Chemical modulation of ephaptic activation of CA3 hippocampal pyramids

In rats under urethane anaesthesia, antidromic population spikes were evoked in CA3 pyramidal layer by fimbrial/commissural stimulation at a very low frequency (approximately 0.5 Hz). Submaximal population spikes--between 20 and 90% of maximum--were enhanced by 8-38% by applications of acetylcholine and bicuculline, or by medial septal stimulation. Noradrenaline had a less pronounced and regular facilitatory action, whereas gamma-aminobutyrate and glutamate only depressed population spikes. Maximal enhancement by acetylcholine or bicuculline was observed when the antidromic population spike was initially at 38-53% of maximum amplitude. A simple explanation of these results is that acetylcholine and bicuculline, by raising their excitability, facilitate the excitation of non-invaded pyramidal cells by antidromic field potentials. They are fully in keeping with previous intracellular observations on ephaptic interactions between CA3 neurons, and provide a further illustration, in situ, of the importance of increased excitability and disinhibition--whether caused by drugs or synaptic action--in promoting synchronized excitation by ephaptic currents.     

Advances in stroke neuroprotection: hyperoxia and beyond

Refinements in patient selection, improved methods of drug delivery, use of more clinically relevant animal stroke models, and the use of combination therapies that target the entire neurovascular unit make stroke neuroprotection an achievable goal. This article provides an overview of the major mechanisms of neuronal injury and the status of neuroprotective drug trials and reviews emerging strategies for treatment of acute ischemic stroke. Advances in the fields of stem cell transplantation, stroke recovery, molecular neuroimaging, genomics, and proteomics will provide new therapeutic avenues in the near future. These and other developments over the past decade raise expectations that successful stroke neuroprotection is imminent.     

Glutamate and glycine induce a negative wave on hippocampal field response through NMDA receptors

In rats under urethane anesthesia, iontophoresis of large amounts (30-300 nA) of glutamate in the hippocampus induced a negative wave on the field potential evoked by stimulation of fimbria/commissura or perforant pathway. The amplitudes of the negative waves ranged between 0.2 and 9.8 mV and their mean duration was 341 +/- 12 ms. This activity was antagonized by iontophoresis of N-methyl-D-aspartate (NMDA) antagonists: Mg2+ (80-100 nA), ketamine (50-150 nA), MK-801 (50-150 nA) and by systemic ketamine (5 mg/kg, i.v.) administration. Iontophoresis of N-methyl-DL-aspartate (NMDLA) (20-40 nA) and glycine (25-100 nA) also elicited a negative wave which was blocked by NMDA antagonists. The negative waves were induced in all hippocampal layers except the dentate hilus by glutamate, NMDLA and glycine. Pyramidal regions were found to be as sensitive as dendritic layers; the mean amplitudes of glutamate-induced negative waves on the field response were 4.1 +/- 0.6 and 4.2 +/- 0.5 mV for CA1 stratum pyramidale and radiatum, respectively. These data suggest that large amounts of glutamate activate NMDA receptor/ion channels causing appearance of a long-lasting negative wave on the hippocampal field response. The data also demonstrate that glycine leads to a significant participation of NMDA receptors during glutamatergic transmission which is largely mediated through non-NMDA receptors.     

Cortical spreading depression can be triggered by sensory stimulation in primed wild type mouse brain: a mechanistic insight to migraine aura generation

BackgroundUnlike the spontaneously appearing aura in migraineurs, experimentally, cortical spreading depression (CSD), the neurophysiological correlate of aura is induced by non-physiological stimuli. Consequently, neural mechanisms involved in spontaneous CSD generation, which may provide insight into how migraine starts in an otherwise healthy brain, remain largely unclear. We hypothesized that CSD can be physiologically induced by sensory stimulation in primed mouse brain.MethodsCortex was made susceptible to CSD with partial inhibition of Na⁺/K⁺-ATPase by epidural application of a low concentration of Na⁺/K⁺-ATPase blocker ouabain, allowing longer than 30-min intervals between CSDs or by knocking-down α2 subunit of Na⁺/K⁺-ATPase, which is crucial for K⁺ and glutamate re-uptake, with shRNA. Stimulation-triggered CSDs and extracellular K⁺ changes were monitored in vivo electrophysiologically and a K⁺-sensitive fluoroprobe (IPG-4), respectively.ResultsAfter priming with ouabain, photic stimulation significantly increased the CSD incidence compared with non-stimulated animals (44.0 vs. 4.9%, p < 0.001). Whisker stimulation also significantly increased the CSD incidence, albeit less effectively (14.9 vs. 2.4%, p = 0.02). Knocking-down Na⁺/K⁺-ATPase (50% decrease in mRNA) lowered the CSD threshold in all mice tested with KCl but triggered CSDs in 14.3% and 16.7% of mice with photic and whisker stimulation, respectively. Confirming Na⁺/K⁺-ATPase hypofunction, extracellular K⁺ significantly rose during sensory stimulation after ouabain or shRNA treatment unlike controls. In line with the higher CSD susceptibility observed, K⁺ rise was more prominent after ouabain. To gain insight to preventive mechanisms reducing the probability of stimulus-evoked CSDs, we applied an A1-receptor antagonist (DPCPX) to the occipital cortex, because adenosine formed during stimulation from ATP can reduce CSD susceptibility. DPCPX induced spontaneous CSDs but only small-DC shifts along with suppression of EEG spikes during photic stimulation, suggesting that the inhibition co-activated with sensory stimulation could limit CSD ignition when K⁺ uptake was not sufficiently suppressed as with ouabain.ConclusionsNormal brain is well protected against CSD generation. For CSD to be ignited under physiological conditions, priming and predisposing factors are required as seen in migraine patients. Intense sensory stimulation has potential to trigger CSD when co-existing conditions bring extracellular K⁺ and glutamate concentrations over CSD-ignition threshold and stimulation-evoked inhibitory mechanisms are overcome.     

Suppression of cervical carcinoma cell growth by intracytoplasmic codelivery of anti-oncoprotein E6 antibody and small interfering RNA

Cervical cancer is caused by high-risk types of human papillomaviruses (HPV) that encode the E6 and E7 oncogenes. Silencing of E6 gene expression in HPV-positive cell lines by transfection of small interfering RNA (siRNA) with cationic lipids restores the dormant p53 tumor suppressor pathway. Because cationic lipids can also be used for intracytoplasmic delivery of proteins, we tested whether the delivery of monoclonal antibodies that bind to HPV16 E6 and neutralize its biological activity in vitro could restore p53 function in tumor cells. Here, we show that the 4C6 antibody is efficiently delivered into the cell cytoplasm using a lipidic reagent used for siRNA transfection. The delivery of 4C6 resulted in the nuclear accumulation of p53 protein in CaSki and SiHa cells but not in HeLa cells. Furthermore, the antibody-mediated p53 response was dramatically increased when a peptide corresponding to the 4C6 epitope and bearing a COOH-terminal cysteine residue was added to the transduction mixture. We found that a fraction of the added peptides were dimers that allowed the formation of antibody polymers adsorbed onto the lipidic matrix. With this system, the proliferation of CaSki and SiHa cells was strongly diminished, but no apoptosis was detectable. Remarkably, cell growth was almost totally suppressed by the addition of E6-specific siRNA to the transduction complex. The results indicate that the activity of E6 oncoprotein can be down-regulated in vivo by lipid-mediated antibody delivery and that antibodies and siRNA act synergistically when codelivered. This novel targeting strategy is simple to implement and may find therapeutic applications.     

Synthesis,anticonvulsant screening,and molecular modeling studies of new arylalkylimidazole oxime ether derivatives

In this study, 15 new oxime ether derivatives were synthesized and their anticonvulsant activities were screened in vivo. The compounds were synthesized by the reaction of various alkyl halides with 1-(2-naphthyl)-2-(1H-imidazol-1-yl)ethanone oxime. Their anticonvulsant activities were determined using acute (maximal electroshock, subcutaneous metrazol [SCM], and 6 Hz seizure test) and chronic (corneal-kindled mouse) seizure models, their neurotoxic effects were evaluated by models of behavioral toxicity according to the Epilepsy Therapy Screening Program protocol of the NIH. All our compounds were protective in at least one of the tests. Quantification studies were applied to some of the active compounds and the intraperitoneal ED₅₀ values in mice were found between 25.48 and 99.56 mg/kg. Some pharmacokinetic properties of the compounds were predicted in silico and molecular docking studies were performed to provide insights into their possible anticonvulsant mechanism regarding their SCM activity.     

Validity and reliability of the Turkish Migraine Disability Assessment (MIDAS) questionnaire

OBJECTIVES: The aim of this study is to assess the comprehensibility, internal consistency, patient-physician reliability, test-retest reliability, and validity of Turkish version of Migraine Disability Assessment (MIDAS) questionnaire in patients with headache. BACKGROUND: MIDAS questionnaire has been developed by Stewart et al and shown to be reliable and valid to determine the degree of disability caused by migraine. DESIGN AND METHODS: This study was designed as a national multicenter study to demonstrate the reliability and validity of Turkish version of MIDAS questionnaire. Patients applying to 17 Neurology Clinics in Turkey were evaluated at the baseline (visit 1), week 4 (visit 2), and week 12 (visit 3) visits in terms of disease severity and comprehensibility, internal consistency, test-retest reliability, and validity of MIDAS. Since the severity of the disease has been found to change significantly at visit 2 compared to visit 1, test-retest reliability was assessed using the MIDAS scores of a subgroup of patients whose disease severity remained unchanged (up to +/-3 days difference in the number of days with headache between visits 1 and 2). RESULTS: A total of 306 patients (86.2% female, mean age: 35.0 +/- 9.8 years) were enrolled into the study. A total of 65.7%, 77.5%, 82.0% of patients reported that "they had fully understood the MIDAS questionnaire" in visits 1, 2, and 3, respectively. A highly positive correlation was found between physician and patient and the applied total MIDAS scores in all three visits (Spearman correlation coefficients were R= 0.87, 0.83, and 0.90, respectively, P <.001). Internal consistency of MIDAS was assessed using Cronbach's alpha and was found at acceptable (>0.7) or excellent (>0.8) levels in both patient and physician applied MIDAS scores, respectively. Total MIDAS score showed good test-retest reliability (R= 0.68). Both the number of days with headache and the total MIDAS scores were positively correlated at all visits with correlation coefficients between 0.47 and 0.63. There was also a moderate degree of correlation (R= 0.54) between the total MIDAS score at week 12 and the number of days with headache at visit 2 + visit 3, which quantify headache-related disability over a 3-month period similar to MIDAS questionnaire. CONCLUSION: These findings demonstrated that the Turkish translation is equivalent to the English version of MIDAS in terms of internal consistency, test-retest reliability, and validity. Physicians can reliably use the Turkish translation of the MIDAS questionnaire in defining the severity of illness and its treatment strategy when applied as a self-administered report by migraine patients themselves.