Coupling between NMDA receptor and acid-sensing ion channel contributes to ischemic neuronal death

Acid-sensing ion channels (ASICs) composed of ASIC1a subunit exhibit a high Ca2+ permeability and play important roles in synaptic plasticity and acid-induced cell death. Here, we show that ischemia enhances ASIC currents through the phosphorylation at Ser478 and Ser479 of ASIC1a, leading to exacerbated ischemic cell death. The phosphorylation is catalyzed by Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, as a result of activation of NR2B-containing N-methyl-D-aspartate subtype of glutamate receptors (NMDARs) during ischemia. Furthermore, NR2B-specific antagonist, CaMKII inhibitor, or overexpression of mutated form of ASIC1a with Ser478 or Ser479 replaced by alanine (ASICla-S478A, ASIC1a-S479A) in cultured hippocampal neurons prevented ischemia-induced enhancement of ASIC currents, cytoplasmic Ca2+ elevation, as well as neuronal death. Thus, NMDAR-CaMKII cascade is functionally coupled to ASICs and contributes to acidotoxicity during ischemia. Specific blockade of NMDAR/CaMKII-ASIC coupling may reduce neuronal death after ischemia and other pathological conditions involving excessive glutamate release and acidosis.

A Balance Equation Determines a Switch in Neuronal Excitability

We use the qualitative insight of a planar neuronal phase portrait to detect an excitability switch in arbitrary conductance-based models from a simple mathematical condition. The condition expresses a balance between ion channels that provide a negative feedback at resting potential (restorative channels) and those that provide a positive feedback at resting potential (regenerative channels). Geometrically, the condition imposes a transcritical bifurcation that rules the switch of excitability through the variation of a single physiological parameter. Our analysis of six different published conductance based models always finds the transcritical bifurcation and the associated switch in excitability, which suggests that the mathematical predictions have a physiological relevance and that a same regulatory mechanism is potentially involved in the excitability and signaling of many neurons. © 2013 Franci et al.

An organizing center in a planar model of neuronal excitability

This paper studies the excitability properties of a generalized FitzHugh-Nagumo model. The model differs from the classical FitzHugh-Nagumo model in that it accounts for the effect of cooperative gating variables such as activation of calcium currents. Excitability is explored by unfolding a pitchfork bifurcation that is shown to organize five different types of excitability. In addition to the three classical types of neuronal excitability, two novel types are described and distinctly associated to the presence of cooperative variables. © 2012 Society for Industrial and Applied Mathematics.

A novel phase portrait for neuronal excitability

Fifty years ago, FitzHugh introduced a phase portrait that became famous for a twofold reason: it captured in a physiological way the qualitative behavior of Hodgkin-Huxley model and it revealed the power of simple dynamical models to unfold complex firing patterns. To date, in spite of the enormous progresses in qualitative and quantitative neural modeling, this phase portrait has remained a core picture of neuronal excitability. Yet, a major difference between the neurophysiology of 1961 and of 2011 is the recognition of the prominent role of calcium channels in firing mechanisms. We show that including this extra current in Hodgkin-Huxley dynamics leads to a revision of FitzHugh-Nagumo phase portrait that affects in a fundamental way the reduced modeling of neural excitability. The revisited model considerably enlarges the modeling power of the original one. In particular, it captures essential electrophysiological signatures that otherwise require non-physiological alteration or considerable complexification of the classical model. As a basic illustration, the new model is shown to highlight a core dynamical mechanism by which calcium channels control the two distinct firing modes of thalamocortical neurons. © 2012 Drion et al.

How neurons migrate: a dynamic in-silico model of neuronal migration in the developing cortex.

BACKGROUND: Neuronal migration, the process by which neurons migrate from their place of origin to their final position in the brain, is a central process for normal brain development and function. Advances in experimental techniques have revealed much about many of the molecular components involved in this process. Notwithstanding these advances, how the molecular machinery works together to govern the migration process has yet to be fully understood. Here we present a computational model of neuronal migration, in which four key molecular entities, Lis1, DCX, Reelin and GABA, form a molecular program that mediates the migration process. RESULTS: The model simulated the dynamic migration process, consistent with in-vivo observations of morphological, cellular and population-level phenomena. Specifically, the model reproduced migration phases, cellular dynamics and population distributions that concur with experimental observations in normal neuronal development. We tested the model under reduced activity of Lis1 and DCX and found an aberrant development similar to observations in Lis1 and DCX silencing expression experiments. Analysis of the model gave rise to unforeseen insights that could guide future experimental study. Specifically: (1) the model revealed the possibility that under conditions of Lis1 reduced expression, neurons experience an oscillatory neuron-glial association prior to the multipolar stage; and (2) we hypothesized that observed morphology variations in rats and mice may be explained by a single difference in the way that Lis1 and DCX stimulate bipolar motility. From this we make the following predictions: (1) under reduced Lis1 and enhanced DCX expression, we predict a reduced bipolar migration in rats, and (2) under enhanced DCX expression in mice we predict a normal or a higher bipolar migration. CONCLUSIONS: We present here a system-wide computational model of neuronal migration that integrates theory and data within a precise, testable framework. Our model accounts for a range of observable behaviors and affords a computational framework to study aspects of neuronal migration as a complex process that is driven by a relatively simple molecular program. Analysis of the model generated new hypotheses and yet unobserved phenomena that may guide future experimental studies. This paper thus reports a first step toward a comprehensive in-silico model of neuronal migration.

A qualidade como fator de diferenciação na logística farmacêutica e de produtos de saúde

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Development and characterization of novel transgenic techniques for the study of neuronal circuitry

Modern neuroscience relies heavily on sophisticated tools that allow us to visualize and manipulate cells with precise spatial and temporal control. Transgenic mouse models, for example, can be used to manipulate cellular activity in order to draw conclusions about the molecular events responsible for the development, maintenance and refinement of healthy and/or diseased neuronal circuits. Although it is fairly well established that circuits respond to activity-dependent competition between neurons, we have yet to understand either the mechanisms underlying these events or the higher-order plasticity that synchronizes entire circuits. In this thesis we aimed to develop and characterize transgenic mouse models that can be used to directly address these outstanding biological questions in different ways. We present SLICK-H, a Cre-expressing mouse line that can achieve drug-inducible, widespread, neuron-specific manipulations in vivo. This model is a clear improvement over existing models because of its particularly strong, widespread, and even distribution pattern that can be tightly controlled in the absence of drug induction. We also present SLICK-V::Ptox, a mouse line that, through expression of the tetanus toxin light chain, allows long-term inhibition of neurotransmission in a small subset (<1%) of fluorescently labeled pyramidal cells. This model, which can be used to study how a silenced cell performs in a wildtype environment, greatly facilitates the in vivo study of activity-dependent competition in the mammalian brain. As an initial application we used this model to show that tetanus toxin-expressing CA1 neurons experience a 15% - 19% decrease in apical dendritic spine density. Finally, we also describe the attempt to create additional Cre-driven mouse lines that would allow conditional alteration of neuronal activity either by hyperpolarization or inhibition of neurotransmission. Overall, the models characterized in this thesis expand upon the wealth of tools available that aim to dissect neuronal circuitry by genetically manipulating neurons in vivo.

Modified cyclodextrins as novel non-viral vectors for neuronal siRNA delivery: focus on Huntington’s disease

Huntington’s Disease (HD) is a rare autosomal dominant neurodegenerative disease caused by the expression of a mutant Huntingtin (muHTT) protein. Therefore, preventing the expression of muHTT by harnessing the specificity of the RNA interference (RNAi) pathway is a key research avenue for developing novel therapies for HD. However, the biggest caveat in the RNAi approach is the delivery of short interfering RNA (siRNAs) to neurons, which are notoriously difficult to transfect. Indeed, despite the great advances in the field of nanotechnology, there remains a great need to develop more effective and less toxic carriers for siRNA delivery to the Central Nervous System (CNS). Thus, the aim of this thesis was to investigate the utility of modified amphiphilic β-cyclodextrins (CDs), oligosaccharide-based molecules, as non-viral vectors for siRNA delivery for HD. Modified CDs were able to bind and complex siRNAs forming nanoparticles capable of delivering siRNAs to ST14A-HTT120Q cells and to human HD fibroblasts, and reducing the expression of the HTT gene in these in vitro models of HD. Moreover, direct administration of CD.siRNA nanoparticles into the R6/2 mouse brain resulted in significant HTT gene expression knockdown and selective alleviation of rotarod motor deficits in this mouse model of HD. In contrast to widely used transfection reagents, CD.siRNA nanoparticles only induced limited cytotoxic and neuroinflammatory responses in multiple brain-derived cell-lines, and also in vivo after single direct injections into the mouse brain. Alternatively, we have also described a PEGylation-based formulation approach to further stabilise CD.siRNA nanoparticles and progress towards a systemic delivery nanosystem. Resulting PEGylated CD.siRNA nanoparticles showed increased stability in physiological saltconditions and, to some extent, reduced protein-induced aggregation. Taken together, the work outlined in this thesis identifies modified CDs as effective, safe and versatile siRNA delivery systems that hold great potential for the treatment of CNS disorders, such as HD.

The balance between the pro-inflammatory effect of plasma noradrenaline and the anti-inflammatory effect of neuronal noradrenaline determines the peripheral effect of noradrenaline

Perfusion experiments on an isolated, canine lateral saphenous vein segment preparation have shown that noradrenaline causes potent, flow dependent effects, at a threshold concentration comparable to that of plasma noradrenaline, when it stimulates the segment by diffusion from its microcirculation (vasa vasorum). The effects caused are opposite to those neuronal noradrenaline causes in vivo and that, in the light of the principle that all information is transmitted in patterns that need contrast to be detected – star patterns need darkness, sound patterns, quietness – has generated the hypothesis that plasma noradrenaline provides the obligatory contrast tissues need to detect and respond to the regulatory information encrypted in the diffusion pattern of neuronal noradrenaline. Based on the implications of that hypothesis, the controlled variable of the peripheral noradrenergic system is believed to be the maintenance of a set point balance between the contrasting effects of plasma and neuronal noradrenaline on a tissue. The hypothalamic sympathetic centres are believed to monitor that balance through the level of afferent sympathetic traffic they receive from a tissue and to correct any deviation it detects in the balance by adjusting the level of efferent sympathetic input it projects to the tissue. The failure of the centres to maintain the correct balance, for reasons intrinsic or extrinsic to themselves, is believed to be responsible for degenerative and genetic disorders. When the failure causes the balance to be polarised in favour of the effect of plasma noradrenaline that is believed to cause inflammatory diseases like dilator cardiac failure, renal hypertension, varicose veins and aneurysms; when it causes it to be polarised in favour of the effect of neuronal noradrenaline that is believed to cause genetic diseases like hypertrophic cardiopathy, pulmonary hypertension and stenoses and when, in pregnancy, a factor causes the polarity to favour plasma noradrenaline in all the maternal tissues except the uterus and conceptus, where it favours neuronal noradrenaline, that is believed to cause preeclampsia.

Effects of neuronal PIK3C3/Vps34 deletion on autophagy and beyond.

PIK3C3/Vps34 plays important roles in the endocytic and autophagic pathways, both of which are essential for maintaining neuronal integrity. However, it is unclear how inactivating PIK3C3 may affect neuronal endosomal versus autophagic processes in vivo. We generated a conditional null allele of the Pik3c3 gene in mouse, and specifically deleted it in postmitotic sensory neurons. Subsequent analyses reveal several interesting and surprising findings.

Mechanisms of specificity in neuronal activity-regulated gene transcription.

The brain is a highly adaptable organ that is capable of converting sensory information into changes in neuronal function. This plasticity allows behavior to be accommodated to the environment, providing an important evolutionary advantage. Neurons convert environmental stimuli into long-lasting changes in their physiology in part through the synaptic activity-regulated transcription of new gene products. Since the neurotransmitter-dependent regulation of Fos transcription was first discovered nearly 25 years ago, a wealth of studies have enriched our understanding of the molecular pathways that mediate activity-regulated changes in gene transcription. These findings show that a broad range of signaling pathways and transcriptional regulators can be engaged by neuronal activity to sculpt complex programs of stimulus-regulated gene transcription. However, the shear scope of the transcriptional pathways engaged by neuronal activity raises the question of how specificity in the nature of the transcriptional response is achieved in order to encode physiologically relevant responses to divergent stimuli. Here we summarize the general paradigms by which neuronal activity regulates transcription while focusing on the molecular mechanisms that confer differential stimulus-, cell-type-, and developmental-specificity upon activity-regulated programs of neuronal gene transcription. In addition, we preview some of the new technologies that will advance our future understanding of the mechanisms and consequences of activity-regulated gene transcription in the brain.

Neuronal Survival of the Fittest: The Importance of Aerobic Capacity in Exercise-Induced Neurogenesis and Cognition

It is commonly accepted that aerobic exercise increases hippocampal neurogenesis, learning and memory, as well as stress resiliency. However, human populations are widely variable in their inherent aerobic fitness as well as their capacity to show increased aerobic fitness following a period of regimented exercise. It is unclear whether these inherent or acquired components of aerobic fitness play a role in neurocognition. To isolate the potential role of inherent aerobic fitness, we exploited a rat model of high (HCR) and low (LCR) inherent aerobic capacity for running. At a baseline, HCR rats have two- to three-fold higher aerobic capacity than LCR rats. We found that HCR rats also had two- to three- fold more young neurons in the hippocampus than LCR rats as well as rats from the heterogeneous founder population. We then asked whether this enhanced neurogenesis translates to enhanced hippocampal cognition, as is typically seen in exercise-trained animals. Compared to LCR rats, HCR rats performed with high accuracy on tasks designed to test neurogenesis-dependent pattern separation ability by examining investigatory behavior between very similar objects or locations. To investigate whether an aerobic response to exercise is required for exercise-induced changes in neurogenesis and cognition, we utilized a rat model of high (HRT) and low (LRT) aerobic response to treadmill training. At a baseline, HRT and LRT rats have comparable aerobic capacity as measured by a standard treadmill fit test, yet after a standardized training regimen, HRT but not LRT rats robustly increase their aerobic capacity for running. We found that sedentary LRT and HRT rats had equivalent levels of hippocampal neurogenesis, but only HRT rats had an elevation in the number of young neurons in the hippocampus following training, which was positively correlated with accuracy on pattern separation tasks. Taken together, these data suggest that a significant elevation in aerobic capacity is necessary for exercise-induced hippocampal neurogenesis and hippocampal neurogenesis-dependent learning and memory. To investigate the potential for high aerobic capacity to be neuroprotective, doxorubicin chemotherapy was administered to LCR and HCR rats. While doxorubicin induces a progressive decrease in aerobic capacity as well as neurogenesis, HCR rats remain at higher levels on those measures compared to even saline-treated LCR rats. HCR and LCR rats that received exercise training throughout doxorubicin treatment demonstrated positive effects of exercise on aerobic capacity and neurogenesis, regardless of inherent aerobic capacity. Overall, these findings demonstrate that inherent and acquired components of aerobic fitness play a crucial role not only in the cardiorespiratory system but also the fitness of the brain.

Night-time neuronal activation of Cluster N in a day- and night-migrating songbird.

Magnetic compass orientation in a night-migratory songbird requires that Cluster N, a cluster of forebrain regions, is functional. Cluster N, which receives input from the eyes via the thalamofugal pathway, shows high neuronal activity in night-migrants performing magnetic compass-guided behaviour at night, whereas no activation is observed during the day, and covering up the birds' eyes strongly reduces neuronal activation. These findings suggest that Cluster N processes light-dependent magnetic compass information in night-migrating songbirds. The aim of this study was to test if Cluster N is active during daytime migration. We used behavioural molecular mapping based on ZENK activation to investigate if Cluster N is active in the meadow pipit (Anthus pratensis), a day- and night-migratory species. We found that Cluster N of meadow pipits shows high neuronal activity under dim-light at night, but not under full room-light conditions during the day. These data suggest that, in day- and night-migratory meadow pipits, the light-dependent magnetic compass, which requires an active Cluster N, may only be used during night-time, whereas another magnetosensory mechanism and/or other reference system(s), like the sun or polarized light, may be used as primary orientation cues during the day.

Spatial and temporal scales of neuronal correlation in visual area V4.

The spiking activity of nearby cortical neurons is correlated on both short and long time scales. Understanding this shared variability in firing patterns is critical for appreciating the representation of sensory stimuli in ensembles of neurons, the coincident influences of neurons on common targets, and the functional implications of microcircuitry. Our knowledge about neuronal correlations, however, derives largely from experiments that used different recording methods, analysis techniques, and cortical regions. Here we studied the structure of neuronal correlation in area V4 of alert macaques using recording and analysis procedures designed to match those used previously in primary visual cortex (V1), the major input to V4. We found that the spatial and temporal properties of correlations in V4 were remarkably similar to those of V1, with two notable differences: correlated variability in V4 was approximately one-third the magnitude of that in V1 and synchrony in V4 was less temporally precise than in V1. In both areas, spontaneous activity (measured during fixation while viewing a blank screen) was approximately twice as correlated as visual-evoked activity. The results provide a foundation for understanding how the structure of neuronal correlation differs among brain regions and stages in cortical processing and suggest that it is likely governed by features of neuronal circuits that are shared across the visual cortex.

Neuronal correlates of visual time perception at brief timescales.

Successful interaction with the world depends on accurate perception of the timing of external events. Neurons at early stages of the primate visual system represent time-varying stimuli with high precision. However, it is unknown whether this temporal fidelity is maintained in the prefrontal cortex, where changes in neuronal activity generally correlate with changes in perception. One reason to suspect that it is not maintained is that humans experience surprisingly large fluctuations in the perception of time. To investigate the neuronal correlates of time perception, we recorded from neurons in the prefrontal cortex and midbrain of monkeys performing a temporal-discrimination task. Visual time intervals were presented at a timescale relevant to natural behavior (<500 ms). At this brief timescale, neuronal adaptation--time-dependent changes in the size of successive responses--occurs. We found that visual activity fluctuated with timing judgments in the prefrontal cortex but not in comparable midbrain areas. Surprisingly, only response strength, not timing, predicted task performance. Intervals perceived as longer were associated with larger visual responses and shorter intervals with smaller responses, matching the dynamics of adaptation. These results suggest that the magnitude of prefrontal activity may be read out to provide temporal information that contributes to judging the passage of time.