) \u003c/strong\u003eHeart tissue expression of cTnT (A marker of myocardial damage)in each group. (n=6 mice per group, Ordinary one-way ANOVA with Tukey’s post-test). All data are represented by mean±SEM. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4473024/v1/0e793d993e9604dce906491d.png"},{"id":62600292,"identity":"fa1fff6d-08e1-4cd2-b72e-20150974ead4","added_by":"auto","created_at":"2024-08-16 09:50:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":569369,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHT7 (Shenmen) acupoint afferent nerves drive a brain-to-heart circuit through the M1L5 in normal mice. (A) \u003c/strong\u003eSchematic of PRV-EGFP injection into the Left ventricular wall. \u003cstrong\u003e(B, C) \u003c/strong\u003eConfocal image showing PRV-infected neurons (green) in the cortex with DAPI (blue) as the counterstaining. Left/Right: enlarged view of the confocal image(B). Three-dimensional view of PRV-infected cortical neurons(C). This experiment was repeated independently in 8 male mice with similar results. Scale bars, 200μm (overview) and 50μm (zoom).\u003cstrong\u003e (D) \u003c/strong\u003eProportion of PRV-infected cortical neurons in M1L5/S1L5 after injection. (n = 6 male mice per group, Unpaired t-test).\u003cstrong\u003e (E, F)\u003c/strong\u003e Representative images and summarized data showing that PRV-infected cortical neurons within the M1L5 are co-localized with glutamatergic neurons. (n=6 male mice per group. Scale bars, 50μm (overview) and 25 μm (zoom). Unpaired t-test).\u003cstrong\u003e (G, H)\u003c/strong\u003e PRV was dripped into the chest, and no PRV-infected neurons were found in the MIL5 (excluding the possibility of PRV-EGFP cardiac injection leakage). (Scale bars, 200μm. This experiment was repeated independently in 3 male mice with similar results).\u003cstrong\u003e (I)\u003c/strong\u003e Schematic diagram showing the injection of H129-EGFP into left thoracic 3 DRG and injection of CTB into HT7 point of wild-type mice. \u003cstrong\u003e(J) \u003c/strong\u003eH129-infected neurons within the DRG are co-localized with CTB-infected neurons. (Scale bars, 100μm (overview) and 50 μm (zoom). \u0026nbsp;This experiment was repeated independently in 4 male mice with similar results).\u003cstrong\u003e(K) \u003c/strong\u003eRepresentative images showing EGFP-labeled neurons in the M1L5 after H129-EGFP injected about 72h. (Scale bars, 50 μm (overview) and 25 μm (zoom). n=4 mice).\u003cstrong\u003e (L) \u003c/strong\u003eProportion of H129-infected cortical neurons in M1L5/S1L5 after injection. (n=4 male miceper group, Unpaired t-test).\u003cstrong\u003e (M)\u003c/strong\u003e Schematic diagram showing rabies virus-based retrogradely trans-monosynaptic tracing from M1L5.\u003cstrong\u003e (N, O)\u003c/strong\u003e An image showing the start neurons in cortical layer 5(N) (Scale bars, 200 μm). eGFP-labeled neurons are located in the lamina Ⅳof contralateral spinal cord and co-localized with Fluorogold (FG)-infected neurons(O). (Scale bars, 100 μm (overview) and 50 μm (zoom). n=4 mice).\u003cstrong\u003e (P, Q) \u003c/strong\u003eSchematic for injection of RV-EvnA-eGFP into the M1L5(P). The representative image shows no labeled neurons observed in dorsal horn after virus injection (Q). (Scale bars, 100 μm.n=4 mice.) All data are represented by mean ± SEM. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, and ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4473024/v1/68d9f5a0df928323a4d165f7.png"},{"id":62600293,"identity":"0428b07c-8349-438e-bcf6-3cf3f7fb33a0","added_by":"auto","created_at":"2024-08-16 09:50:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":362823,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOptogenetic activation of M1L5 excitatory neurons induces enhanced cardiac sympathetic tone. (A)\u003c/strong\u003e Schematic of optogenetic experiment. \u003cstrong\u003e(B) \u003c/strong\u003eLeft: optical fibers implanted above M1 L5 neurons for optogenetic stimulation. Right: enlarged image showing CaMKII-ChR2-mCherry expression (red) in M1 L5 neurons. (Scale bars, 200 mm (overview) and 50 mm (zoom). \u003cstrong\u003e(C, D) \u003c/strong\u003eTypical traces of heart rate(HR), R-R interval(R-R) and sample ECG traces in mice stimulated by optogenetic stimulation of M1L5 excitatory neurons. \u003cstrong\u003e(E, F, G)\u003c/strong\u003e LF/HF ratio (E), PNS index(F) and HRV frequency domain analysis(G) showing changes in cardiac sympathetic tone in mice before and during light photostimulation. (n=7 malemice per group, Unpaired t-test). \u003cstrong\u003e(H) \u003c/strong\u003eHeart tissue expression of NE level in mice before and during light photostimulation. (n=7 male mice per group, Unpaired t-test).All data are represented by mean±SEM. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, and ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4473024/v1/187d3827c6497ac065460a42.png"},{"id":62599844,"identity":"01000b73-e353-4044-ba0b-953d36044e96","added_by":"auto","created_at":"2024-08-16 09:42:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":436687,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEA HT7 inhibits the M1L5\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eGlu\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e neurons to suppresses PVCs occurring post-myocardial infarction. (A)\u003c/strong\u003eSchematic diagram for c-Fos expression of M1L5 pyramidal neurons upstream of the heart in MI mice treated EA. \u003cstrong\u003e(B, C)\u003c/strong\u003e Images(B) and data(C) showing the expression of c-Fos-positive neurons in the M1L5 (pyramidal neurons upstream of the heart) in MI mice treated with EA HT7. (Scale bars, 100 mm (overview), 50 mm (zoom) and 25 mm (zoom). n=18 slice from 6 mice for each group. Ordinary one-way ANOVA with Tukey’s post-test). \u003cstrong\u003e(D)\u003c/strong\u003e Schematic diagram of in vivo electrophysiological study in mice. Sample traces of action potentials recorded from M1L5 F-INs neuron and EPs neuron of each group. \u003cstrong\u003e(E)\u003c/strong\u003e Representative graph of waveform differentiation and 3D clusters based on an offline sorter, Spikes originating from individual units were sorted using three-dimensional principal-component analysis(left). Representative cross-correlogram performed between a putative inhibitory IN/PN and a non-identified neuron(right). \u003cstrong\u003e(F)\u003c/strong\u003e Firing rate data of action potentials recorded from M1L5\u003csup\u003eEps\u003c/sup\u003e neurons in mice of each group. (SH: n=53 units; MI: n=27 units; EA: n=37 units, Ordinary one-way ANOVA with Tukey’s post-test). \u003cstrong\u003e(G, H) \u003c/strong\u003eSchematic for EA stimulation and fiber photometric recording in MI mice(G). A typical image showing the GCaMP6m fluorescence and track of fiber in the M1L5(H) (Scale bar, 50 μm). \u003cstrong\u003e(I)\u003c/strong\u003eHeatmaps and representative traces of M1L5\u003csup\u003eGlu\u003c/sup\u003e GCaMP6m signals at the EA 0mA and the EA 0.5mA treatment; the thick lines and gray shadows in the middle of the image represent the mean±SEM. \u003cstrong\u003e(J, K)\u003c/strong\u003e Comparison of the averaged ΔF/F(J) and calcium events(K) of M1L5\u003csup\u003eGlu\u003c/sup\u003e GCaMP6m signals at the EA 0mA and the EA 0.5mA treatment. (n=7 mice per group. Unpaired t-test). \u003cstrong\u003e(L, M, N)\u003c/strong\u003e Schematic for viral injection and microendoscopic calcium imaging(L). Image of GCaMP6m-expressing M1L5 neurons (M), (Scale bar, 20 μm) and independent-component-analysis-derived neuron activity traces (N). \u003cstrong\u003e(O, P)\u003c/strong\u003e Comparison of the ΔF/F(O) and calcium events(P) of M1L5\u003csup\u003eGlu\u003c/sup\u003e GCaMP6m signals.ΔF/F0, the change in fluorescence (ΔF) over the baseline fluorescence (F0) of calcium spikes.(SH, n=47 cells from four mice; MI, n=95 cells from eight mice; EA, n=122 cells from eleven mice. Ordinary one-way ANOVA with Tukey’s post-test). \u003cstrong\u003e(Q)\u003c/strong\u003e Chemogenetic experimental timeline. \u003cstrong\u003e(R, S, T, U)\u003c/strong\u003e Effects of chemogenetic activitation of M1L5\u003csup\u003eGlu\u003c/sup\u003e neurons on the counts of PVCs(R), LF/HF(S), QRS width(T) and NE level(U) in EA treatment mice. (n=6 mice per group, Ordinary one-way ANOVA with Tukey’s post-test). All data are represented by mean±SEM. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, and ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4473024/v1/ee83df3fdeba977942c84bc8.png"},{"id":62599841,"identity":"f5c41e73-3b4e-455d-9e9b-768373e5af04","added_by":"auto","created_at":"2024-08-16 09:42:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":413734,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDissection of the M1L5\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eGlu\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e→ZI circuit. (A, B)\u003c/strong\u003e Schematic for the anterograde tracing from the M1L5 to the ZI(A). Representative images showing mCherry+ never fibers in the ZI(B). (Scale bars, 200 μm (overview) and 50 μm (zoom). n=3 mice per group).\u003cstrong\u003e (C, D, E) \u003c/strong\u003eSchematic of the Cre-dependent retrograde trans-monosynaptic RV tracing strategy(C). Representative image of the injection site and viral expression within the M1L5 (left). Starter cells (yellow) co-express AAV-DIO-TVA-mCherry(red), AAV-DIO-RVG, and RV-EnvA-DG-eGFP (green); mCherry-labeled neurons within the M1L5 (right)(D). mCherry-labeled neurons within the M1L5 traced from the ZI are co-localized with glutamatergic neurons(E). (Scale bars, 200μm (overview) and 50μm (zoom). n=3 mice per group).\u003cstrong\u003e\u0026nbsp; (F, G)\u003c/strong\u003e Strategy for viral trans-synaptic YFP labeling of ZI neurons receiving M1L5 afferent projections(F) and analysis of non-GABAergic neurons(G). (Scale bars, 100 μm (overview) and 50 μm (zoom). n = 7 mice per group. Unpaired t-test). \u003cstrong\u003e(H, I)\u003c/strong\u003e c-Fos expression in GABAergic/non-GABAergic neurons in ZI domains after optogenetic activation of M1L5\u003csup\u003eGlu\u003c/sup\u003e neurons(n = 4 mice per group. Unpaired t-test). \u003cstrong\u003e(J, K) \u003c/strong\u003ec-Fos expression in GABAergic neurons in ZI domains after optogenetic inhibition of M1L5Glu-ZI circuit (Scale bars, 200 mm (overview), 50μm (zoom), 20μm (zoom),n=4 mice per group. Unpaired t-test). \u003cstrong\u003e(L) \u003c/strong\u003eThree-dimensional pattern of M1L5 projecting ZI. \u003cstrong\u003e(M)\u003c/strong\u003e In vivo fiber photometry recordings of calcium transients through GCaMP6f recording Ca2+ signal in the ZI upon optogenetic activitation of M1-ZI projections. (n=5 mice per group). \u003cstrong\u003e(N)\u003c/strong\u003e Strategy (left) for retrograde labeling of M1L5 neurons (right) after injection in ZI. (n=4 mice per group). \u003cstrong\u003e(O)\u003c/strong\u003e In vivo fiber photometry recordings of calcium transients through GCaMP6f recording Ca2+ signal in the ZI upon optogenetic inhibition of M1-ZI projections. (n=4 mice per group). \u003cstrong\u003e(P)\u003c/strong\u003e A model of M1L5\u003csup\u003eGlu\u003c/sup\u003e→ZId\u003csup\u003enon-GABA\u003c/sup\u003e→ZIv\u003csup\u003eGABA\u003c/sup\u003e circuit. GluRs, glutamate receptors. All data are represented by mean ± SEM. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4473024/v1/175730ae059e07a974c5f242.png"},{"id":62599845,"identity":"3245b072-8751-46b4-8beb-67b9d4f4d088","added_by":"auto","created_at":"2024-08-16 09:42:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":450925,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe M1L5-ZI-RVLM neural circuit was involved in the regulation of heart function and mediated the protective effect of EA-HT7 against PVCs. (A, B) \u003c/strong\u003eSchematic for vial injection and Optical-fiber calcium recording(A). A typical image showing the GCaMP6m fluorescence and track of fiber in the ZI(B). (Scale bars, 200 mm).\u003cstrong\u003e(C, D, E)\u003c/strong\u003e The typical trace(C), the ΔF/F (D) and calcium events(E) show that Ca2+ signals of ZIGABA rapidly increased in EA HT7 0.5mA compared with 0mA with MI mice. (n=6 male mice per group. Unpaired t-test).\u003cstrong\u003e (F)\u003c/strong\u003eThe schematic diagram shows the Fos-TRAP labeling strategy, which blocks the expression of the TRE-tight promoter by replenishing Dox-supplemented drinking water.\u003cstrong\u003e(G) \u003c/strong\u003eThe schedule of neuron experiments marked with the Fos-TRAP labeling system.\u003cstrong\u003e(H) \u003c/strong\u003eComparison of drinking volumes of Dox-supplemented water and non-Dox water in mice. (n=7 male mice per group. Unpaired t-test).\u003cstrong\u003e (I)\u003c/strong\u003ePercentage of neurons expressing mCherry in ZI. (n=6 male mice per group, One-way ANOVA with Tukey’ post-test).\u003cstrong\u003e (J)\u003c/strong\u003eThe representative images of mCherry expression in FN under five different experimental schemes. (Scale bar, 20 μm).\u003cstrong\u003e(K)\u003c/strong\u003eOn day 3 after EA treatment, mCherry-labeled neurons in the ZI are mainly co-located with GABA immunofluorescent-positive cells. (Scale bar, 20 µm. n=4 per group, Unpaired t-test).\u003cstrong\u003e (L) \u003c/strong\u003eSchematic of ZI injection of rAAV-EF1a-DIO-Ypet-2A-mGFP-WPRE-pA in mice(left). representative image of mGFP labeling neurons by ZI infusion of rAAV-EF1a-DIO-Ypet-2A-mGFP-WPRE-pA(middle). (Scale bars, 200 μm). Images representative of mGFP+ fibers in RVLM(right). (Scale bars, 50 μm). (n=4 male mice).\u003cstrong\u003e (M)\u003c/strong\u003eNormalized distributions of virals-labeled output neurons across different brain areas in ipsilateral to the injection site.\u003cstrong\u003e(N) \u003c/strong\u003eSchematic of the Cre-dependent anterograde trans-monosynaptic tracing strategy(left). Typical image showing the RVLM by AAV-DIO–eGFP(right). n=4 mice , Scale bars, 50 μm)\u003cstrong\u003e. (O) \u003c/strong\u003eSchematic of the cre-dependent anterograde trans-monosynaptic HSV-ΔTK tracing strategy(left). Typical image showing the infusion site within the ZI(middle) ( Scale bars, 100 mm) and the tdTomato-labeled neurons within the RVLM traced from the ZIv-RVLM(right) (Scale bars, 20 mm, n=4 mice). \u003cstrong\u003e(P)\u003c/strong\u003e Schematic of the Cre-dependent retrograde trans-monosynaptic RV tracing strategy(left). Representative image of the injection site(middle) ( Scale bars, 100 mm) \u0026nbsp;and viral expression within the ZI(right) (Scale bars, 20 mm). Starter cells (yellow) co-express AAV-DIO-TVA-mCherry(red), AAV-DIO-RVG, and RV-EnvA-DG-eGFP (green); eGFP-labeled neurons within the ZI traced from the RVLM. (n=4 mice). All data are represented by mean ± SEM. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4473024/v1/3ebee1f9ad71d9573396bfd8.png"},{"id":62599848,"identity":"2ff7da1b-650e-4874-a263-ef555a0a6c81","added_by":"auto","created_at":"2024-08-16 09:42:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":164110,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eactivitation of M1L5-projecting ZI circuit abolishes EA HT7-induced suppresses PVCs. (A)\u003c/strong\u003e Schematic diagram showing the strategy used to determine whether neuronal activity in the RVLM was modulated by the M1 and ZI. Retro-AAV-CaMKII-ChR2 virus was injected into theVZI, AAV-Vgat-hM3dq-mCherry was injected into the ZI and a optical fiber was implanted into the M1L5 and RVLM. The timeline shown below demonstrates the experimental procedure. First, 5 s of blue light was delivered to optogenetically activate the M1L5-ZI circuit, and then neuronal. activity recorded in the RVLM was recorded for 5min. 30min after CNO injection to inhibit ZIGABA neurons, another light stimulus was given, and neuronal activity was recorded simultaneously for another 5min. \u003cstrong\u003e(B, C, D)\u003c/strong\u003e Representative typical trace(R), photometric heatmaps(S) and ΔF/F (%)(T)show that Ca\u003csup\u003e2+\u003c/sup\u003e signals rapidly decreased in EA HT7 mice compared with control mice. But these phenomena can be blocked by chemogenetic activation of ZI\u003csup\u003eGABA\u003c/sup\u003e neurons. (n=6 mice per group, Ordinary one-way ANOVA with Tukey’s post-test). \u003cstrong\u003e(E)\u003c/strong\u003e Chemogenetic experimental timeline. \u003cstrong\u003e(F, G, H, I)\u003c/strong\u003e Effects of optogenetic activitation of M1L5-ZI circuit and chemogenetic activitation of ZI\u003csup\u003eGABA\u003c/sup\u003e on the counts of PVCs(V), LF/HF(W), QRS width(X) and NE level(Y) in EA treatment mice. (n=6 mice per group, Ordinary one-way ANOVA with Tukey’s post-test). All data are represented by mean ± SEM. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4473024/v1/6133ce74205739bfbdbd3050.png"},{"id":62601108,"identity":"a6d3d205-7584-4b60-8142-cdffccc2181d","added_by":"auto","created_at":"2024-08-16 09:58:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3771699,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4473024/v1/3ba2b1a9-807a-48a8-98c8-1e44a221014f.pdf"},{"id":62599842,"identity":"a1a07d64-de2a-40d6-853e-55fdf73b9d0e","added_by":"auto","created_at":"2024-08-16 09:42:04","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12302039,"visible":true,"origin":"","legend":"Supplemental data FigureS1-S9","description":"","filename":"SupplementaldataFigureS1S9.docx","url":"https://assets-eu.researchsquare.com/files/rs-4473024/v1/04564ab376f39b3b35bdb631.docx"},{"id":62599839,"identity":"4914b404-2281-4057-b578-928cb14898e7","added_by":"auto","created_at":"2024-08-16 09:42:03","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":47573,"visible":true,"origin":"","legend":"Supplemental Methods and Material","description":"","filename":"SupplementalMethodsandMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4473024/v1/2c5e727b5626289cbbf2e71c.docx"},{"id":62599849,"identity":"5ffa5e95-5196-4ec7-9009-210c8a872146","added_by":"auto","created_at":"2024-08-16 09:42:05","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":196413,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-4473024/v1/c2af69d88ec2219f3d2e60ec.png"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Electroacupuncture Suppresses Premature Ventricular Complexes Occurring Post-myocardial Infarction through corticothalamic circuit","fulltext":[{"header":"Highlights","content":"\u003cp\u003eEA suppresses PVCs occurring post-MI\u003c/p\u003e\n\u003cp\u003eHT7 (Shenmen) acupoint afferent nerves drive a brain-to-heart circuit through M1L5\u003c/p\u003e\n\u003cp\u003eEA reduces hyperactivity of M1L5 neurons in MI model mice\u003c/p\u003e\n\u003cp\u003eEA-induced suppression of PVCs occurring post-MI via M1L5-ZI-RVLM circuit\u003c/p\u003e"},{"header":"INTRODUCTION","content":"\u003cp\u003ePremature ventricular complexes (PVCs) following myocardial infarction (MI) pose a significant risk factor for subsequent sudden cardiac death (SCD)\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. While treatments over the past decade have encompassed antiarrhythmic drug therapy, defibrillation, or ablation, early-stage use of antiarrhythmic drugs such as encainide, flecainide, or moricizine has been associated with increased rates of SCD and overall mortality\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Consequently, effectively mitigating PVCs post-MI remains a formidable scientific challenge.\u003c/p\u003e \u003cp\u003eBioelectronic medicine therapy, which includes nerve electrical stimulation, has emerged as a promising alternative. Acupuncture, a practice with roots dating back over 2000 years in China and now globally recognized, is increasingly utilized as a form of bioelectronic medicine therapy. Electroacupuncture (EA), in particular, has demonstrated efficacy in alleviating PVCs post-MI, ameliorating disorders of the sympathetic system, and reducing the risk of SCD\u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The tachycardias precipitating cardiac arrest are primarily driven by abnormal sympathetic excitation\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Human studies have shown that electroacupuncture can regulate abnormal states of autonomic nerves\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Furthermore, expert consensus recommends acupuncture as an effective adjunctive therapy for regulating heartbeat rhythm in arrhythmia patients\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Leveraging the somatosensory autonomic nervous system, acupuncture's effects can extend to distant areas\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The inherent characteristics of EA effects have led to the hypothesis that they improve PVCs post-MI via modulation of the autonomic nervous system. However, the precise mechanisms underlying these effects remain to be elucidated.\u003c/p\u003e \u003cp\u003e \u003cem\u003eShenmen\u003c/em\u003e (HT7), an acupoint situated in the anterior carpal region at the ulnar end of the distal metacarpal crease and along the radial margin of the flexor carpi ulnaris tendon \u003cb\u003e(Fig. S1A)\u003c/b\u003e, has been specifically targeted for the treatment of myocardial ischemia, particularly post-myocardial infarction PVCs \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Alterations in activities in various brain regions involved in sensation and visceral motor processing have been revealed in functional magnetic resonance imaging (fMRI) studies with individuals receiving EA treatment at HT7\u003csup\u003e18\u003c/sup\u003e. The thalamus serves as a relay for multimodal sensory information and reciprocally connects with the sympathetic nervous system\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Within the thalamus, the zona incerta (ZI) receives diverse inputs from cortical and subcortical regions\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, suggesting a potential role for the ZI as a conduit for somatosensory processing during EA. Nevertheless, the specific organization of cell types and the functions of corticothalamic circuits underlying EA-induced suppression of post-myocardial infarction PVCs remain largely unexplored.\u003c/p\u003e \u003cp\u003eIn the present study, we investigated the functional organization of the M1L5-ZI-RVLM circuit and elucidated its role in suppressing post-MI PVCs in response to EA stimulation. Artificial manipulation of this circuitry can replicate or abolish the effects of EA-induced suppression of post-MI PVCs. Our findings indicate that inhibition of the M1L5-ZI-RVLM circuit is essential for the manifestation of EA's suppressive effects on post-MI PVCs.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eEA suppresses PVCs depends on balance sympathetic nervous system in mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated whether stimulation of the HT7 acupoint by EA suppresses\u0026nbsp;post-myocardial infarction PVCs in mice.\u0026nbsp;Mouse models of\u0026nbsp;premature ventricular complexes were induced by ligation\u0026nbsp;of the left anterior descending coronary artery\u0026nbsp;(MI) \u003cstrong\u003e(Fig. 1A)\u003c/strong\u003e. Male mice were the focus of our study unless otherwise specified. At 3 hours post-ligation,\u0026nbsp;mice\u0026nbsp;exhibited\u0026nbsp;a significant\u0026nbsp;increase in\u0026nbsp;PVCs\u0026nbsp;and ST-segment elevation compared to sham-operated mice \u003cstrong\u003e(Fig. S1B,\u003c/strong\u003e \u003cstrong\u003eS1C)\u003c/strong\u003e. To assess the effect of different EA intensities on post-MI PVC suppression in mice, we evaluated the impact of EA treatment at two intensities (0.5mA or 3mA). We found that only an EA intensity of 0.5mA elicited PVC suppression in MI mice \u003cstrong\u003e(Fig. 1B-D)\u003c/strong\u003e. Additionally, the 0.5mA EA intensity reduced the QRS width in MI mice \u003cstrong\u003e(Fig. 1E)\u003c/strong\u003e. It is noteworthy that QRS width reflects ventricular electrical activity and exhibits abnormal prolongation during ventricular arrhythmias\u003csup\u003e21\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe investigated whether EA suppresses PVCs in MI mice by potentially reducing sympathetic nerve activation. The autonomic nervous system significantly contributes to the development of PVCs\u003csup\u003e22,23\u003c/sup\u003e. In human studies, inhibiting abnormal sympathetic excitation after myocardial ischemia effectively reduces PVC occurrence\u003csup\u003e24\u003c/sup\u003e. We conducted heart rate variability (HRV) analysis and detected cardiac norepinephrine (NE) content to assess the potential effects of EA (0.5mA, 2Hz, pulse width of 50 μs) on the cardiac sympathetic component of PVCs occurring post-MI in mice. Our findings indicate that compared to the MI group, EA treatment significantly attenuated cardiac sympathetic excitability \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e1F,\u003c/strong\u003e \u003cstrong\u003e1G)\u0026nbsp;\u003c/strong\u003e(\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.0045, Sham: \u003cem\u003en\u0026nbsp;\u003c/em\u003e= 7; MI: \u003cem\u003en\u003c/em\u003e = 7; EA: \u003cem\u003en\u003c/em\u003e = 6), reduced cardiac NE content\u003cstrong\u003e\u0026nbsp;(Fig.\u003c/strong\u003e \u003cstrong\u003e1H)\u003c/strong\u003e (\u003cem\u003ep\u003c/em\u003e = 0.0053, \u003cem\u003en\u003c/em\u003e = 6), improved survival rate in MI mice \u003cstrong\u003e(Fig. 1I)\u003c/strong\u003e (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.006, Sham: \u003cem\u003en\u003c/em\u003e = 8; MI: \u003cem\u003en\u003c/em\u003e = 11; EA: \u003cem\u003en\u003c/em\u003e = 13),\u0026nbsp;and\u0026nbsp;decreased\u0026nbsp;myocardial damage in MI mice \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e1J)\u003c/strong\u003e (AAR/LV: \u003cem\u003ep\u003c/em\u003e = 0.0027; \u003cem\u003en\u0026nbsp;\u003c/em\u003e= 13). We also observed that EA 0.5mA reduced QRS width (\u003cem\u003ep\u003c/em\u003e = 0.026; \u003cem\u003en\u003c/em\u003e = 6) and ST deviation (\u003cem\u003ep\u003c/em\u003e = 0.0319;\u003cem\u003e\u0026nbsp;n\u003c/em\u003e = 6) in MI mice during\u0026nbsp;electrocardiogram\u0026nbsp;(ECG) analysis. However, it did not affect RR interval, heart rate, PR interval, QTc interval, and R amplitude \u003cstrong\u003e(Fig\u003c/strong\u003e.\u003cstrong\u003e\u0026nbsp;S1H, S1I)\u003c/strong\u003e. Increased\u0026nbsp;cardiac sympathetic activity can induce severe PVCs during MI\u003csup\u003e25,26\u003c/sup\u003e, which worsens damage to myocardial cells and fibrosis. Furthermore, activated myocardial fibrosis perpetuates PVC occurrence\u003csup\u003e27,28\u003c/sup\u003e. Several studies have shown that reducing excessive cardiac sympathetic nerve activation is crucial for improving cardiac function and reducing PVCs\u003csup\u003e9,29\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eWe then examined ventricular infarction and fibrosis levels through\u0026nbsp;histological analysis in MI mice following EA treatment. We observed reductions in cell atrophy and nuclear shrinkage proportions, cross-section of cardiomyocytes, interstitial congestion, and inflammatory cell infiltration \u003cstrong\u003e(Fig\u003c/strong\u003e. \u003cstrong\u003eS1D)\u003c/strong\u003e, and a decrease in cell fibrosis in the MI+EA 0.5mA group compared to the MI group \u003cstrong\u003e(Fig\u003c/strong\u003e. \u003cstrong\u003eS1E,\u003c/strong\u003e \u003cstrong\u003eS1F)\u003c/strong\u003e (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.0076, \u003cem\u003en\u003c/em\u003e = 6). Furthermore, considering the unique structure and restricted expression of cardiac troponin T (cTnT) and creatine kinase (CK)-MB by heart myocytes, they are utilized as biomarkers for acute myocardial injury. Hence, we assessed the expression of cTnT and CK-MB in myocardial tissue. Our results indicated significantly lower expression of cTnT \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e1K)\u003c/strong\u003e (\u003cem\u003ep\u003c/em\u003e = 0.0088, \u003cem\u003en\u0026nbsp;\u003c/em\u003e= 6) and CK-MB \u003cstrong\u003e(Fig\u003c/strong\u003e. \u003cstrong\u003eS1G)\u0026nbsp;\u003c/strong\u003e(\u003cem\u003ep\u003c/em\u003e = 0.0083, \u003cem\u003en\u003c/em\u003e = 6) in the MI+EA 0.5mA group compared to the MI group. These findings suggest that EA at 0.5mA effectively reduces PVCs and substantially mitigates risk of sudden cardiac death in MI mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHT7 (Shenmen) acupoint afferent nerves drive a brain-to-heart circuit\u003c/strong\u003e\u003cstrong\u003ethrough the Layer 5 neurons in motor cortex (M1L5)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe then investigated the neural circuitry underlying the observed suppression of PVCs following post-MI induced by EA. Given the pivotal role of the primary cortex in processing and integrating afferent somatosensory inputs\u003csup\u003e30,31\u003c/sup\u003e, we examined the neurons receiving somatosensory input from EA (HT7 acupoint) sites to the primary cortex. We employed herpes simplex virus (HSV) as a tracer, as it selectively targets somatosensory neurons and can traverse the somatosensory circuit. Considering the widespread distribution of somatosensory neurons at the HT7 acupoint in the dorsal root ganglion (DRG) of the T3 spinal cord segment \u003cstrong\u003e(Fig\u003c/strong\u003e. \u003cstrong\u003eS2C,\u003c/strong\u003e \u003cstrong\u003eS2D,\u003c/strong\u003e \u003cstrong\u003eS2E,\u003c/strong\u003e \u003cstrong\u003eS2F)\u003c/strong\u003e, we performed microinjections to directly administer the virus into the T3 DRG, ensuring a high concentration around the soma \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e2A,\u003c/strong\u003e \u003cstrong\u003e2B)\u003c/strong\u003e. Additionally, to mitigate immune elimination by the host, we combined DRG microinjection with bortezomib to enhance HSV infection in T3 DRG neurons\u003csup\u003e32\u003c/sup\u003e \u003cstrong\u003e(Fig\u003c/strong\u003e. \u003cstrong\u003eS3A)\u003c/strong\u003e. Our observations revealed that HSV requires a minimum of 48 hours to transport from the soma of DRG neurons to the central terminals \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003eS3B)\u003c/strong\u003e. Secondary spinal cord neurons can be labeled approximately 72 hours after viral injection \u003cstrong\u003e(Fig\u003c/strong\u003e. \u003cstrong\u003eS3C\u003c/strong\u003e), after which the virus spreads along the ascending pathway and gradually labels the downstream neurons \u003cstrong\u003e(Fig\u003c/strong\u003e. \u003cstrong\u003eS3D)\u003c/strong\u003e. We found that S1 and M1 neurons of the cortical area were labeled by HSV (green) \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e2C,\u003c/strong\u003e \u003cstrong\u003e2D)\u003c/strong\u003e (\u003cem\u003ep\u003c/em\u003e = 0.9383, \u003cem\u003en\u003c/em\u003e = 6).\u003c/p\u003e\n\u003cp\u003eThe M1 serves as a crucial command center in somatosensory signal processing. Recent studies have suggested the possibility that dorsal root ganglion neurons directly transmit sensory signals to the motor cortex through ascending transmission of spinal cord projection neurons\u003csup\u003e33,34\u003c/sup\u003e. To verify whether afferent somatosensory nerves from the HT7 acupoint project to the M1L5 via a direct spino-cortical circuit, we injected the EnvA-pseudotyped glycoprotein (G)-deleted rabies virus (EnvA-RV-ΔG-eGFP)21 after expressing AAV2/9-DIO-RVG-TVA-mCherry for 3 weeks in the M1 of mice, with fluorogold (FG) injected at the HT7 acupoint site \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e2E)\u003c/strong\u003e. We observed a cluster of eGFP-labeled neurons located in the IV to VI layers of the T3 thoracic spinal cord. Remarkably, in the same animals, FG-positive fibers were also identified in the IV to VI layers of the T3 thoracic spinal cord \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e2F,\u003c/strong\u003e \u003cstrong\u003e2G)\u003c/strong\u003e. Conversely, no labeled neurons were observed in the dorsal horn of the T3 spinal cord after injecting AAV2/9-DIO-TVA-mCherry alone into the layer 5 neurons in M1 \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e2H,\u003c/strong\u003e \u003cstrong\u003e2I)\u003c/strong\u003e. These findings suggest that afferent somatosensory nerves from the HT7 acupoint relay information to the M1L5.\u0026nbsp;Furthermore, the direct spinal cortical pathway may be implicated in the transmission of HT7 acupoint afferent somatosensory nerves.\u003c/p\u003e\n\u003cp\u003eAlthough the HT7 acupoint's afferent somatosensory nerves sending projections to the M1L5 in mice are well established, it remains unknown whether the M1L5 can modulate sympathetic outflow to the heart. To identify cortical neurons involved in heart control and sympathetic nervous regulation, we initiated experiments by injecting pseudorabies virus (PRV) 531 encoding EGFP into the left ventricular wall of C57BL/6J mice to label upstream neurons retrogradely and trans-synaptically \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e2J)\u003c/strong\u003e. Validating this method, we began observations 80-90 hours post PRV injection, revealing the emergence of viral labeling in subcortical areas previously implicated in cardiac function \u003cstrong\u003e(Fig. S2A)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eCritically, at 110-120 hours post PRV injection, A small cluster of EGFP-positive neurons was consistently observed in the bilateral primary motor cortex (M1; bregma: 0.20 to –0.85 mm, lateral from midline: ±0.78 to ±1.36 mm) \u003cstrong\u003e(Fig. 2K)\u003c/strong\u003e. Cell counting indicated that the labeled cortical region was situated in M1 rather than the primary sensory cortex (S1) \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e2L,\u003c/strong\u003e \u003cstrong\u003e2M)\u003c/strong\u003e (p \u0026lt; 0.0001, n = 8). In contrast, control animals receiving the same PRV injection into the chest or other viscera showed no labeled neurons in these regions \u003cstrong\u003e(Fig. 2N, 2O, and Fig. S2B)\u003c/strong\u003e. Moreover, the overwhelming majority of EGFP+ neurons in M1 (~95%) were localized in layer 5 and exhibited the characteristic morphology of projecting pyramidal neurons. Through immunofluorescence identification, we found that the upstream M1L5 pyramidal neurons of the heart primarily co-labeled with glutamatergic antibodies rather than GABAergic antibodies \u003cstrong\u003e(Fig. 2P, 2Q)\u003c/strong\u003e (p \u0026lt; 0.0001, n = 6).\u0026nbsp;Based on these findings, the M1L5\u0026nbsp;glutamatergic neuron (M1L5\u003csup\u003eGlu\u003c/sup\u003e) links HT7 acupoint afferent\u0026nbsp;somatosensory nerves\u0026nbsp;to the mice heart sympathetic outflow center.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe necessity of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ecardiac sympathetic\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;regulation by M1L5\u003c/strong\u003e \u003cstrong\u003e\u003csup\u003eGlu\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor a long time, the ventrolateral region of the rostral medulla (RVLM), housing cardiac sympathetic premotor neurons, has been considered pivotal in cardiovascular disease\u003csup\u003e35\u003c/sup\u003e. Subsequently, we investigated c-Fos expression in the RVLM, observing an increase in c-Fos levels in the RVLM of post-MI mice compared to sham mice, with this phenomenon reversed by EA treatment \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e3A,\u003c/strong\u003e \u003cstrong\u003e3B)\u003c/strong\u003e (p = 0.0439, n = 6). To further analyze whether M1L5 activation can induce RVLM activity and augment sympathetic outflow, we chemogenetically activated glutamatergic (Glu) neurons in M1L5, significantly increasing c-Fos expression in RVLM neurons \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e3C,\u003c/strong\u003e \u003cstrong\u003e3D)\u003c/strong\u003e (p = 0.0019, n = 4).\u003c/p\u003e\n\u003cp\u003eNext, we investigated whether stimulation of M1L5\u003csup\u003eGlu\u003c/sup\u003e input to the heart alters cardiac sympathetic activity. Specifically, we conducted optogenetic manipulations that were temporally restricted using the CaMKIIα promoter-driven expression of Channelrhodopsin (ChR2). Optogenetic stimulation of Glu neurons in M1L5 in WT mice injected with AAV1-CaMKIIα-ChR2-mCherry (20Hz, 5ms, 3-5mw/mm\u003csup\u003e3\u003c/sup\u003e) significantly increased. We also verified ChR2 cells co-expression and anti-glutamate positive cells \u003cstrong\u003e(Fig. S4A, S4B)\u003c/strong\u003e, heart rate \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e3E, 3F)\u003c/strong\u003e, and the HRV LF/HF ratio (an indication of cardiac sympathetic nerve excitation) \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e3G, S4C)\u0026nbsp;\u003c/strong\u003ein male mice. Additionally, the score of cardiac sympathetic nerve activity \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003eS4E)\u003c/strong\u003e and the content of NE in cardiac tissue increased after optogenetic stimulation\u003cstrong\u003e\u0026nbsp;(Fig.\u003c/strong\u003e \u003cstrong\u003e3H)\u003c/strong\u003e (p = 0.0034, n = 7). Conversely, there was no change in heart NE levels in mice injected with the control virus at the end of 20 minutes of optogenetic stimulation. These data suggest that M1L5\u003csup\u003eGlu\u003c/sup\u003e represents a potential target for EA to suppress ventricular extrasystole after myocardial infarction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe effect of electroacupuncture on PVCs occurring post-MI mice depends on the M1L5\u003c/strong\u003e\u003csup\u003eGlu\u003c/sup\u003e\u003cstrong\u003e\u0026nbsp;neurons\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on our aforementioned findings, we sought to examine changes in M1L5\u003csup\u003eGlu\u003c/sup\u003e activity in MI mice induced by EA stimulation. We conducted c-Fos staining, a marker for neuronal activation, on M1L5 neurons upstream of the heart \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e4A)\u003c/strong\u003e. We observed increased c-Fos expression in M1L5 pyramidal neurons from MI mice. Interestingly, c-Fos expression was reduced in MI+EA mice compared with MI mice \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e4B,\u003c/strong\u003e \u003cstrong\u003e4C)\u003c/strong\u003e (p = 0.0010, n = 18). To further investigate M1L5 neuron activity in MI+EA mouse models, we performed \u003cem\u003ein vivo\u003c/em\u003e electrophysiological recordings \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e4D)\u003c/strong\u003e. Recorded M1L5 neurons were classified as putative excitatory pyramidal neurons (Eps,\u0026nbsp;trough to peak duration 406.4 ± 3.312 μs, n = 154) and putative inhibitory interneurons (INs, trough to peak duration 593.3 ± 9.399 μs, n = 113) using unsupervised clustering\u0026nbsp;techniques \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e4E)\u003c/strong\u003e and cross-correlogram analyses \u003cstrong\u003e(Fig. S5A,\u003c/strong\u003e \u003cstrong\u003eS5B)\u003c/strong\u003e based on the area under peak, trough to peak duration, and firing rate\u003csup\u003e36\u003c/sup\u003e. Interestingly, we observed an increase in the spike action potential firing rate of M1L5\u003csup\u003eEps\u003c/sup\u003e neurons in MI mice compared to sham mice, and this effect was reversed by EA treatment \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e4F)\u003c/strong\u003e (p = 0.0044, Sham: n = 53; MI: n = 27; MI+EA: n = 37).\u003c/p\u003e\n\u003cp\u003eTo delve deeper into the \u003cem\u003ein vivo\u003c/em\u003e neuronal activity of M1L5 glutamatergic neurons following EA treatment, we conducted fiber photometry recordings in MI+EA mice by infusing an adeno-associated virus (AAV) expressing the fluorescent Ca\u003csup\u003e2+\u003c/sup\u003e indicator GCaMP6m (AAV-CaMKIIα-GCaMP6m) into M1L5 \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e4G,\u003c/strong\u003e \u003cstrong\u003e4H)\u003c/strong\u003e.\u0026nbsp;We observed that under EA treatment (0.5mA, 2Hz, pulse width of 50 μs), the fluorescence intensity of GCaMP6m-expressing neurons in MI mice was lower than before treatment (mean ΔF/F(%), p = 0.0107; calcium events, p = 0.0062, n = 7) \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eFig.\u003c/strong\u003e \u003cstrong\u003e4I,\u003c/strong\u003e \u003cstrong\u003e4J,\u003c/strong\u003e \u003cstrong\u003e4K)\u003c/strong\u003e, but this effect was not observed under 3mA EA intensity \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003eS5D,\u003c/strong\u003e \u003cstrong\u003eS5E,\u003c/strong\u003e \u003cstrong\u003eS5F)\u003c/strong\u003e. Additionally, to mitigate the effects of isoflurane, we conducted fiber photometry recordings in freely moving MI mice \u003cstrong\u003e(Fig. S5G)\u003c/strong\u003e. We found that the average ΔF/F(%) and calcium events of M1L5 neurons in MI+EA mice were significantly lower than those in MI model mice (mean ΔF/F(%), p = 0.0021; calcium events, p = 0.0024, n = 7) \u003cstrong\u003e(Fig\u003c/strong\u003e. \u003cstrong\u003eS5H,\u003c/strong\u003e \u003cstrong\u003eS5I,\u003c/strong\u003e \u003cstrong\u003eS5J,\u003c/strong\u003e \u003cstrong\u003eS5K)\u003c/strong\u003e. Microendoscopic calcium imaging \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e4L,\u003c/strong\u003e \u003cstrong\u003e4M)\u003c/strong\u003e revealed that the fluorescence intensity (p = 0.0221, Sham: n = 47; MI: n = 122; EA+MI: n = 95) \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e4N,\u003c/strong\u003e \u003cstrong\u003e4O)\u003c/strong\u003e and spontaneous calcium event rates (p = 0.0434) \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e4P)\u003c/strong\u003e from M1L5 neurons were significantly enhanced in MI mice compared with those in sham mice, and this enhancement was reversed by EA treatment.\u003c/p\u003e\n\u003cp\u003eThe aforementioned studies consistently demonstrate a heightened firing rate in M1L5 Glu neurons during PVCs, which was effectively reversed by EA treatment. To ascertain the necessity of M1L5 neurons in EA-mediated suppression of PVCs in MI mice, we employed a pharmacogenetic approach to selectively activate bilateral M1L5\u003csup\u003eGlu\u003c/sup\u003e neurons \u003cstrong\u003e(Fig. 4Q)\u003c/strong\u003e. Specifically, we utilized an AAV vector to deliver hM3Dq, a designer receptor exclusively activated by the inert agonist clozapine-n-oxide (CNO) into the M1L5 region of mice (AAV-CaMKIIα-hM3Dq-mCherry)\u003csup\u003e37\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eSubsequently, we assessed the therapeutic efficacy of EA in MI mice following the pharmacogenetic activation of M1L5\u003csup\u003e\u0026nbsp;Glu\u003c/sup\u003e neurons by analyzing PVC counts (refer to \u003cstrong\u003eFig. 4R)\u003c/strong\u003e, LF/HF ratio \u003cstrong\u003e(Fig. 4S)\u003c/strong\u003e, QRS width \u003cstrong\u003e(Fig. 4T\u003c/strong\u003e), and NE levels \u003cstrong\u003e(Fig. 4U)\u003c/strong\u003e. Intriguingly, our findings indicate that the EA-induced reduction in PVCs and attenuation of cardiac sympathetic excitability in MI mice were significantly impeded upon activation of M1L5\u003csup\u003e\u0026nbsp;Glu\u003c/sup\u003e excitatory neurons.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe ZI receives direct inputs from M1L5\u003csup\u003eGlu\u003c/sup\u003e neurons\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe primary motor cortex (M1) has been extensively studied for its role in regulating the autonomic nervous system and its involvement in motor initiation processes\u003csup\u003e38,39\u003c/sup\u003e. Notably, the cerebral cortex appears to be crucial in activating somatic-visceral reflexes induced by electroacupuncture. Studies utilizing intracortical recordings and transcranial magnetic stimulation have revealed that the perception of cardiac activity triggers responses in the human primary motor cortex\u003csup\u003e40\u003c/sup\u003e. One of the most distinctive features of the cerebral cortex is its reciprocal connections with the thalamus and midbrain\u003csup\u003e41,42\u003c/sup\u003e. In order to elucidate the acupoint (HT7)-heart pathway between these\u0026nbsp;regions,\u0026nbsp;we investigated the functional connections of various cortical circuits. Initially, an AAV expressing channelrhodopsin-2 (AAV-CAMKIIα–ChR2–mCherry) was administered into the M1L5 of wild-type mice \u003cstrong\u003e(Fig. 5A)\u003c/strong\u003e. Subsequently, three weeks later,\u0026nbsp;we observed mCherry\u003csup\u003e+\u003c/sup\u003e fibers in\u0026nbsp;the\u0026nbsp;ZI \u003cstrong\u003e(Fig. 5B)\u003c/strong\u003e and\u0026nbsp;in the ventrolateral periaqueductal gray (vlPAG) of the midbrain \u003cstrong\u003e(Fig. S6A)\u003c/strong\u003e. Notably, anterograde viral tracing experiments revealed that the RVLM does not receive direct fiber projections from M1L5\u003csup\u003eGlu\u003c/sup\u003e \u003cstrong\u003e(Fig. S6B)\u003c/strong\u003e, indicating indirect downstream modulation.\u003c/p\u003e\n\u003cp\u003eTo further elucidate this pathway, we initially expressed ChR2 in all Glu neurons of the M1L5 and optogenetically stimulated M1 projection fibers to the ZI \u003cstrong\u003e(Fig. S6C)\u003c/strong\u003e or the vlPAG \u003cstrong\u003e(Fig. S6D)\u003c/strong\u003e. Remarkably, we observed that optogenetic stimulation of M1L5\u003csup\u003eGlu\u003c/sup\u003e-ZI, but not M1L5\u003csup\u003eGlu\u003c/sup\u003e-vlPAG, increased the expression of c-Fos (a neuronal activity marker) in the RVLM \u003cstrong\u003e(Fig. S6E)\u003c/strong\u003e. Additionally, optogenetic stimulation of M1 projection fibers to the ZI \u003cstrong\u003e(Fig. S6F)\u003c/strong\u003e elevated the HRV LF/HF ratio in male mice \u003cstrong\u003e(Fig. S6G, S6H\u003c/strong\u003e), and increased the content of NE in cardiac tissue \u003cstrong\u003e(Fig. S6I, S6J)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTo elucidate the organization of the M1L5-ZI connection, we utilized a retrograde trans-monosynaptic tracing system employing a modified rabies virus (EnvA-pseudotyped RV-△G-eGFP) and Cre-dependent helper viruses (AAV-Ef1a-DIO-TVA-GFP and AAV-Ef1a-DIO-RVG) in mice \u003cstrong\u003e(Fig.5C)\u003c/strong\u003e.\u0026nbsp;Incorporation of these helper viruses facilitated the monosynaptic retrograde spread of the RV. Neurons intensely labeled with eGFP were identified in several brain regions, including the anterior cingulate cortex (ACC), primary somatosensory cortex (S1L4), primary visual cortex (V1L5), and bilateral primary motor cortex (M1L5) \u003cstrong\u003e(Fig.S7A-C)\u003c/strong\u003e. Notably, the eGFP+ signal was predominantly observed in layer 5 of M1, co-localizing with the glutamate-specific antibody signal, while being absent in layers 1, 2, 3, 4, and 6 \u003cstrong\u003e(Fig.5D,E)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eDespite these observations, there exists a paucity of knowledge regarding the cellular circuitry underlying the modulation of the ZI by M1, a field which remains unexplored. The ZI, a subthalamic structure conserved across mammals, primarily comprises GABAergic neurons exerting widespread inhibitory influence throughout the brain\u003csup\u003e43\u003c/sup\u003e. ZI neurons display functional heterogeneity, exhibiting specificity in modulating various behaviors such as defensive behaviors\u003csup\u003e44,45\u003c/sup\u003e, binge eating\u003csup\u003e46\u003c/sup\u003e, hunting\u003csup\u003e47\u003c/sup\u003e, and sleep\u003csup\u003e48,49\u003c/sup\u003e, contingent upon their neurochemical properties and subdivisions across different anatomical domains. Considering potential tracer biases and limitations associated with viral tropism, we conducted retrograde tracing using retro-AAV-YFP virus injected into the ZI. Subsequently, we observed labeled neurons in layer 5\u0026nbsp;of the M1 \u003cstrong\u003e(Fig. 5N)\u003c/strong\u003e, while layer 6 of the M1 remained devoid of labeled neurons.\u003c/p\u003e\n\u003cp\u003eSeveral observations collectively suggest that M1 activation primarily enhances output from the ZI to non-GABAergic neurons. Firstly, employing a viral strategy for fluorescent labeling of synaptic projection targets and GABAergic neurons, we identified that GABA neurons in the ZI predominantly reside in ZIv. Most ZI neurons receiving monosynaptic inputs from the M1 mainly consist of non-GABAergic neurons from ZId and are not co-labeled with GABA neurons in ZIv \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e5F,\u003c/strong\u003e \u003cstrong\u003e5G)\u003c/strong\u003e. Notably, employing an anterograde trans-monosynaptic tracing system to track downstream neurons of M1L5\u003csup\u003eGlu\u003c/sup\u003e-ZId\u003csup\u003enon-GABA\u003c/sup\u003ecircuit revealed that ZIv\u003csup\u003eGABA\u0026nbsp;\u003c/sup\u003eneurons received fiber projections from M1L5\u003csup\u003eGlu\u003c/sup\u003e-ZId\u003csup\u003enon-GABA\u003c/sup\u003e. Secondly, optogenetic stimulation of the M1 in mice with MI led to increased c-Fos expression in Zid but\u0026nbsp;not in Ziv, which was non-GABAergic neuron-specific \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e5H,\u003c/strong\u003e \u003cstrong\u003e5I) (\u003c/strong\u003e\u003cstrong\u003eFig. S7D,\u003c/strong\u003e \u003cstrong\u003eS7E)\u003c/strong\u003e. In summary, GABAergic neurons exhibited a decrease in c-Fos expression following M1 stimulation, suggesting modulation within the local ZI microcircuit.\u003c/p\u003e\n\u003cp\u003eTo elucidate the reciprocal modulation of neuronal microcircuits in ZI \u003cstrong\u003e(Fig. 5J)\u003c/strong\u003e, we administered AAV carrying eNpHR4.0 (AAV-CAMKIIα-eNpHR-mCherry) into the M1L5 of MI mice, enabling optical inhibition of M1L5\u003csup\u003eGlu\u003c/sup\u003e terminals in ZI \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e5K)\u003c/strong\u003e. A significant presence of mCherry nerve fibers in ZI was observed. Immunostaining revealed that c-Fos primarily colocalized with the GABA antibody \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e5L)\u003c/strong\u003e. Furthermore, we selectively monitored the responses of ZI\u003csup\u003eGABA\u003c/sup\u003e neurons in freely behaving mice following optogenetic activation or inhibition of the M1L5\u003csup\u003eGlu\u003c/sup\u003e-ZI circuit. To achieve this, we delivered Retro-AAV1-CAMKIIα-NpHR4.0-mCherry/Retro-AAV1-CAMKIIα-ChR2-mCherry and AAV-GAD2-GCaMP6m virus into the ZI of mice. Fiber photometry recordings demonstrated a notable decrease in fluorescence intensity observed in GCaMP6m-expressing ZI\u003csup\u003eGABA\u003c/sup\u003e neurons after optogenetic stimulation of ChR2-expressing M1L5\u003csup\u003eGlu\u003c/sup\u003e neurons \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e5M)\u003c/strong\u003e. Concurrently, the activity of ZI\u003csup\u003eGABA\u003c/sup\u003e neurons increased after optogenetic inhibition of M1L5\u003csup\u003eGlu\u003c/sup\u003e-ZI \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e5O)\u003c/strong\u003e. These findings unveil modulation within local ZId-ZIv microcircuits \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e5P)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eM1L5-ZI-RVLM circuit regulates cardiac sympathetic and mediates EA to reduce PVCs occurring post-MI in mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated the impact of EA on ZI\u003csup\u003eGABA\u003c/sup\u003e neurons in mice with MI \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e6A)\u003c/strong\u003e. AAV-GAD2-GCaMP6m virus was introduced into the ZI, and optic fibers were implanted above these regions in MI mice \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e6B)\u003c/strong\u003e. By examining the fluorescence intensity of GCaMP6m-expressing ZI\u003csup\u003eGABA\u003c/sup\u003e neurons, we established a direct correlation between their activity and EA stimulation. Notably, EA significantly increased the fluorescence intensity of these neurons (mean △F/F (%), \u003cem\u003ep\u003c/em\u003e = 0.0002; calcium events, \u003cem\u003ep\u003c/em\u003e = 0.0048; \u003cem\u003en\u003c/em\u003e = 6) \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e6C,\u003c/strong\u003e \u003cstrong\u003e6D,\u003c/strong\u003e \u003cstrong\u003e6E)\u003c/strong\u003e. Subsequently, we bilaterally administered AAV2/9-CAMKIIα-hM3dq-mCherry into the M1L5 region, activating M1L5\u003csup\u003eGlu\u003c/sup\u003e neurons chemogenetically with CNO via intraperitoneal injection prior to EA stimulation\u0026nbsp;\u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003eS9A,\u003c/strong\u003e \u003cstrong\u003eS9B)\u003c/strong\u003e. Interestingly, we observed that the EA-induced increase in ZI\u003csup\u003eGABA\u003c/sup\u003e neuron\u0026nbsp;activity in MI mice was\u0026nbsp;inhibited\u0026nbsp;(mean\u0026nbsp;△F/F (%), \u003cem\u003ep\u003c/em\u003e = 0.0004;\u0026nbsp;calcium\u0026nbsp;events, \u003cem\u003ep\u003c/em\u003e = 0.0028;\u0026nbsp;\u003cem\u003en\u003c/em\u003e = 7) \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eFig.\u003c/strong\u003e \u003cstrong\u003eS9C,\u003c/strong\u003e \u003cstrong\u003eS9D,\u003c/strong\u003e \u003cstrong\u003eS9E,\u003c/strong\u003e \u003cstrong\u003eS9F)\u003c/strong\u003e.\u0026nbsp;Concurrently, the EA-induced reduction in\u0026nbsp;c-Fos expression in RVLM neurons\u0026nbsp;was abolished\u0026nbsp;\u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003eS9G)\u003c/strong\u003e.\u0026nbsp;Given\u0026nbsp;previous\u0026nbsp;findings suggesting the presence\u0026nbsp;of non-GABA neurons\u0026nbsp;in the ZI, we\u0026nbsp;deemed\u0026nbsp;it\u0026nbsp;crucial\u0026nbsp;to\u0026nbsp;verify\u0026nbsp;that\u0026nbsp;ZIGABA neuron\u0026nbsp;activation\u0026nbsp;resulted from\u0026nbsp;EA stimulation rather than technical\u0026nbsp;artifacts or\u0026nbsp;isoflurane\u0026nbsp;effects.\u003c/p\u003e\n\u003cp\u003eTo address this, we utilized\u0026nbsp;Fos-targeted recombination in active populations (Fos-TRAP) labeling technology to\u0026nbsp;selectively identify\u0026nbsp;EA-activated neurons\u0026nbsp;in\u0026nbsp;a time-dependent manner.\u0026nbsp;Recombinant adeno-associated viruses rAAV-TRE-tight-mCherry and rAAV-c-Fos-tTA were injected into the ZI\u0026nbsp;\u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e6F)\u003c/strong\u003e. To validate \u0026nbsp;Fos-TRAP labeling system reliability, we compared mCherry expression in neurons across different conditions\u0026nbsp;\u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e6G)\u003c/strong\u003e. Following a 30-day period of drinking non-Dox water, approximately 57.27±4.09% of ZI neurons exhibited mCherry expression. Conversely, Dox treatment resulted in only about 1.87±0.15% of ZI neurons expressing mCherry, confirming effective TRE promoter suppression by Dox. Furthermore, 12.72±2.09% of ZI neurons expressed mCherry three days after EA stimulation at the HT7 acupoint in MI mice\u0026nbsp;\u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e6I, 6J)\u003c/strong\u003e. Notably, there was no significant difference in water consumption between Dox water-drinking mice and non-Dox water-drinking mice\u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e6H)\u003c/strong\u003e. Subsequent immunofluorescence staining revealed that approximately 60.27±9.64% of the mCherry signal colocalized with a specific GABA antibody\u0026nbsp;\u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e6K\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e, confirming the activation of ZI\u003csup\u003eGABA\u003c/sup\u003e neurons following EA.\u003c/p\u003e\n\u003cp\u003eWe then proceeded to examine the intricate circuits connecting the ZI to the medulla. Employing sparse neuronal type-specific labeling, we introduced rAAV-EF1a-DIO-Ypet-2A-mGFP-WPRE-pA and AAV2/9-Vgat-Cre virus into the ZI, facilitating selective labeling of ZI\u003csup\u003eGABA\u003c/sup\u003e dendritic spines. Subsequently, mGFP\u003csup\u003e+\u003c/sup\u003e fibers were observed in various\u0026nbsp;brain regions\u0026nbsp;\u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e6M and Fig.\u003c/strong\u003e \u003cstrong\u003eS8A)\u003c/strong\u003e. Given our previous investigation demonstrating the reversal of cardiac sympathetic nerve hyperexcitability in MI mice through EA, we correlated this with the well-documented projections of the M1, particularly emphasizing the\u0026nbsp;RVLM \u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e6L)\u003c/strong\u003e.\u0026nbsp;This discovery is intriguing, given the ZI's role as an integrative hub for modulating sensory integration, behavioral control, and visceral activity regulation\u003csup\u003e43,50,51\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo verify the ZI-RVLM projection, we infused AAV-DIO–eGFP virus into the ZI and AAV-hsyn-Cre virus into the RVLM of C57BL/6J mice, leading to the identification of eGFP-expressing neurons in the RVLM\u0026nbsp;\u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e6N)\u003c/strong\u003e. However, the specific neuronal types in the ZI projecting to the RVLM remained unknown. Therefore, we conducted anterograde trans-monosynaptic tracing by administering AAV1-Vgat-cre/AAV-DIO-TK-mRFP helper virus and HSV-△TK-eGFP into the ZI of mice. Remarkably, we found that RVLM primarily receives fiber projections from ZI\u003csup\u003eGABA\u003c/sup\u003e neurons\u0026nbsp;\u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e6O\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and fig.\u003c/strong\u003e \u003cstrong\u003eS8B)\u003c/strong\u003e. Subsequently, we employed a retrograde trans-monosynaptic tracing system, introducing modified rabies virus (EnvA-pseudotyped RV-△G-eGFP) into the RVLM along with Cre-dependent helper viruses (AAV-Ef1a-DIO-TVA-mCherry and AAV-Ef1a-DIO-RVG) in mice. Our results revealed relatively abundant eGFP signals in the ventral part of the Zona Incerta (ZIv) but scarcity in the dorsal part (ZId) \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eFig.\u003c/strong\u003e \u003cstrong\u003e6P)\u003c/strong\u003e. Notably, no eGFP signal was detected in M1L5.\u003c/p\u003e\n\u003cp\u003eTo explore the functional connections within the M1L5\u003csup\u003eGlu\u003c/sup\u003e-ZI\u003csup\u003eGABA\u003c/sup\u003e-RVLM circuit,\u0026nbsp;we administered Retro-AAV-CaMKIIα-ChR2 virus and AAV-Vgat-hM3dq-mCherry virus into the ZI of C57 mice\u0026nbsp;\u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e7A)\u003c/strong\u003e.\u0026nbsp;Using\u0026nbsp;fiber photometry recordings, we stimulated ChR2-containing upstream ZI cells in M1L5 and observed an increase in △F/F and calcium events in the RVLM. Notably, these responses were attenuated by chemogenetic activation of ZI\u003csup\u003eGABA\u003c/sup\u003e neurons\u0026nbsp;\u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003e7B-D)\u003c/strong\u003e. These observations elucidate the microcircuitry wherein ZI\u003csup\u003eGABA\u003c/sup\u003e neurons receive innervation from local ZI non\u003csup\u003e-GABA\u003c/sup\u003e interneurons, both of which directly receive inputs from M1L5\u003csup\u003eGlu\u003c/sup\u003e neurons. Given that premotor neurons for cardiac sympathetic activity are located in the RVLM, and M1L5 mediates the somatosensory effects of acupuncture at the HT7 point, we investigated whether the functional connection of the M1L5\u003csup\u003eGlu\u003c/sup\u003e-ZI\u003csup\u003eGABA\u003c/sup\u003e-RVLM\u0026nbsp;circuit contributes to the anti-PVCs effects induced by 0.5mA EA in MI mice.\u003c/p\u003e\n\u003cp\u003eTo this end, we infused AAV2/9-Vgat-hM3Dq-mCherry \u0026amp; Retro-AAV-CaMKIIα-ChR2 virus into the bilateral ZI and implanted optic fibers above the ipsilateral M1L5 regions in MI mice \u003cstrong\u003e(Fig. 7E)\u003c/strong\u003e. Upon optogenetic activation of the M1L5Glu-ZI pathway, we observed a blockade in the EA-induced anti-PVCs effect in MI mice \u003cstrong\u003e(Fig\u003c/strong\u003e. \u003cstrong\u003e7F)\u003c/strong\u003e, along with a blockage in the reduction of cardiac sympathetic excitability induced by EA. However, simultaneous activation of both M1L5Glu and ZIGABA neurons reduced PVCs in MI mice subjected to EA \u003cstrong\u003e(Fig\u003c/strong\u003e. \u003cstrong\u003e7G-I)\u003c/strong\u003e. Subsequently, we investigated the potential physiological characteristics of the M1L5\u003csup\u003eGlu\u003c/sup\u003e-ZI\u003csup\u003eGABA\u003c/sup\u003e-RVLM pathway in anti-PVCs in MI mice \u003cstrong\u003e(Fig\u003c/strong\u003e. \u003cstrong\u003eS9H, S9I)\u003c/strong\u003e. The 0.5mA EA-induced reduction in PVC counts in MI mice was replicated upon optogenetic inhibition of the M1L5Glu-ZI pathway and was blocked upon neural activation of the RVLM \u003cstrong\u003e(Fig\u003c/strong\u003e. \u003cstrong\u003eS9J-S9O)\u003c/strong\u003e.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe neural pathway underlying the regulation of cardiac function via acupuncture at the HT7 point has long been enigmatic, with an intricate interplay between somatosensory systems and internal organs. Our investigation has delineated a complex polysynaptic circuit comprising M1L5-ZI-RVLM neurons, pivotal in conveying HT7 afferent signals to modulate sympathetic outflow and, consequently, heart function. Specifically, this M1L5-ZI-RVLM pathway we've elucidated suppresses post-myocardial infarction PVCs in response to EA stimulation, underscoring its role in the cardioprotective effects of HT7 activation in MI-afflicted mice.\u003c/p\u003e \u003cp\u003eEmploying HSV for polysynaptic tracing of HT7 acupoint somatosensory neural networks has proven highly effective. HSV's broad host range, particularly its robust amplification and propagation efficiency in mice\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, establishes it as the optimal tracer for such investigations. Nonetheless, variations in host immunity necessitated the use of the immunosuppressant bortezomib\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e to enhance HSV infection rates consistently. While our focus primarily centered on HSV-labeled cases within the S1 and M1 regions, intriguingly, our observations revealed the involvement of not just the S1 but also the M1 in the reception and processing of sensory input from the HT7 acupoint.\u003c/p\u003e \u003cp\u003eFor tracking autonomic motor networks, PRV emerged as the preferred tracer. Our investigation identified a distinct cluster of M1L5 pyramidal neurons, primarily located upstream of the heart in the cortex, emphasizing the sensory-cardiac autonomic motor connection specifically within the M1L5 region. Notably, recent human imaging studies have corroborated a parallel pattern of sensory-motor integration within the M1L5 cortex, further substantiating our findings. In essence, the intricate intertwining\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e of sensory and motor pathways, forged through evolutionary processes, emerges as a fundamental mechanism underpinning life's development.\u003c/p\u003e \u003cp\u003eRecent research has shed light on the influence of transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS) on the modulation of sympathetic outflow to the heart through motor cortex activity\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. While the corticothalamic circuit appears to be a key player in PVCs\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, the specific organization of corticothalamic connections by cell types and their roles in PVCs remain largely unexplored.\u003c/p\u003e \u003cp\u003eThis study aims to investigate the involvement of M1 neurons in PVCs using a mouse model of myocardial ischemia-induced left ventricular PVCs. Our findings reveal heightened activity in the RVLM and M1L5\u003csup\u003eGlu\u003c/sup\u003e neurons, along with reduced activity in ZIGABA neurons in MI mice. Notably, inhibition of either M1L5 or ZI\u003csup\u003eGABA\u003c/sup\u003e neuronal activity leads to the suppression of PVCs induced by MI, underscoring their respective roles. Our data provide compelling evidence for the modulation of PVCs by the M1L5-ZI circuit via the RVLM.\u003c/p\u003e \u003cp\u003eSupport for this hypothesis comes from human imaging studies suggesting distinct roles for M1 and ZI neurons in regulating sympathetic outflow to the heart\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Compared to extensively studied autonomic nervous pathways activated by acupoints\u003csup\u003e\u003cspan additionalcitationids=\"CR60\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e, the specific central pathway transmitting afferent nerve signals from HT7 acupoints to sympathetic outflow towards the heart remains uncharted. Employing virus tracing techniques, we identified neurons that were innervated by spinal afferents from HT7 acupoints, predominantly congregating in M1L5. These glutamatergic neurons within M1L5 project to the ZI, and subsequently to the RVLM, serving as a pivotal conduit for regulating sympathetic outflow to the heart. While the somatosensory cortex is conventionally recognized as the primary pathway for receiving somatosensory signals, relying on thalamocortical relay pathways in the thalamus\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e, our findings reveal a novel route. We discovered that layer 5 of the somatic motor cortex (M1L5) directly receives somatosensory signals, bypassing the thalamus, thus providing morphological evidence for the regulation of visceral function, particularly the heart, through bioelectrical stimulation at HT7.\u003c/p\u003e \u003cp\u003eIt is noteworthy that the neural mechanism underlying the regulation of cardiac function by human acupuncture at HT7 is undoubtedly more intricate than observed in mice. Human studies have indicated the involvement of multiple brain regions in autonomic regulation, including the hypothalamus, cerebellum, prefrontal cortex, and amygdala, in response to acupuncture\u003csup\u003e\u003cspan additionalcitationids=\"CR65\" citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. Consequently, further exploration is necessary to clarify how other downstream brain regions receiving projections from M1L5 (excluding ZI and PAG), or transmitting afferent nerve signals from HT7 acupoints, may elucidate the acupuncture treatment for MI. While our findings underscore the significant role of the brain in processing acupuncture signals, we acknowledge the potential for acupuncture to directly modulate the autonomic nervous system of the heart through sensory transmission in the spinal cord.\u003c/p\u003e \u003cp\u003eOverall, our study has identified a neural pathway linking the brain and the heart, implicating the role of HT7 in the processing of heart function, which could catalyze research into the connection between the body surface and internal organs. In the future, these discoveries hold promise for inspiring the development of alternative interventions for myocardial diseases.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLimitations of the study\u003c/h2\u003e \u003cp\u003eIn this investigation, we primarily observed cortical involvement in the inhibitory effect of EA at HT7 on PVCs. However, our findings also indicate widespread distribution of somatosensory neurons at HT7 within the dorsal root ganglion of the T3 spinal cord. Given that the cardiac sympathetic nerve is distributed in the T3-T4 spinal cord, further investigation is warranted to ascertain whether a direct connection exists between HT7 and the cardiac sympathetic nerve at the spinal cord level, thereby contributing to the effect of EA at HT7. Simultaneously, somatosensory neural projections from the HT7 acupoint extend to multiple brain regions, including the periaqueductal gray, lateral hypothalamic area, and parvocellular medial vestibular nucleus. However, whether these projections are implicated in the suppression of PVCs in MI mice by EA remains unknown.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (82074536) awarded to RLC, Natural Science Foundation of Anhui Province (2108085Y30) awarded to RLC, Distinguished Young Youth Scientific Research Project in Universities of Anhui Province (2022AH020043) awarded to RLC, Research Funds of Center for Xin\u0026apos;an Medicine and Modernization of Traditional Chinese Medicine of IHM (2023CXMMTCM019) awarded to RLC, The Plans for Major Provincial Science \u0026amp; Technology Projects (202303a07020002) to ZZ, National Natural Science Foundation of China (82104999) awarded to QY, Natural Science Foundation of Anhui Province (2108085QH364) awarded to QY, Excellent Young Youth Scientific Research Project in Universities of Anhui Province (2022AH030062) awarded to QY, Higher Education Teaching Quality and Teaching Reform Project of Anhui Provincial (2023xscx092) awarded to FZ, National Natural Science Foundation of China (81973757) awarded to LH, College Natural Science Project of Anhui Provincial(2022AH050514).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: Fan ZHANG, Rong-lin CAI, Qing YU,\u0026nbsp;Zhi ZHANG,\u0026nbsp;Xia ZHU. Data curation: Fan ZHANG,\u0026nbsp;Qian-yi WANG, Li-bin WU,\u0026nbsp;Jie ZHOU,\u0026nbsp;Liu YANG, Wen-xiu DUAN. Formal analysis: Fan ZHANG, Qian-yi WANG, Xiang ZHOU,\u0026nbsp;Bin ZHANG. Funding acquisition: Ling HU, Qing YU, Rong-lin CAI, Zhi ZHANG. Investigation: Fan ZHANG, Li-bin WU,\u0026nbsp;Qi SHU. \u0026nbsp;Methodology: Fan ZHANG, Xia WEI, Qing YU, Hui-min CHANG. Project administration: Zhi ZHANG, Ling HU,\u0026nbsp;Rong-lin CAI, Qing YU. Resources: Rong-lin CAI, Qing YU, Xia ZHU. Software: Fan ZHANG, Yan WU, Qian-yi WANG,\u0026nbsp;Wen-jing SHAO,\u0026nbsp;Zheng-jie LUO. Supervision: Fan ZHANG, Ling HU, Qing YU, Rong-lin CAI,\u0026nbsp;Zhi ZHANG.\u0026nbsp;Validation: Qing YU, Rong-lin CAI. Visualization: Fan ZHANG, Rong-lin CAI. Writing\u0026ndash;original draft: Fan ZHANG, Qian-yi WANG,\u0026nbsp;Xia WEI. Writing \u0026ndash; review \u0026amp; editing: Fan ZHANG, Rong-lin CAI. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDECLARATION OF INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author will unreservedly provide the raw data to support the conclusions of this article. All data generated in this study can be found at: https://data.mendeley.com/datasets/vs34h73gr3/1, or through contact of the lead author, Rong-lin CAI ([email protected]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe obtained six-week-old male C57BL/6J mice from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd., for inclusion in this study. The production license for experimental animals is SCXK (ZHE) 2019-0004. The mice were housed in cages with ad libitum access to food and water, and the ambient temperature was maintained at a relative range of 23\u0026ndash;25 \u0026deg;C. All animal experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and received approval from the Animal Nursing and Utilization Committee of Anhui University of Chinese Medicine (AHUCM-mouse-2022083).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAl-Khatib, S. 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Effects of acupuncture stimulation on brain activation induced by cue-elicited alcohol craving. \u003cem\u003eNeural regeneration research\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 1059-1064, doi:10.4103/1673-5374.324849 (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"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":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Electroacupuncture, myocardial ischemia, Primary motor cortex, premature ventricular complex, CNS","lastPublishedDoi":"10.21203/rs.3.rs-4473024/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4473024/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eElectroacupuncture (EA) has been shown to suppress premature ventricular complexes (PVCs) following myocardial infarction (MI) in humans. However, the specific neural circuitry and causal mechanisms underlying this effect remain unclear. Here, we reveal a previously unrecognized connection from the primary motor cortex (M1) to the nucleus rostral ventrolateral medulla (RVLM) circuitry via the layer 5 of the primary motor cortex (M1L5)-zona incerta (ZI) pathway, which selectively suppresses PVCs in post-MI mice. Utilizing viral tracing, fiber photometry recordings, and optogenetic stimulation, we demonstrate that EA inhibits glutamatergic projections from M1L5 to ZI, leading to the activation of local GABAergic neurons and subsequent inhibition of RVLM (M1L5-ZI-RVLM). Furthermore, optogenetic or chemogenetic inhibition of the M1L5-ZI-RVLM circuit replicates the anti-PVC effects observed with EA in MI mice. Artificial activation of M1L5-projecting ZI neurons reverses the suppressive effects of EA on PVCs in MI mice. Overall, our findings highlight the M1L5-ZI-RVLM circuit as a crucial mediator of EA-induced suppression of PVCs following myocardial infarction. Additionally, this newly identified corticothalamic circuit may represent a promising target for mitigating PVCs post-myocardial infarction.\u003c/p\u003e","manuscriptTitle":"Electroacupuncture Suppresses Premature Ventricular Complexes Occurring Post-myocardial Infarction through corticothalamic circuit","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-16 09:41:58","doi":"10.21203/rs.3.rs-4473024/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"62d56c30-bd60-48a2-a82a-2b74f8f670d3","owner":[],"postedDate":"August 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":32878067,"name":"Biological sciences/Neuroscience/Neural circuits"},{"id":32878068,"name":"Health sciences/Neurology"}],"tags":[],"updatedAt":"2024-08-16T09:41:58+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-16 09:41:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4473024","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4473024","identity":"rs-4473024","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}