Recruitment of cortical silent responses by forskolin in the anterior cingulate cortex of adult mice.

Recent studies using different experimental approaches demonstrate that silent synapses may exist in the adult cortex including the sensory cortex and anterior cingulate cortex (ACC). The postsynaptic form of long-term potentiation (LTP) in the ACC recruits some of these silent synapses and the activity of calcium-stimulated adenylyl cyclases (ACs) is required for such recruitment. It is unknown if the chemical activation of ACs may recruit silent synapses. In this study, we found that activation of ACs contributed to synaptic potentiation in the ACC of adult mice. Forskolin, a selective activator of ACs, recruited silent responses in the ACC of adult mice. The recruitment was long-lasting. Interestingly, the effect of forskolin was not universal, some silent synapses did not undergo potentiation or recruitment. These findings suggest that these adult cortical synapses are not homogenous. The application of a selective calcium-permeable AMPA receptor inhibitor 1-naphthyl acetyl spermine (NASPM) reversed the potentiation and the recruitment of silent responses, indicating that the AMPA receptor is required. Our results strongly suggest that the AC-dependent postsynaptic AMPA receptor contributes to the recruitment of silent responses at cortical LTP.

Alleviation of migraine related pain and anxiety by inhibiting calcium-stimulating AC1-dependent CGRP in the insula of adult rats.

BACKGROUND: Recent animal and clinical findings consistently highlight the critical role of calcitonin gene-related peptide (CGRP) in chronic migraine (CM) and related emotional responses. CGRP antibodies and receptor antagonists have been approved for CM treatment. However, the underlying CGRP-related signaling pathways in the pain-related cortex remain poorly understood. METHODS: The SD rats were used to establish the CM model by dural infusions of inflammatory soup. Periorbital mechanical thresholds were assessed using von-Frey filaments, and anxiety-like behaviors were observed via open field and elevated plus maze tests. Expression of c-Fos, CGRP and NMDA GluN2B receptors was detected using immunofluorescence and western blotting analyses. The excitatory synaptic transmission was detected by whole-cell patch-clamp recording. A human-used adenylate cyclase 1 (AC1) inhibitor, hNB001, was applied via insula stereotaxic and intraperitoneal injections in CM rats. RESULTS: The insular cortex (IC) was activated in the migraine model rats. Glutamate-mediated excitatory transmission and NMDA GluN2B receptors in the IC were potentiated. CGRP levels in the IC significantly increased during nociceptive and anxiety-like activities. Locally applied hNB001 in the IC or intraperitoneally alleviated periorbital mechanical thresholds and anxiety behaviors in migraine rats. Furthermore, CGRP expression in the IC decreased after the hNB001 application. CONCLUSIONS: Our study indicated that AC1-dependent IC plasticity contributes to migraine and AC1 may be a promising target for treating migraine in the future.

Activation of the glutamatergic cingulate cortical-cortical connection facilitates pain in adult mice.

The brain consists of the left and right cerebral hemispheres and both are connected by callosal projections. Less is known about the basic mechanism of this cortical-cortical connection and its functional importance. Here we investigate the cortical-cortical connection between the bilateral anterior cingulate cortex (ACC) by using the classic electrophysiological and optogenetic approach. We find that there is a direct synaptic projection from one side ACC to the contralateral ACC. Glutamate is the major excitatory transmitter for bilateral ACC connection, including projections to pyramidal cells in superficial (II/III) and deep (V/VI) layers of the ACC. Both AMPA and kainate receptors contribute to synaptic transmission. Repetitive stimulation of the projection also evoked postsynaptic Ca2+ influx in contralateral ACC pyramidal neurons. Behaviorally, light activation of the ACC-ACC connection facilitated behavioral withdrawal responses to mechanical stimuli and noxious heat. In an animal model of neuropathic pain, light inhibitory of ACC-ACC connection reduces both primary and secondary hyperalgesia. Our findings provide strong direct evidence for the excitatory or facilitatory contribution of ACC-ACC connection to pain perception, and this mechanism may provide therapeutic targets for future treatment of chronic pain and related emotional disorders.

Ionizing radiation alters functional neurotransmission in Drosophila larvae.

Introduction: Patients undergoing cranial ionizing radiation therapy for brain malignancies are at increased risk of long-term neurocognitive decline, which is poorly understood and currently untreatable. Although the molecular pathogenesis has been intensively researched in many organisms, whether and how ionizing radiation alters functional neurotransmission remains unknown. This is the first study addressing physiological changes in neurotransmission after ionizing radiation exposure. Methods: To elucidate the cellular mechanisms of radiation damage, using calcium imaging, we analyzed the effects of ionizing radiation on the neurotransmitter-evoked responses of prothoracicotropic hormone (PTTH)-releasing neurons in Drosophila larvae, which play essential roles in normal larval development. Results: The neurotransmitters dopamine and tyramine decreased intracellular calcium levels of PTTH neurons in a dose-dependent manner. In gamma irradiated third-instar larvae, a dose of 25 Gy increased the sensitivity of PTTH neurons to dopamine and tyramine, and delayed development, possibly in response to abnormal functional neurotransmission. This irradiation level did not affect the viability and arborization of PTTH neurons and successful survival to adulthood. Exposure to a 40-Gy dose of gamma irradiation decreased the neurotransmitter sensitivity, physiological viability and axo-dendritic length of PTTH neurons. These serious damages led to substantial developmental delays and a precipitous reduction in the percentage of larvae that survived to adulthood. Our results demonstrate that gamma irradiation alters neurotransmitter-evoked responses, indicating synapses are vulnerable targets of ionizing radiation. Discussion: The current study provides new insights into ionizing radiation-induced disruption of physiological neurotransmitter signaling, which should be considered in preventive therapeutic interventions to reduce risks of neurological deficits after photon therapy.

Multiple modulatory roles of serotonin in chronic pain and injury-related anxiety.

Chronic pain is long-lasting pain that often persists during chronic diseases or after recovery from disease or injury. It often causes serious side effects, such as insomnia, anxiety, or depression which negatively impacts the patient's overall quality of life. Serotonin (5-HT) in the central nervous system (CNS) has been recognized as an important neurotransmitter and neuromodulator which regulates various physiological functions, such as pain sensation, cognition, and emotions-especially anxiety and depression. Its widespread and diverse receptors underlie the functional complexity of 5-HT in the CNS. Recent studies found that both chronic pain and anxiety are associated with synaptic plasticity in the anterior cingulate cortex (ACC), the insular cortex (IC), and the spinal cord. 5-HT exerts multiple modulations of synaptic transmission and plasticity in the ACC and the spinal cord, including activation, inhibition, and biphasic actions. In this review, we will discuss the multiple actions of the 5-HT system in both chronic pain and injury-related anxiety, and the synaptic mechanisms behind them. It is likely that the specific 5-HT receptors would be new promising therapeutic targets for the effective treatment of chronic pain and injury-related anxiety in the future.

Lamina feedback neurons regulate the bandpass property of the flicker-induced orientation response in Drosophila.

Natural scenes contain complex visual cues with specific features, including color, motion, flicker, and position. It is critical to understand how different visual features are processed at the early stages of visual perception to elicit appropriate cellular responses, and even behavioral output. Here, we studied the visual orientation response induced by flickering stripes in a novel behavioral paradigm in Drosophila melanogaster. We found that free walking flies exhibited bandpass orientation response to flickering stripes of different frequencies. The most sensitive frequency spectrum was confined to low frequencies of 2-4 Hz. Through genetic silencing, we showed that lamina L1 and L2 neurons, which receive visual inputs from R1 to R6 neurons, were the main components in mediating flicker-induced orientation behavior. Moreover, specific blocking of different types of lamina feedback neurons Lawf1, Lawf2, C2, C3, and T1 modulated orientation responses to flickering stripes of particular frequencies, suggesting that bandpass orientation response was generated through cooperative modulation of lamina feedback neurons. Furthermore, we found that lamina feedback neurons Lawf1 were glutamatergic. Thermal activation of Lawf1 neurons could suppress neural activities in L1 and L2 neurons, which could be blocked by the glutamate-gated chloride channel inhibitor picrotoxin (PTX). In summary, lamina monopolar neurons L1 and L2 are the primary components in mediating flicker-induced orientation response. Meanwhile, lamina feedback neurons cooperatively modulate the orientation response in a frequency-dependent way, which might be achieved through modulating neural activities of L1 and L2 neurons.

Neurotransmitters Affect Larval Development by Regulating the Activity of Prothoracicotropic Hormone-Releasing Neurons in Drosophila melanogaster.

Ecdysone, an essential insect steroid hormone, promotes larval metamorphosis by coordinating growth and maturation. In Drosophila melanogaster, prothoracicotropic hormone (PTTH)-releasing neurons are considered to be the primary promoting factor in ecdysone biosynthesis. Recently, studies have reported that the regulatory mechanisms of PTTH release in Drosophila larvae are controlled by different neuropeptides, including allatostatin A and corazonin. However, it remains unclear whether neurotransmitters provide input to PTTH neurons and control the metamorphosis in Drosophila larvae. Here, we report that the neurotransmitters acetylcholine (ACh) affect larval development by modulating the activity of PTTH neurons. By downregulating the expression of different subunits of nicotinic ACh receptors in PTTH neurons, pupal volume was significantly increased, whereas pupariation timing was relatively unchanged. We also identified that PTTH neurons were excited by ACh application ex vivo in a dose-dependent manner via ionotropic nicotinic ACh receptors. Moreover, in our Ca2+ imaging experiments, relatively low doses of OA caused increased Ca2+ levels in PTTH neurons, whereas higher doses led to decreased Ca2+ levels. We also demonstrated that a low dose of OA was conveyed through OA ß-type receptors. Additionally, our electrophysiological experiments revealed that PTTH neurons produced spontaneous activity in vivo, which provides the possibility of the bidirectional regulation, coming from neurons upstream of PTTH cells in Drosophila larvae. In summary, our findings indicate that several different neurotransmitters are involved in the regulation of larval metamorphosis by altering the activity of PTTH neurons in Drosophila.

Chlorogenic acid-enriched extract from Eucommia ulmoides leaves inhibits hepatic lipid accumulation through regulation of cholesterol metabolism in HepG2 cells.

CONTEXT: Eucommia ulmoides Oliver (Eucommiaceae) leaf exhibits beneficial lipid-lowering and anti-obesity effects. However, the mechanisms remain unknown. OBJECTIVE: The objective of this study is to investigate the lipid-lowering effects of chlorogenic acid (CGA)-enriched extract from this plant (CAEF) in human hepatoma HepG2 cells, focusing on cholesterol metabolism. MATERIALS AND METHODS: HepG2 cells were treated with CAEF (10, 20, 25, 40, 60, and 80 mg/L), CGA (0.3, 3, 30, 300, and 600 µmol/L), and simvastatin (0.1, 1, 10, 50, and 100 µmol/L) for 24 or 48 h. The cytotoxicity, Oil red O staining, total cholesterol, and triacylglycerol in supernatants were determined. The mRNA expression of genes involved in cholesterol metabolism was determined with RT-PCR. The protein expression of HMG-CoA reductase (HMGCR) was examined by immunocytochemistry and western-blot. RESULTS: The IC50 values were 59.2 mg/L for CAEF, 335.9 µmol/L for CGA, and 10.5 µmol/L for simvastatin. By treating cells with CAEF (25 mg/L), CGA (30 µmol/L), or simvastatin (10 µmol/L) for 48 h, the efflux of total cholesterol and triacylglycerol was increased (CAEF, 4.06- and 31.00-folds; CGA, 2.94- and 2.17-folds; and simvastatin, 3.94- and 24.67-folds), and the cellular lipid droplets were reduced in Oil red O staining. CAEF and CGA increased mRNA expression of ABCA1, CYP7A1, and AMPKα2, while CAEF and simvastatin decreased SREBP2. However, their effects on LXRα mRNA expression were variable. Importantly, all drugs significantly inhibited protein expression of HMGCR at mRNA and protein levels. DISCUSSION AND CONCLUSION: CAEF is a promising dietary supplement to prevent obesity and dyslipidemia and the effects appear to be due, at least in part, to regulating cholesterol metabolism through inhibition of HMGCR in HepG2 cells.

Use of an in vivo animal model for assessing the role of integrin α(6)ß(4) and syndecan-1 in early steps in papillomavirus infection.

Human papillomaviruses (HPV) are small DNA tumor viruses. HPV infection requires entry of virions into epithelial host cells that support the viral life cycle. Here, we used an in vivo mouse model, in which HPV pseudoviruses (PVs) are scored for their ability to transduce reporter genes, to test the role of various cellular proteins in entry. We initially investigated the role of integrin α(6)ß(4) in mediating early steps of HPV infection. Deficiency of integrin α(6)ß(4) is modestly but significantly suppressed reporter-gene transduction by PVs in conditional integrin ß(4) knockout mice. We also investigated the role of syndecan 1, a heparin sulfate proteoglycan (HSPG) for its role in HPV infection. We did not see a significant reduction in reporter-gene transduction by PVs in syndecan-1 null mice. This indicates that this HSPG is not essential for early steps in HPV infection, but does not discount a need of other HSPGs in mediating HPV infection.

Novel antivirals inhibit early steps in HPV infection.

The future incidence of cervical cancer is forecast to decline because of the remarkably effective prophylactic vaccines against human papillomaviruses. However, lack of access to these expensive vaccines in the developing countries where cervical cancer is most frequent, and the restricted genotypes these vaccines protect against, will limit their impact. Clearly, there is still a need for identifying other modalities for preventing HPV infections. Ready access to effective, inexpensive antivirals represents one potentially valuable approach to the prevention of genital HPV infections. We developed a well-validated high throughput screening (HTS) assay for identifying compounds that inhibit HPV infection and applied this assay to identify lead compounds that act by inhibiting an early step in infection. We screened over 40,000 small molecules that were available at the University of Wisconsin Small Molecule Screening Facility (UW-SMSF). The top 22 compounds were chosen for further analyses based upon the pharmacological property, scaffold diversity, strength of the inhibitory activity and lack of nonspecific cytotoxicity. Of these compounds, #13 and #14 had the most acceptable properties of low to submicromolar IC(50)'s and low cytotoxicity. Optimal antiviral activities were elicited by exposure of cells to the #13 and #14 during the initial 12 h following infection. Twenty-nine #13-like and 15 #14-like analogs were identified in silico and tested for their antiviral activities corresponded to the altered structures comparing to #13 and #14, informing on the pharmacophore structure of each compound. Studies indicate that both compounds inhibit infection post-entry.

Inhibition of gamma secretase blocks HPV infection.

Human papillomaviruses (HPV) are common sexually transmitted pathogens that predispose women to cervical and other anogenital cancers. HPV vaccines can prevent infection by some but not other sexually transmitted HPVs but are too costly for use in much of the world at greatest risk to HPV-associated cancers. Microbicides provide an inexpensive alternative to vaccines. In a high throughput screen, drugs that inhibit the cellular protein complex known as gamma secretase were identified as potential HPV microbicides. gamma Secretase inhibitors (GSIs) inhibited the infectivity of HPV pseudoviruses both in human keratinocytes and in mouse cells, with IC(50) values in the picomolar to the nanomolar range. Using a mouse model, we observed that a GSI could inhibit HPV infection to the same degree as its effectiveness in inhibiting gamma secretase activity in vivo. We conclude that gamma secretase activity is required for HPV infection and that GSIs are effective microbicides against anogenital HPVs.

[The effect of rosiglitazone on the activity of STAT1 in rats with severe acute pancreatitis].

OBJECTIVE: To explore the effect of rosiglitazone on the activity of signal transducer and activator of transcription 1 in rats with severe acute pancreatitis. METHODS: Fifty-four male Wistar rats were randomly allocated into three groups (n = 18). SO group: sham-operated animals served as control, operation was executed and sodium chloride but not sodium taurocholate was injected. SAP group: SAP was induced by the retrograde injection of 5% sodium taurocholate into the biliopancreatic duct. ROSI group: same as SAP group, but rosiglitazone (6 mg/kg) was administered intravenously 30 min before operation. Rats in each group were sacrificed at 3,6 and 12 h after operation. The levels of serum amylase and histologic scores of pancreatic tissue were measured. The expression of TNF-alpha mRNA in pancreatic tissue were determined by reverse transcription-polymerase chain reaction (RT-PCR). The expression of phosphorylated STAT1 in pancreatic tissue was assayed by immunohistochemistry. RESULTS: Compared to SO group, the levels of serum amylase and phosphorylated STAT1, TNF-alpha mRNA and histologic scores of pancreatic tissue were significantly elevated at the same time points after SAP (P < 0.01). The levels of these detection in ROSI group were lower than those of the SAP group at the same time points (P < 0.05), but higher than SO group (P < 0.05). CONCLUSIONS: STAT1 was activated in severe acute pancreatitis. Rosiglitazone has a protective effects in rats with severe acute pancreatitis. The mechanism of its protective effects maybe that it inhibits the activation of JAK/STAT pathway, which can down-regulate the expression of TNF-alpha mRNA and block the the inflammatory cascade partially.

[Expression of human phosphodiesterase 3A gene using baculovirus expression system in insect cell].

AIM: To investigate the expression of recombinant human phosphodiesterase 3A (HPDE3A) using baculovirus expression system in Tn cell line. METHODS: The HPDE3A cDNA was recombined with baculovirus, and then the recombinant was transfected into Tn cell line. The expression of HPDE3A in Tn cell line was detected and identified by the RT-PCR, SDS-PAGE and Western blotting. RESULTS: The recombinant HPDE3A protein was stably expressed in Tn cell line and detected by the distinct morphological changes of Tn cell, RT-PCR, SDS-PAGE and Western blotting using polyclonal antibody. The M(w) of the recombinant protein was about 120 kD. CONCLUSION: Recombinant HPDE3A can be expressed in Tn cell line using the baculovirus expression system, and thus provided the basic material for studying its bioactivity and application in screening for HPDE3A inhibitor.