Mutated Measles Virus Matrix and Fusion Protein Influence Viral Titer In Vitro and Neuro-Invasion in Lewis Rat Brain Slice Cultures

Measles virus (MV) can cause severe acute diseases as well as long-lasting clinical deteriorations due to viral-induced immunosuppression and neuronal manifestation. How the virus enters the brain and manages to persist in neuronal tissue is not fully understood. Various mutations in the viral genes were found in MV strains isolated from patient brains. In this study, reverse genetics was used to introduce mutations in the fusion, matrix and polymerase genes of MV. The generated virus clones were characterized in cell culture and used to infect rat brain slice cultures. A mutation in the carboxy-terminal domain of the matrix protein (R293Q) promoted the production of progeny virions. This effect was observed in Vero cells irrespective of the expression of the signaling lymphocyte activation molecule (SLAM). Furthermore, a mutation in the fusion protein (I225M) induced syncytia formation on Vero cells in the absence of SLAM and promoted viral spread throughout the rat brain slices. In this study, a solid ex vivo model was established to elucidate the MV mutations contributing to neural manifestation.

Image-based stroke rat brain atrophy volume and infarct volume computation

Stroke is one of the leading causes of death as well as results in a massive economic burden for society. Stroke is a cerebrovascular disease mainly divided into two types: ischemic stroke and hemorrhagic stroke, which, respectively, refer to the partial blockage and bleeding inside brain blood vessels. Both stroke types lead to nutrient and oxygen deprivation in the brain, which ultimately cause brain damage or death. This study focuses on ischemic stroke in rats with middle cerebral artery occlusion (MCAO) as experimental subjects, and the volumes of infarct and atrophy are calculated based on the brain slice images of rat brains stained with 2,3,5-triphenyl tetrazolium chloride. In this study, a stroke rat brain infarct and atrophy volumes computation system (SRBIAVC system) is developed to segment the infarcts and atrophies from the rat brain slice images. Based on the segmentation results, the infarct and atrophy volumes of a rat brain can be computed. In this study, 168 images of brain slices cut from 28 rat brains with MCAO are used as the test samples. The experimental results show that the segmentation results obtained by the SRBIAVC system are close to those obtained by experts.

Targeting the mDia Formin-Assembled Cytoskeleton Is an Effective Anti-Invasion Strategy in Adult High-Grade Glioma Patient-Derived Neurospheres

High-grade glioma (HGG, WHO Grade III–IV) accounts for the majority of adult primary malignant brain tumors. Failure of current therapies to target invasive glioma cells partly explains the minimal survival advantages: invasive tumors lack easily-defined surgical margins, and are inherently more chemo- and radioresistant. Much work centers upon Rho GTPase-mediated glioma invasion, yet downstream Rho effector roles are poorly understood and represent potential therapeutic targets. The roles for the mammalian Diaphanous (mDia)-related formin family of Rho effectors have emerged in invasive/metastatic disease. mDias assemble linear F-actin to promote protrusive cytoskeletal structures underlying tumor cell invasion. Small molecule mDia intramimic (IMM) agonists induced mDia functional activities including F-actin polymerization. mDia agonism inhibited polarized migration in Glioblastoma (WHO Grade IV) cells in three-dimensional (3D) in vitro and rat brain slice models. Here, we evaluate whether clinically-relevant high-grade glioma patient-derived neuro-sphere invasion is sensitive to formin agonism. Surgical HGG samples were dissociated, briefly grown as monolayers, and spontaneously formed non-adherent neuro-spheres. IMM treatment dramatically inhibited HGG patient neuro-sphere invasion, both at neuro-sphere embedding and mid-invasion assay, inducing an amoeboid morphology in neuro-sphere edge cells, while inhibiting actin- and tubulin-enriched tumor microtube formation. Thus, mDia agonism effectively disrupts multiple aspects of patient-derived HGG neuro-sphere invasion.

Blockade of in vitro ictogenesis by low-frequency stimulation coincides with increased epileptiform response latency

Low-frequency stimulation, delivered through transcranial magnetic or deep-brain electrical procedures, reduces seizures in patients with pharmacoresistant epilepsy. A similar control of ictallike discharges is exerted by low-frequency electrical stimulation in rodent brain slices maintained in vitro during convulsant treatment. By employing field and “sharp” intracellular recordings, we analyzed here the effects of stimuli delivered at 0.1 or 1 Hz in the lateral nucleus of the amygdala on ictallike epileptiform discharges induced by the K+ channel blocker 4-aminopyridine in the perirhinal cortex, in a rat brain slice preparation. We found that 1) ictal events were nominally abolished when the stimulus rate was brought from 0.1 to 1 Hz; 2) this effect was associated with an increased latency of the epileptiform responses recorded in perirhinal cortex following each stimulus; and 3) both changes recovered to control values following arrest of the 1-Hz stimulation protocol. The control of ictal activity by 1-Hz stimulation and the concomitant latency increase were significantly reduced by GABAB receptor antagonism. We propose that this frequency-dependent increase in latency represents a short-lasting, GABAB receptor-dependent adaptive mechanism that contributes to decrease epileptiform synchronization, thus blocking seizures in epileptic patients and animal models.

Posttetanic enhancement of striato-pallidal synaptic transmission

The striato (Str)-globus pallidus external segment (GPe) projection plays major roles in the control of neuronal activity in the basal ganglia under both normal and pathological conditions. The present study used rat brain slice preparations to characterize the enhancement of Str-GPe synapses observed after repetitive conditioning stimuli (CS) of Str with the whole cell patch-clamp recording technique. The results show that 1) the Str-GPe synapses have a posttetanic enhancement (PTE) mechanism, which is considered to be a combination of an augmentation and a posttetanic potentiation; 2) the degree of PTE observed in GPe neurons had a wide range and was positively correlated with a wide range of paired-pulse ratios assessed before application of CS; 3) a wide range of CS, from frequencies as low as 2 Hz with as few as 5 pulses to as high as 100 Hz with 100 pulses, could induce PTE; 4) the decay time constant of PTE was dependent on the strength of CS and was prolonged greatly, up to 120 s, when strong CS were applied; and 5) the level of postsynaptic Cl− became a limiting factor for the degree of PTE when strong CS were applied. These results imply that Str-GPe synapses transmit inhibitions in a nonlinear activity-weighted manner, which may be suited for scaling timing and force of repeated or sequential body movements. Other possible factors controlling the induction of PTE and functional implications are also discussed.

Short-term plasticity shapes activity pattern-dependent striato-pallidal synaptic transmission

The cortico-striato (Str)-globus pallidus external segment (GPe) projection plays major roles in the control of neuronal activity in the basal ganglia under both normal and pathological conditions. The present study used rat brain-slice preparations to address our hypothesis that the gain of this disynaptic projection is dynamically controlled by activations of short-term plasticity mechanisms of Str-GPe synapses. The Str-GPe projection neurons fire with very different frequency and firing patterns in vivo depending on the condition of the animal. The results show that the Str-GPe synapses have very strong short-term enhancement mechanisms and that repetitive burst activation of the Str-GPe synapses, which mimic oscillatory burst firing of Str neurons, can sustain enhanced states of synaptic transmission for tens of seconds. The results reveal that the short-term enhancement of Str-GPe synapses contributes to the generation of pauses in the firing of GPe neurons and that signal transfer function in the Str-GPe projection is highly dependent on the firing pattern of Str neurons.

NMDA-induced burst firing in a model subthalamic nucleus neuron

Experiments in rat brain slice show that hyperpolarized subthalamic nucleus (STN) neurons engage in slow, regular burst firing when treated with an N-methyl-d-aspartate (NMDA) bath. A depolarization-activated inward current (DIC) has been hypothesized to contribute to this bursting activity. To explore the mechanism for STN burst firing in this setting, we augmented a previously published conductance-based computational model for single rat STN neurons to include both DIC and NMDA currents, fit to data from published electrophysiological recordings. Simulations show that with these additions, the model engages in bursting activity at <1 Hz in response to hyperpolarizing current injection and that this bursting exhibits several features observed experimentally in STN. Furthermore, a reduced model is used to show that the combination of NMDA and DIC currents, but not either alone, suffices to generate oscillations under hyperpolarizing current injection. STN neurons show enhanced burstiness in Parkinson's disease patients and experimental models of parkinsonism, and the burst mechanism studied presently could contribute to this effect.