Increased GluK1 Subunit Receptors in Corticostriatal Projection from the Anterior Cingulate Cortex Contributed to Seizure-Like Activities.

The corticostriatal connection plays a crucial role in cognitive, emotional, and motor control. However, the specific roles and synaptic transmissions of corticostriatal connection are less studied, especially the corticostriatal transmission from the anterior cingulate cortex (ACC). Here, a direct glutamatergic excitatory synaptic transmission in the corticostriatal projection from the ACC is found. Kainate receptors (KAR)-mediated synaptic transmission is increased in this corticostriatal connection both in vitro and in vivo seizure-like activities. GluK1 containing KARs and downstream calcium-stimulated adenylyl cyclase subtype 1 (AC1) are involved in the upregulation of KARs following seizure-like activities. Inhibiting the activities of ACC or its corticostriatal connection significantly attenuated pentylenetetrazole (PTZ)-induced seizure. Additionally, injection of GluK1 receptor antagonist UBP310 or the AC1 inhibitor NB001 both show antiepileptic effects. The studies provide direct evidence that KARs are involved in seizure activity in the corticostriatal connection and the KAR-AC1 signaling pathway is a potential novel antiepileptic strategy.

A novel electrocatalyst from TOCN/CGG hydrogel-supported Fe-rich sludge and its performance in treating azo dyes-contaminated water.

Monolithic electrocatalysts are desired for the electro-Fenton oxidation system. We used a hydrogel consisting of TEMPO-oxidized cellulose nanofibers (TOCN) and cationic guar gum (CGG) to disperse and support Fe-rich sludge and finally obtained a Fe-doped biochar (denoted as C-Sludge@TOCN/CGG) after the freeze-drying and carbonization. This C-Sludge@TOCN/CGG exhibited a porous structure with evenly-distributed Fe due to the inherently three-dimensional porous structure of TOCN/CGG hydrogel and the abundant carbon content. Importantly, Fe and FeO existed in C-Sludge@TOCN/CGG due to the presence of TOCN and CGG during the pyrolysis. The electrochemical properties of C-Sludge@TOCN/CGG demonstrated its good electrocatalytic activity and stability with few side reactions. It had good performance in the electrocatalytic degradation of various azo dyes, attributed to the synergistic integration of TOCN/CGG-derived carbon matrix and carbonized Fe-rich sludge particles. Specifically, two transient radicals (i.e. ·OH and ·O2-) primarily improved the electrocatalytic degradation performance of C-Sludge@TOCN/CGG. This C-Sludge@TOCN/CGG also efficiently degraded a papermill-sourced wastewater containing direct red 23, direct yellow 11, direct black 19 and toner, in which the COD value decreased from 365.12 to 179.13 mg/L within 9 h. This work provides an example of utilizing renewable materials and solid waste to design electrocatalysts to address the wastewater issue.

Cortical Tagged Synaptic Long-Term Depression in the Anterior Cingulate Cortex of Adult Mice.

The anterior cingulate cortex (ACC) is a key cortical region for pain perception and emotion. Different forms of synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD), have been reported in the ACC. Synaptic tagging of LTP plays an important role in hippocampus-related associative memory. In this study, we demonstrate that synaptic tagging of LTD is detected in the ACC of adult male and female mice. This form of tagged LTD requires the activation of metabotropic glutamate receptor subtype 1 (mGluR1). The induction of tagged LTD is time-related with the strongest tagged LTD appearing when the interval between two independent stimuli is 30 min. Inhibitors of mGluR1 blocked the induction of tagged LTD; however, blocking N-methyl-d-aspartate receptors did not affect the induction of tagged LTD. Nimodipine, an inhibitor of L-type voltage-gated calcium channels, also blocked tagged LTD. In an animal model of amputation, we found that tagged LTD was either reduced or completely blocked. Together with our previous report of tagged LTP in the ACC, this study strongly suggests that excitatory synapses in the adult ACC are highly plastic. The biphasic tagging of synaptic transmission provides a new form of heterosynaptic plasticity in the ACC which has functional and pathophysiological significance in phantom pain.

Cortical nitric oxide required for presynaptic long-term potentiation in the insular cortex.

Nitric oxide (NO) is a key diffusible messenger in the mammalian brain. It has been proposed that NO may diffuse retrogradely into presynaptic terminals, contributing to the induction of hippocampal long-term potentiation (LTP). Here, we present novel evidence that NO is required for kainate receptor (KAR)-dependent presynaptic form of LTP (pre-LTP) in the adult insular cortex (IC). In the IC, we found that inhibition of NO synthase erased the maintenance of pre-LTP, while the induction of pre-LTP required the activation of KAR. Furthermore, NO is essential for pre-LTP induced between two pyramidal cells in the IC using the double patch-clamp recording. These results suggest that NO is required for homosynaptic pre-LTP in the IC. Our results present strong evidence for the critical roles of NO in pre-LTP in the IC. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Inhibition of calcium-stimulated adenylyl cyclase subtype 1 (AC1) for the treatment of pain and anxiety symptoms in Parkinson's disease mice model.

Pain and anxiety are two common and undertreated non-motor symptoms in Parkinson's disease (PD), which affect the life quality of PD patients, and the underlying mechanisms remain unclear. As an important subtype of adenylyl cyclases (ACs), adenylyl cyclase subtype 1 (AC1) is critical for the induction of cortical long-term potentiation (LTP) and injury induced synaptic potentiation in the cortical areas including anterior cingulate cortex (ACC) and insular cortex (IC). Genetic deletion of AC1 or pharmacological inhibition of AC1 improved chronic pain and anxiety in different animal models. In this study, we proved the motor deficit, pain and anxiety symptoms of PD in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice model. As a lead candidate AC1 inhibitor, oral administration (1 dose and seven doses) of NB001 (20 and 40 mg/kg) showed significant analgesic effect in MPTP-treated mice, and the anxiety behavior was also reduced (40 mg/kg). By using genetic knockout mice, we found that AC1 knockout mice showed reduced pain and anxiety symptoms after MPTP administration, but not AC8 knockout mice. In summary, genetic deletion of AC1 or pharmacological inhibition of AC1 improved pain and anxiety symptoms in PD model mice, but didn't affect motor function. These results suggest that NB001 is a potential drug for the treatment of pain and anxiety symptoms in PD patients by inhibiting AC1 target.

Glutamatergic synapses from the insular cortex to the basolateral amygdala encode observational pain.

Empathic pain has attracted the interest of a substantial number of researchers studying the social transfer of pain in the sociological, psychological, and neuroscience fields. However, the neural mechanism of empathic pain remains elusive. Here, we establish a long-term observational pain model in mice and find that glutamatergic projection from the insular cortex (IC) to the basolateral amygdala (BLA) is critical for the formation of observational pain. The selective activation or inhibition of the IC-BLA projection pathway strengthens or weakens the intensity of observational pain, respectively. The synaptic molecules are screened, and the upregulated synaptotagmin-2 and RIM3 are identified as key signals in controlling the increased synaptic glutamate transmission from the IC to the BLA. Together, these results reveal the molecular and synaptic mechanisms of a previously unidentified neural pathway that regulates observational pain in mice.

Mapping thalamic-anterior cingulate monosynaptic inputs in adult mice.

The anterior cingulate cortex (ACC) is located in the frontal part of the cingulate cortex, and plays important roles in pain perception and emotion. The thalamocortical pathway is the major sensory input to the ACC. Previous studies have show that several different thalamic nuclei receive projection fibers from spinothalamic tract, that in turn send efferents to the ACC by using neural tracers and optical imaging methods. Most of these studies were performed in monkeys, cats, and rats, few studies were reported systematically in adult mice. Adult mice, especially genetically modified mice, have provided molecular and synaptic mechanisms for cortical plasticity and modulation in the ACC. In the present study, we utilized rabies virus-based retrograde tracing system to map thalamic-anterior cingulate monosynaptic inputs in adult mice. We also combined with a new high-throughput VISoR imaging technique to generate a three-dimensional whole-brain reconstruction, especially the thalamus. We found that cortical neurons in the ACC received direct projections from different sub-nuclei in the thalamus, including the anterior, ventral, medial, lateral, midline, and intralaminar thalamic nuclei. These findings provide key anatomic evidences for the connection between the thalamus and ACC.

Synaptic potentiation of anterior cingulate cortex contributes to chronic pain of Parkinson's disease.

Parkinson's disease (PD) is a multi-system neurodegenerative disorder. Patients with PD often suffer chronic pain. In the present study, we investigated motor, sensory and emotional changes in three different PD mice models. We found that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treatment caused significant changes in all measurements. Mechanical hypersensitivity of PD model induced by MPTP peaked at 3 days and persisted for at least 14 days. Using Fos transgenic mice, we found that neurons in the anterior cingulate cortex (ACC) were activated after MPTP treatment. Inhibiting ACC by bilateral microinjection of muscimol significantly reduced mechanical hypersensitivity and anxiety-like responses. By contrast, MPTP induced motor deficit was not affected, indicating ACC activity is mostly responsible for sensory and emotional changes. We also investigated excitatory synaptic transmission and plasticity using brain slices of MPTP treated animals. While L-LTP was blocked or significantly reduced. E-LTP was not significantly affected in slices of MPTP treated animals. LTD induced by repetitive stimulation was not affected. Furthermore, we found that paired-pulse facilitation and spontaneous release of glutamate were also altered in MPTP treated animals, suggesting presynaptic enhancement of excitatory transmission in PD. Our results suggest that ACC synaptic transmission is enhanced in the animal model of PD, and cortical excitation may play important roles in PD related pain and anxiety.

Oxytocin in the anterior cingulate cortex attenuates neuropathic pain and emotional anxiety by inhibiting presynaptic long-term potentiation.

Oxytocin is a well-known neurohypophysial hormone that plays an important role in behavioral anxiety and nociception. Two major forms of long-term potentiation, presynaptic LTP (pre-LTP) and postsynaptic LTP (post-LTP), have been characterized in the anterior cingulate cortex (ACC). Both pre-LTP and post-LTP contribute to chronic-pain-related anxiety and behavioral sensitization. The roles of oxytocin in the ACC have not been studied. Here, we find that microinjections of oxytocin into the ACC attenuate nociceptive responses and anxiety-like behavioral responses in animals with neuropathic pain. Application of oxytocin selectively blocks the maintenance of pre-LTP but not post-LTP. In addition, oxytocin enhances inhibitory transmission and excites ACC interneurons. Similar results are obtained by using selective optical stimulation of oxytocin-containing projecting terminals in the ACC in animals with neuropathic pain. Our results demonstrate that oxytocin acts on central synapses and reduces chronic-pain-induced anxiety by reducing pre-LTP.

Inhibition of calcium-stimulated adenylyl cyclase subtype 1 (AC1) for the treatment of neuropathic and inflammatory pain in adult female mice.

Cortical long-term potentiation (LTP) serves as a cellular model for chronic pain. As an important subtype of adenylyl cyclases (ACs), adenylyl cyclase subtype 1 (AC1) is critical for the induction of cortical LTP in the anterior cingulate cortex (ACC). Genetic deletion of AC1 or pharmacological inhibition of AC1 blocked behavioral allodynia in animal models of neuropathic and inflammatory pain. Our previous experiments have identified a lead candidate AC1 inhibitor, NB001, which is highly selective for AC1 over other AC isoforms, and found that NB001 is effective in inhibiting behavioral allodynia in animal models of chronic neuropathic and inflammatory pain. However, previous experiments were carried out in adult male animals. Considering the potential gender difference as an important issue in researches of pain and analgesia, we investigated the effect of NB001 in female chronic pain animal models. We found that NB001, when administered orally, has an analgesic effect in female animal models of neuropathic and inflammatory pain without any observable side effect. Genetic deletion of AC1 also reduced allodynia responses in models of neuropathic pain and chronic inflammation pain in adult female mice. In brain slices of adult female mice, bath application of NB001(20 µM) blocked the induction of LTP in ACC. Our results indicate that calcium-stimulated AC1 is required for injury-related cortical LTP and behavioral allodynia in both sexes of adult animals, and NB001 can be used as a potential therapeutic drug for treating neuropathic and inflammatory pain in man and woman.

Cellular and synaptic mechanisms for Parkinson's disease-related chronic pain.

Parkinson's disease is the second most common neurodegenerative disorder after Alzheimer's disease. Chronic pain is experienced by the vast majority of patients living with Parkinson's disease. The degeneration of dopaminergic neuron acts as the essential mechanism of Parkinson's disease in the midbrain dopaminergic pathway. The impairment of dopaminergic neurons leads to dysfunctions of the nociceptive system. Key cortical areas, such as the anterior cingulate cortex (ACC) and insular cortex (IC) that receive the dopaminergic projections are involved in pain transmission. Dopamine changes synaptic transmission via several pathway, for example the D2-adenly cyclase (AC)-cyclic AMP (cAMP)-protein kinase A (PKA) pathway and D1-G protein-coupled receptor kinase 2 (GRK2)-fragile X mental retardation protein (FMRP) pathway. The management of Parkinson's disease-related pain implicates maintenance of stable level of dopaminergic drugs and analgesics, however a more selective drug targeting at key molecules in Parkinson's disease-related pain remains to be investigated.

Contagious itch can be induced in humans but not in rodents.

Itch contagion has been reported in human when people watch someone scratching in a video. The basic mechanism of contagious itch induced by scratching video is still being investigated. A recent study has reported that adult mice showed itch like responses after watching itch-like video or mice showing itching responses. However, such contagious itch behaviors failed to be reproduced by another study by repeating the same experiments of viewing itching mice. It is unclear if contagious itch induced by seeing itching video may be reproducible. In the present study, we used a four-iPad paradigm to repeat these experiments, and found that mice showed no significant itch-like responses after watching itching video of mice. To test if mice actually can see the video, we placed mirrors at the same location. Interestingly, mice showed altered activities in the open field with the mirrors. Finally, in healthy subjects, we found that viewing human itch video did cause itch sensation or responses. Our results indicate that the mouse model may not appropriate for studying contagious itch in humans.

Pharmacokinetic evaluation of the interaction between oral kaempferol and ethanol in rats.

This study was aimed at investigating the effect of ethanol on oral bioavailability of kaempferol in rats, namely, at disclosing their possible interaction. Kaempferol (100 or 250 mg kg-1 bm) was administered to the rats by oral gavage with or without ethanol (600 mg kg-1 bm) co-administration. Intravenous administration (10 and 25 mg kg-1 bm) of kaempferol was used to determine the bioavailability. The concentration of kaempferol in plasma was estimated by ultra high performance liquid chromatography. During coadministration, a significant increase of the area under the plasma concentration-time curve as well as the peak concentration were observed, along with a dramatic decrease in total body clearance. Consequently, the bioavailability of kaempferol in oral control groups was 3.1 % (100 mg kg-1 bm) and 2.1 % (250 mg kg-1 bm). The first was increased by 4.3 % and the other by 2.8 % during ethanol co-administration. Increased permeability of cell membrane and ethanolkaempferol interactions on CYP450 enzymes may enhance the oral bioavailability of kaempferol in rats.

An improved in vitro method for screening toxin and medicine targeting CYP2E1.

The cytochrome P450 enzyme 2E1 (CYP2E1) presents in both microsome and mitochondrion, which influences the metabolism of many xenobiotics. The mice active liver homogenate was prepared for the medicinal incubation and mitochondrion was extracted for chemical screening targeting CYP2E1 enzyme. Representative CYP2E1 inducers (ethanol and pyrazole) and inhibitors (diallyldisulfide and kaempferol) were applied to evaluate the effectiveness of homogenate-mitochondrial system. In parallel, the in-vitro microsomal method targeting CYP2E1 was also operated for comparison. The results showed that in homogenate-mitochondrial method, the protein level and activity of CYP2E1 were increased by ethanol and pyrazole; reduced by diallyldisulfide and kaempferol, and this homogenate-mitochondrial method is convenient with good repeatability and reproducibility in screening chemicals targeting CYP2E1, especially for the inducers. Thus, the homogenate-mitochondrial method might be effective in screening both CYP2E1 inhibitor and inducer.

Neurological Toxicity of Individual and Mixtures of Low Dose Arsenic, Mono and Di (n-butyl) Phthalates on Sub-Chronic Exposure to Mice.

The objective of this study was to evaluate the toxicity of individual and mixtures of di(n-butyl) phthalates (DBP) and their active metabolite monobutyl phthalate (MBP) and arsenic (As) on spatial cognition associated with hippocampal apoptosis in mice. Mice were exposed, individually or in combination, to DBP (50 mg/kg body weight, intragastrically), MBP (50 mg/kg body weight, intragastrically), and As (10 mg/L, per os) for 8 weeks. The Morris water maze test showed that mice exposed to DBP/MBP combined with As exhibited longer escape latencies and the lower average number of crossing the platform. The As content in the hippocampus after As exposure increased as compared to those without As exposure. In mice exposed to DBP/MBP combined with As, pathological alterations and oxidative damage to the hippocampus were found. Expression of apoptosis-related protein: Bax and caspase-3 were significantly increased in the hippocampus, while there was no significant change in expression of Bcl-2. The results suggested that DBP and MBP combined with As can induce spatial cognitive deficits through altering the expression of apoptosis-related protein and As played a critical role in cognition impairments. And the joint exposure has antagonistic effect.

A multivariate assessment of innate immune-related gene expressions due to exposure to low concentration individual and mixtures of four kinds of heavy metals on zebrafish (Danio rerio) embryos.

Concerns over the potential health effects of mixtures of low concentration heavy metals on living organisms keep growing by the day. However, the toxicity of low concentration metal mixtures on the immune system of fish species has rarely been investigated. In this study, the zebrafish model was employed to investigate the effect on innate immune and antioxidant-related gene expressions, on exposure to environmentally relevant concentrations of individual and mixtures of Pb (0.01 mg/L), Hg (0.001 mg/L), As (0.01 mg/L) and Cd (0.005 mg/L). Messenger-RNA (mRNA) levels of IL1ß, TNF-α, IFNγ, Mx, Lyz, C3B and CXCL-Clc which are closely associated with the innate immune system were affected after exposing zebrafish embryos to metals for 120 h post fertilization (hpf). Individual and mixtures of metals exhibited different potentials to modulate innate-immune gene transcription. IL1ß genes were significantly up regulated on exposure to Pb + As (2.01-fold) and inhibited on exposure to Pb + Hg + Cd (0.13-fold). TNF-α was significantly inhibited on exposure to As (0.40-fold) and Pb + As (0.32-fold) compared to control. Metal mixtures generally up regulated IFNγ compared to individual metals. Additionally, antioxidant genes were affected, as CAT and GPx gene expressions generally increased, whiles Mn-SOD and Zn/Cu-SOD reduced. Multivariate analysis showed that exposure to individual metals greatly influenced modulation of innate immune genes; whiles metal mixtures influenced antioxidant gene expressions. This suggests that beside oxidative stress, there may be other pathways influencing gene expressions of innate immune and antioxidant-related genes. Low concentration heavy metals also affect expression of development-related (wnt8a and vegf) genes. Altogether, the results of this study clearly demonstrate that low concentration individual and mixtures of metals in aquatic systems will greatly influence the immune system. It is indicative that mechanisms associated with toxicity of metal mixtures is complex, however, further studies to elucidate them are ongoing in our research laboratory.

Protective Effect of Porcine Cerebral Hydrolysate Peptides on Learning and Memory Deficits and Oxidative Stress in Lead-Exposed Mice.

In this study, lead acetate solution and porcine cerebral hydrolysate peptides (PCHPs) were administered to developing mice. Porcine cerebral protein pretreated by ultrasound was hydrolyzed with alcalase, and 11 peptide fragments were obtained by Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of PCHPs. Our data showed that PCHPs significantly decreased Pb2+-induced spontaneous locomotor activity, latencies to reach the platform, and the time in target quadrant. It also decreased the accumulation of lead in the blood and brain of Pb2+-exposed developing mice. Co-administration of PCHPs and dimercaptosuccinic acid (DMSA) did not only reduce the accumulation of lead in blood but also increased the absorption of zinc and iron in Pb2+-exposed mice. Administration of PCHPs individually significantly enhanced hematopoietic parameters compared with the Pb2+-exposed group. PCHPs significantly reduced the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) but increased glutathione (GSH) content and anti-oxidant enzymes and nitric oxide synthase (NOS) activities in Pb2+-exposed brain. Our findings suggest that PCHPs have the ability to protect against Pb2+-exposed learning and memory deficits and oxidative damage.