Browsing by Author "Xiong, Wenhui"
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Item Characterization of dendritic morphology and neurotransmitter phenotype of thoracic descending propriospinal neurons after complete spinal cord transection and GDNF treatment(Elsevier, 2016-03) Deng, Lingxiao; Ruan, Yiwen; Chen, Chen; Frye, Christian Corbin; Xiong, Wenhui; Jin, Xiaoming; Jones, Kathryn; Sengelaub, Dale; Xu, Xiao-Ming; Department of Anatomy & Cell Biology, IU School of MedicineAfter spinal cord injury (SCI), poor regeneration of damaged axons of the central nervous system (CNS) causes limited functional recovery. This limited spontaneous functional recovery has been attributed, to a large extent, to the plasticity of propriospinal neurons, especially the descending propriospinal neurons (dPSNs). Compared with the supraspinal counterparts, dPSNs have displayed significantly greater regenerative capacity, which can be further enhanced by glial cell line-derived neurotrophic factor (GDNF). In the present study, we applied a G-mutated rabies virus (G-Rabies) co-expressing green fluorescence protein (GFP) to reveal Golgi-like dendritic morphology of dPSNs. We also investigated the neurotransmitters expressed by dPSNs after labeling with a retrograde tracer Fluoro-Gold (FG). dPSNs were examined in animals with sham injuries or complete spinal transections with or without GDNF treatment. Bilateral injections of G-Rabies and FG were made into the 2nd lumbar (L2) spinal cord at 3 days prior to a spinal cord transection performed at the 11th thoracic level (T11). The lesion gap was filled with Gelfoam containing either saline or GDNF in the injury groups. Four days post-injury, the rats were sacrificed for analysis. For those animals receiving G-rabies injection, the GFP signal in the T7-9 spinal cord was visualized via 2-photon microscopy. Dendritic morphology from stack images was traced and analyzed using a Neurolucida software. We found that dPSNs in sham injured animals had a predominantly dorsal-ventral distribution of dendrites. Transection injury resulted in alterations in the dendritic distribution with dorsal-ventral retraction and lateral-medial extension. Treatment with GDNF significantly increased the terminal dendritic length of dPSNs. The density of spine-like structures was increased after injury, and treatment with GDNF enhanced this effect. For the group receiving FG injections, immunohistochemistry for glutamate, choline acetyltransferase (ChAT), glycine, and GABA was performed in the T7-9 spinal cord. We show that the majority of FG retrogradely-labeled dPSNs were located in the Rexed Lamina VII. Over 90% of FG-labeled neurons were glutamatergic, with the other three neurotransmitters contributing less than 10% of the total. To our knowledge this is the first report describing the morphologic characteristics of dPSNs and their neurotransmitter expressions, as well as the dendritic response of dPSNs after transection injury and GDNF treatment.Item Enhancing excitatory activity of somatosensory cortex alleviates neuropathic pain through regulating homeostatic plasticity(Nature Publishing group, 2017-10-06) Xiong, Wenhui; Ping, Xingjie; Ripsch, Matthew S.; Chavez, Grace Santa Cruz; Hannon, Heidi Elise; Jiang, Kewen; Bao, Chunhui; Jadhav, Vaishnavi; Chen, Lifang; Chai, Zhi; Ma, Cungen; Wu, Huangan; Feng, Jianqiao; Blesch, Armin; White, Fletcher A.; Jin, Xiaoming; Anatomy and Cell Biology, School of MedicineCentral sensitization and network hyperexcitability of the nociceptive system is a basic mechanism of neuropathic pain. We hypothesize that development of cortical hyperexcitability underlying neuropathic pain may involve homeostatic plasticity in response to lesion-induced somatosensory deprivation and activity loss, and can be controlled by enhancing cortical activity. In a mouse model of neuropathic pain, in vivo two-photon imaging and patch clamp recording showed initial loss and subsequent recovery and enhancement of spontaneous firings of somatosensory cortical pyramidal neurons. Unilateral optogenetic stimulation of cortical pyramidal neurons both prevented and reduced pain-like behavior as detected by bilateral mechanical hypersensitivity of hindlimbs, but corpus callosotomy eliminated the analgesic effect that was ipsilateral, but not contralateral, to optogenetic stimulation, suggesting involvement of inter-hemispheric excitatory drive in this effect. Enhancing activity by focally blocking cortical GABAergic inhibition had a similar relieving effect on the pain-like behavior. Patch clamp recordings from layer V pyramidal neurons showed that optogenetic stimulation normalized cortical hyperexcitability through changing neuronal membrane properties and reducing frequency of excitatory postsynaptic events. We conclude that development of neuropathic pain involves abnormal homeostatic activity regulation of somatosensory cortex, and that enhancing cortical excitatory activity may be a novel strategy for preventing and controlling neuropathic pain.Item Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice(2019) Lin, Xiaojing; Zhao, Tingbao; Xiong, Wenhui; Wen, Shaonan; Jin, Xiaoming; Xu, Xiao-Ming; Neurological Surgery, School of MedicineThe mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.Item Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice(Journal of Visualized Experiments (JoVE), 2019-01-07) Lin, Xiaojing; Zhao, Tingbao; Xiong, Wenhui; Wen, Shaonan; Jin, Xiaoming; Xu, Xiao-Ming; Neurological Surgery, School of MedicineThe mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.Item Impaired Glutamate Receptor Function Underlies Early Activity Loss of Ipsilesional Motor Cortex after Closed-Head Mild Traumatic Brain Injury(Liebert, 2021) Nguyen, Tyler; Al-Juboori, Mohammed Haider; Walerstein, Jakub; Xiong, Wenhui; Jin, Xiaoming; Anatomy and Cell Biology, School of MedicineAlthough mild traumatic brain injury (mTBI) accounts for the majority of TBI patients, the effects and cellular and molecular mechanisms of mTBI on cortical neural circuits are still not well understood. Given the transient and non-specific functional deficits after mTBI, it is important to understand whether mTBI causes functional deficits of the brain and the underlying mechanism, particularly during the early stage after injury. Here, we used in vivo optogenetic motor mapping to determine longitudinal changes in cortical motor map and in vitro calcium imaging to study how changes in cortical excitability and calcium signals may contribute to the motor deficits in a closed-head mTBI model. In channelrhodopsin 2 (ChR2)-expressing transgenic mice, we recorded electromyograms (EMGs) from bicep muscles induced by scanning blue laser on the motor cortex. There were significant decreases in the size and response amplitude of motor maps of the injured cortex at 2 h post-mTBI, but an increase in motor map size of the contralateral cortex in 12 h post-mTBI, both of which recovered to baseline level in 24 h. Calcium imaging of cortical slices prepared from green fluorescent calmodulin proteins–expressing transgenic mice showed a lower amplitude, but longer duration, of calcium transients of the injured cortex in 2 h post-mTBI. Blockade of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or N-methyl-d-aspartate receptors resulted in smaller amplitude of calcium transients, suggesting impaired function of both receptor types. Imaging of calcium transients evoked by glutamate uncaging revealed reduced response amplitudes and longer duration in 2, 12, and 24 h after mTBI. Higher percentages of neurons of the injured cortex had a longer latency period after uncaging than that of the uninjured neurons. The results suggest that impaired glutamate neurotransmission contributes to functional deficits of the motor cortex in vivo, which supports enhancing glutamate neurotransmission as a potential therapeutic approach for the treatment of mTBI.Item An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury(Journal of Visualized Experiments, 2017-12-31) Chen, Chen; Zhang, Yi Ping; Sun, Yan; Xiong, Wenhui; Shields, Lisa B. E.; Shields, Christopher B.; Jin, Xiaoming; Xu, Xiao-Ming; Neurological Surgery, School of MedicineSpinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues throughout the acute injury phase. In fact, endothelial cells appear to be the first to die after a contusive SCI. The early vascular events, including increased permeability of the blood-spinal cord barrier (BSCB), induce vasogenic edema and contribute to detrimental secondary injury events caused by complex injury mechanisms. Targeting the vascular disruption, therefore, could be a key strategy to reduce secondary injury cascades that contribute to histological and functional impairments after SCI. Previous studies were mostly performed on postmortem samples and were unable to capture the dynamic changes of the vascular network. In this study, we have developed an in vivo duo-color two-photon imaging method to monitor acute vascular dynamic changes following contusive SCI. This approach allows detecting blood flow, vessel diameter, and other vascular pathologies at various sites of the same rat pre- and post-injury. Overall, this method provides an excellent venue for investigating vascular dynamics.Item Increased threshold of short-latency motor evoked potentials in transgenic mice expressing Channelrhodopsin-2(PLoS, 2017-05-31) Wu, Wei; Xiong, Wenhui; Zhang, Ping; Chen, Lifang; Fang, Jianqiao; Shields, Christopher; Xu, Xiao-Ming; Jin, Xiaoming; Neurological Surgery, School of MedicineTransgenic mice that express channelrhodopsin-2 or its variants provide a powerful tool for optogenetic study of the nervous system. Previous studies have established that introducing such exogenous genes usually does not alter anatomical, electrophysiological, and behavioral properties of neurons in these mice. However, in a line of Thy1-ChR2-YFP transgenic mice (line 9, Jackson lab), we found that short-latency motor evoked potentials (MEPs) induced by transcranial magnetic stimulation had a longer latency and much lower amplitude than that of wild type mice. MEPs evoked by transcranial electrical stimulation also had a much higher threshold in ChR2 mice, although similar amplitudes could be evoked in both wild and ChR2 mice at maximal stimulation. In contrast, long-latency MEPs evoked by electrically stimulating the motor cortex were similar in amplitude and latency between wild type and ChR2 mice. Whole-cell patch clamp recordings from layer V pyramidal neurons of the motor cortex in ChR2 mice revealed no significant differences in intrinsic membrane properties and action potential firing in response to current injection. These data suggest that corticospinal tract is not accountable for the observed abnormality. Motor behavioral assessments including BMS score, rotarod, and grid-walking test showed no significant differences between the two groups. Because short-latency MEPs are known to involve brainstem reticulospinal tract, while long-latency MEPs mainly involve primary motor cortex and dorsal corticospinal tract, we conclude that this line of ChR2 transgenic mice has normal function of motor cortex and dorsal corticospinal tract, but reduced excitability and responsiveness of reticulospinal tracts. This abnormality needs to be taken into account when using these mice for related optogenetic study.Item Longitudinal Optogenetic Motor Mapping Revealed Structural and Functional Impairments and Enhanced Corticorubral Projection after Contusive Spinal Cord Injury in Mice(Mary Ann Liebert, 2019-02-01) Qian, Jun; Wu, Wei; Xiong, Wenhui; Chai, Zhi; Xu, Xiao-Ming; Jin, Xiaoming; Anatomy and Cell Biology, School of MedicineCurrent evaluation of impairment and repair after spinal cord injury (SCI) is largely dependent on behavioral assessment and histological analysis of injured tissue and pathways. Here, we evaluated whether transcranial optogenetic mapping of motor cortex could reflect longitudinal structural and functional damage and recovery after SCI. In Thy1-Channelrhodopsin2 transgenic mice, repeated motor mappings were made by recording optogenetically evoked electromyograms (EMGs) of a hindlimb at baseline and 1 day and 2, 4, and 6 weeks after mild, moderate, and severe spinal cord contusion. Injuries caused initial decreases in EMG amplitude, losses of motor map, and subsequent partial recoveries, all of which corresponded to injury severity. Reductions in map size were positively correlated with motor performance, as measured by Basso Mouse Scale, rota-rod, and grid walk tests, at different time points, as well as with lesion area at spinal cord epicenter at 6 weeks post-SCI. Retrograde tracing with Fluoro-Gold showed decreased numbers of cortico- and rubrospinal neurons, with the latter being negatively correlated with motor map size. Combined retro- and anterograde tracing and immunostaining revealed more neurons activated in red nucleus by cortical stimulation and enhanced corticorubral axons and synapses in red nucleus after SCI. Electrophysiological recordings showed lower threshold and higher amplitude of corticorubral synaptic response after SCI. We conclude that transcranial optogenetic motor mapping is sensitive and efficient for longitudinal evaluation of impairment and plasticity of SCI, and that spinal cord contusion induces stronger anatomical and functional corticorubral connection that may contribute to spontaneous recovery of motor function.Item The Positive Allosteric Modulator of α2/3-Containing GABAA Receptors, KRM-II-81, Is Active in Pharmaco-Resistant Models of Epilepsy and Reduces Hyperexcitability after Traumatic Brain Injury(American Society for Pharmacology and Experimental Therapeutics, 2020-01) Witkin, Jeffrey M.; Li, Guanguan; Golani, Lalit K.; Xiong, Wenhui; Smith, Jodi L.; Ping, Xingjie; Rashid, Farjana; Jahan, Rajwana; Cerne, Rok; Cook, James M.; Jin, Xiaoming; Neurological Surgery, School of MedicineThe imidizodiazepine, 5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepin-3-yl)oxazole (KRM-II-81), is selective for α2/3-containing GABAA receptors. KRM-II-81 dampens seizure activity in rodent models with enhanced efficacy and reduced motor-impairment compared with diazepam. In the present study, KRM-II-81 was studied in assays designed to detect antiepileptics with improved chances of impacting pharmaco-resistant epilepsies. The potential for reducing neural hyperactivity weeks after traumatic brain injury was also studied. KRM-II-81 suppressed convulsions in corneal-kindled mice. Mice with kainate-induced mesial temporal lobe seizures exhibited spontaneous recurrent hippocampal paroxysmal discharges that were significantly reduced by KRM-II-81 (15 mg/kg, orally). KRM-II-81 also decreased convulsions in rats undergoing amygdala kindling in the presence of lamotrigine (lamotrigine-insensitive model) (ED50 = 19 mg/kg, i.p.). KRM-II-81 reduced focal and generalized seizures in a kainate-induced chronic epilepsy model in rats (20 mg/kg, i.p., three times per day). In mice with damage to the left cerebral cortex by controlled-cortical impact, enduring neuronal hyperactivity was dampened by KRM-II-81 (10 mg/kg, i.p.) as observed through in vivo two-photon imaging of layer II/III pyramidal neurons in GCaMP6-expressing transgenic mice. No notable side effects emerged up to doses of 300 mg/kg KRM-II-81. Molecular modeling studies were conducted: docking in the binding site of the α1β3γ2L GABAA receptor showed that replacing the C8 chlorine atom of alprazolam with the acetylene of KRM-II-81 led to loss of the key interaction with α1His102, providing a structural rationale for its low affinity for α1-containing GABAA receptors compared with benzodiazepines such as alprazolam. Overall, these findings predict that KRM-II-81 has improved therapeutic potential for epilepsy and post-traumatic epilepsy. SIGNIFICANCE STATEMENT: We describe the effects of a relatively new orally bioavailable small molecule in rodent models of pharmaco-resistant epilepsy and traumatic brain injury. KRM-II-81 is more potent and generally more efficacious than standard-of-care antiepileptics. In silico docking experiments begin to describe the structural basis for the relative lack of motor impairment induced by KRM-II-81. KRM-II-81 has unique structural and anticonvulsant effects, predicting its potential as an improved antiepileptic drug and novel therapy for post-traumatic epilepsy.