Regulation of three subtypes of SOD gene in Aleuroglyphus ovatus (Acari:Acaridae) under lead stress.

Superoxide dismutase (SOD) is an important enzyme that acts as the first line of protection in the mite antioxidant defense system, involved in eliminating reactive oxygen species (ROS) under harsh environmental conditions. Nevertheless, the SOD gene family was yet to be reported in stored grain pest mite (Aleuroglyphus ovatus). In this study, A. ovatus was used to evaluate the response of SOD gene during lead stress. A. ovatus were separately exposed to different concentration lead (12.5, 25, 50, and 100 mg/kg), which induce the dynamic trend of SOD enzyme activity initially increased and then reduced with an increase in lead concentration, whereas they were still substantially higher than the control group. Moreover, after lead stress, it was found that all of the three SOD genes showed enhanced relative messenger RNA expression at high concentrations and decreased relative expression at low concentrations, which indicated that lead stress induces the expression of AoSODs. The present work implies that AoSODs play an important role in resisting oxidative damage caused by lead stress.

Effects of Long-Term Exposure to Cadmium on Development, Reproduction and Antioxidant Enzymes of Aleuroglyphus ovatus (Acari: Acaridae).

Grain contaminated by cadmium (Cd) has become a serious food security problem, and it is necessary to determine and evaluate the toxic effect and defense mechanism of long-term heavy metal pollution in grain. In order to evaluate the effects of long-term heavy metal Cd stress on the stored grain pests, Aleuroglyphus ovatus were fed with an artificial diet supplemented with different concentrations of Cd (0, 5, 10, 20 mg/kg). The development, fecundity and detoxification enzymes of A. ovatus were analyzed and observed. In this study, the immature duration of A. ovatus was significantly prolonged under long-term Cd stress. Moreover, the survival duration of female adults was significantly shortened. The total number of eggs laid and the daily number laid per female adult decreased significantly. There were significant differences in protein content at protonymph and tritonymph stages when the concentration of Cd exceeded 10 mg/kg. The protein content of female adults was higher than that of male adults. The activity of detoxification enzymes showed differences in different conditions, such as development stage, Cd concentration and gender. These findings confirmed that A. ovatus were sensitive to Cd, and their offspring were severely affected under long-term Cd stress. Therefore, A. ovatus is a good model for evaluating the toxicity of long-term heavy metal Cd stress. The study provides the basis and enriches the research content of heavy metal pollution on mites, contributing to the harmonious and healthy development between the environment and human beings.

Salmonella Pullorum lacking srfA is attenuated, immunogenic and protective in chickens.

Sallmonella Pullorum is a host-restricted pathogen for poultry and causes severe economic importance in many developing countries. The development of novel vaccines for Salmonella Pullorum is necessary to eradicate the prevalence of the pathogen. In our study, a srfA deletion mutant (C79-13ΔsrfA) of Salmonella Pullorum was constructed, and then the biological characteristics and protective efficacy of the mutant were evaluated. The mutant C79-13ΔsrfA was much less virulent than its parental strain C79-13 in one-day-old HY-line white chickens, immunization with C79-13ΔsrfA (4 × 107 CFU) through oral pathway induced highly specific humoral and cellular immune responses, the growth performance of vaccinated chickens was consistent with that of unvaccinated chickens. The survival percentages of vaccinated chickens reached 90% and 80%, after challenge with Salmonella Pullorum strain C79-13 and Salmonella Gallinarum strain SG9 at 10 days post-immunization (dpi), respectively. Collectively, our results indicate that C79-13ΔsrfA is a live attenuated vaccine candidate.

One-Pot Synthesis of Glucose-Derived Carbon Coated Ni3S2 Nanowires as a Battery-Type Electrode for High Performance Supercapacitors.

We report a novel Ni3S2 carbon coated (denoted as NCC) rod-like structure prepared by a facile one-pot hydrothermal method and employ it as a binder free electrode in supercapacitor. We coated carbon with glucose as carbon source on the surface of samples and investigated the suitable glucose concentration. The as-obtained NCC rod-like structure demonstrated great performance with a huge specific capacity of 657 C g-1 at 1 A g-1, preeminent rate capability of 87.7% retention, the current density varying to 10 A g-1, and great cycling stability of 76.7% of its original value through 3500 cycles, which is superior to the properties of bare Ni3S2. The result presents a facile, general, viable strategy to constructing a high-performance material for the supercapacitor applications.

Carotenoid composition and antioxidant activities of Chinese orange-colored tomato cultivars and the effects of thermal processing on the bioactive components.

To facilitate the production of tomato products with high bioactivity and improve the utilization of orange-colored tomatoes, the carotenoids of 11 tomato cultivars were analyzed using high-performance liquid chromatography with photodiode array detection. Moreover, antioxidant activities were evaluated by four chemical-based assays, and the influences of thermal treatment on the carotenoids in orange-colored tomatoes rich in tetra-cis (7Z, 9Z, 7'Z, and 9'Z)-lycopene, phytofluene, and phytoene were studied. The nine orange-colored tomatoes (OT) were divided into two categories: OT-B, containing five cultivars rich in ß-carotene, and OT-L, containing the other four cultivars that were abundant in tetra-cis-lycopene, phytofluene, and phytoene. The antioxidant activities of OT-L were higher than those of OT-B and the SD-2 cultivar in OT-L showed similar antioxidant activity to the red tomatoes. During thermal processing, tetra-cis-lycopene in SD-2 decreased about 38% after being exposed to heat for 2 hr at 80 °C, while its content was still higher than other lycopene isomers. Other-Z-lycopenes and all-trans (E)-lycopene increased from 2.36 ± 0.19 to 14.73 ± 1.16 µg/g fresh weight (FW) and 0.75 ± 0.10 to 5.91 ± 1.02 µg/g FW, respectively. Thus, thermal treatments at lower temperature, such as cold break and pasteurization, were more suitable for processing OT-L. The results demonstrated that OT-L could be an excellent raw material to produce tomato products with high bioavailability and bioactivity. The results of this research could provide helpful information for the research and development of tomato products using orange tomatoes and benefit planters and consumers. PRACTICAL APPLICATION: Some orange tomato cultivars are promising raw materials for tomato products because of their high contents of bioactive tetra-cis-lycopene, phytofluene, and phytoene. This study demonstrated the carotenoid components and antioxidant activities of the widely planted orange-colored tomatoes in China. The obtained knowledge, including the thermal processing effects on the isomerization and degradation of carotenoids in the cultivars, will offer useful information to food processors and benefit the consumers.

Impaired Glutamate Receptor Function Underlies Early Activity Loss of Ipsilesional Motor Cortex after Closed-Head Mild Traumatic Brain Injury.

Although 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.

Exploring the biophysicochemical alteration of green alga Asterococcus superbus interactively affected by nanoparticles, triclosan and illumination.

Toxic effects on Asterococcus superbus were studied based on different combinations of P25-TiO2, nano-ZnO and triclosan under multiple illumination conditions. A full factorial design (2 × 2×2 × 3) was implemented to explore interactive effects, and to identify significant factors. The results showed illumination is the most important factor with significance and becomes one of the main reasons to affect chlorophyll pigments, photosynthesis activity, unsaturated fatty acids, mitochondria function, and cause oxidative stress. Triclosan considerably affects cell viability, photosynthesis activity, lipid peroxidation and protein structure, for which triclosan is more significant than nano-ZnO. P25 is significant for oxidative stress, antioxidant enzyme, and lipid peroxidation. P25 * nano-ZnO is the only significant interaction of pollutants, affecting macromolecules, lipid peroxidation, and photosynthesis activity. High-order interactions play significant roles in affecting multiple molecular components. Two groups of endpoints are best to reflect alga responses to interactively effects from P25, nano-ZnO, and triclosan. One is ROS, chlorophyll pigments, TBARS, area, MTT, and MMP, and the other one is chlorophyll pigments, ROS, TBARS, CAT, MTT and SOD. Our findings can be instructive for a comprehensive comparison among interactions of multiple pollutants and environmental factors in natural waters, such that more robust environmental toxicity analyses can be performed.

Brønsted Acid-Catalyzed Asymmetric Friedel-Crafts Alkylation of Indoles with Benzothiazole-Bearing Trifluoromethyl Ketone Hydrates.

An efficient Brønsted acid-catalyzed asymmetric Friedel-Crafts alkylation of indoles with benzothiazole-bearing trifluoromethyl ketone hydrates as electrophiles has been developed. The mild organocatalytic reactions proceeded well with low catalyst loading to afford a range of enantioenriched α-trifluoromethyl tertiary alcohols containing both benzothiazole and indole rings with excellent yields and enantioselectivities.

Exploring the use of ceramic disk filter coated with Ag/ZnO nanocomposites as an innovative approach for removing Escherichia coli from household drinking water.

Ceramic water filter is suitable for low-income families and rural communities in developing countries to obtain safe drinking water because of its low cost and good performance. As an innovative effort, the ceramic disk filter coated with Ag/ZnO nanocomposites (AZ-CDF) was proposed in this study. The manufacture of AZ-CDFs was optimized by experiments based on the Box-Behnken design. The results of thermal field emission scanning electron microscopy (TFE-SEM) and very powerful elemental and structural probe employing radiation from a synchrotron (VESPERS) indicated that Ag/ZnO nanocomposites were mainly distributed on the upper surface of AZ-CDF. The antibacterial activity of AZ-CDF was investigated by detecting the variation of cell status and intracellular reactive oxygen species during a period of time using flow cytometry. Both non-photocatalytic and photocatalytic antibacterial activities of Ag/ZnO nanocomposite contributed to the bacterial reduction property of AZ-CDF. During filtration, the initial Escherichia coli (E. coli) concentration and illumination intensity also influenced the E. coli removal performance of AZ-CDF. When the light illumination intensity was 7000 Lux, AZ-CDF was appropriate to treat the water contaminated by E. coli concentration of less than 103 cfu/mL. Increasing illumination intensity resulted in the improvement of E. coli removal performance of AZ-CDF. It was concluded the main mechanisms for the E. coli removal of AZ-CDF were filtration, non-photocatalytic and photocatalytic antibacterial activities.

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.

The 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 α1ß3γ2L 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.

Visible-Light Induction/Brønsted Acid Catalysis in Relay for the Enantioselective Synthesis of Tetrahydroquinolines.

An efficient method merging Brønsted acid catalysis with visible-light induction for the highly enantioselective synthesis of tetrahydroquinolines has been developed. This mild process directly transforms 2-aminoenones into 2-substituted tetrahydroquinolines with excellent enantioselectivities through a relay visible-light-induced cyclization/chiral phosphoric acid-catalyzed transfer hydrogenation reaction.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice.

The 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.

Longitudinal Optogenetic Motor Mapping Revealed Structural and Functional Impairments and Enhanced Corticorubral Projection after Contusive Spinal Cord Injury in Mice.

Current 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.

Transition-metal-free α-arylation of nitroketones with diaryliodonium salts for the synthesis of tertiary α-aryl, α-nitro ketones.

Transition-metal-free α-arylation of α-nitroketones with diaryliodonium salts has been realized for the first time. As an application of this methodology, a concise synthesis of the clinical drug tiletamine was also achieved via a three-step procedure from 2-nitrocyclohexanone without the isolation of intermediates.

Enhancing excitatory activity of somatosensory cortex alleviates neuropathic pain through regulating homeostatic plasticity.

Central 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.

Increased threshold of short-latency motor evoked potentials in transgenic mice expressing Channelrhodopsin-2.

Transgenic 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.

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury.

Spinal 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.

In vivo reprogramming reactive glia into iPSCs to produce new neurons in the cortex following traumatic brain injury.

Traumatic brain injury (TBI) results in a significant amount of cell death in the brain. Unfortunately, the adult mammalian brain possesses little regenerative potential following injury and little can be done to reverse the initial brain damage caused by trauma. Reprogramming adult cells to generate induced pluripotent stem cell (iPSCs) has opened new therapeutic opportunities to generate neurons in a non-neurogenic regions in the cortex. In this study we showed that retroviral mediated expression of four transcription factors, Oct4, Sox2, Klf4, and c-Myc, cooperatively reprogrammed reactive glial cells into iPSCs in the adult neocortex following TBI. These iPSCs further differentiated into a large number of neural stem cells, which further differentiated into neurons and glia in situ, and filled up the tissue cavity induced by TBI. The induced neurons showed a typical neuronal morphology with axon and dendrites, and exhibited action potential. Our results report an innovative technology to transform reactive glia into a large number of functional neurons in their natural environment of neocortex without embryo involvement and without the need to grow cells outside the body and then graft them back to the brain. Thus this technology offers hope for personalized regenerative cell therapies for repairing damaged brain.

Characterization of dendritic morphology and neurotransmitter phenotype of thoracic descending propriospinal neurons after complete spinal cord transection and GDNF treatment.

After 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.

In vivo local dye electroporation for Ca²âº imaging and neuronal-circuit tracing.

A major technical challenge for using optical imaging to analyze neuronal circuit functions is how to effectively load synthetic Ca(2+) dyes or neural tracers into the brain. We introduce here a simple but versatile approach to label many neurons and clearly visualize their axonal and dendritic morphology. The method uses a large-tip patch pipette filled with dextran-conjugated Ca(2+) dyes or fluorescent tracers. By inserting the pipette into a targeted brain area and passing microampere current pulses, dyes or tracers are electroporated into dendrites and axons near the pipette tip. The dyes are then transported to reveal the entire cell morphology, suitable for both functional Ca(2+) imaging and neuronal circuit tracing. This process requires basic physiological equipment normally available in a physiological laboratory.