The Primary Visual Cortex Is Differentially Modulated by Stimulus-Driven and Top-Down Attention

Selective attention can be focused either volitionally, by top-down signals derived from task demands, or automatically, by bottom-up signals from salient stimuli. Because the brain mechanisms that underlie these two attention processes are poorly understood, we recorded local field potentials (LFPs) from primary visual cortical areas of cats as they performed stimulus-driven and anticipatory discrimination tasks. Consistent with our previous observations, in both tasks, we found enhanced beta activity, which we have postulated may serve as an attention carrier. We characterized the functional organization of task-related beta activity by (i) cortical responses (EPs) evoked by electrical stimulation of the optic chiasm and (ii) intracortical LFP correlations. During the anticipatory task, peripheral stimulation that was preceded by high-amplitude beta oscillations evoked large-amplitude EPs compared with EPs that followed low-amplitude beta. In contrast, during the stimulus-driven task, cortical EPs preceded by high-amplitude beta oscillations were, on average, smaller than those preceded by low-amplitude beta. Analysis of the correlations between the different recording sites revealed that beta activation maps were heterogeneous during the bottom-up task and homogeneous for the top-down task. We conclude that bottom-up attention activates cortical visual areas in a mosaic-like pattern, whereas top-down attentional modulation results in spatially homogeneous excitation.     

Left and Right Amygdala - Mediofrontal Cortical Functional Connectivity Is Differentially Modulated by Harm Avoidance

Background

The left and right amygdalae are key regions distinctly involved in emotion-regulation processes. Individual differences, such as personality features, may affect the implicated neurocircuits. The lateralized amygdala affective processing linked with the temperament dimension Harm Avoidance (HA) remains poorly understood. Resting state functional connectivity imaging (rsFC) may provide more insight into these neuronal processes.

Methods

In 56 drug-naive healthy female subjects, we have examined the relationship between the personality dimension HA on lateralized amygdala rsFC.

Results

Across all subjects, left and right amygdalae were connected with distinct regions mainly within the ipsilateral hemisphere. Females scoring higher on HA displayed stronger left amygdala rsFC with ventromedial prefrontal cortical (vmPFC) regions involved in affective disturbances. In high HA scorers, we also observed stronger right amygdala rsFC with the dorsomedial prefrontal cortex (dmPFC), which is implicated in negative affect regulation.

Conclusions

In healthy females, left and right amygdalae seem implicated in distinct mPFC brain networks related to HA and may represent a vulnerability marker for sensitivity to stress and anxiety (disorders).     

Enhancement of Morphological Plasticity in Hippocampal Neurons by a Physically Modified Saline via Phosphatidylinositol-3 Kinase

Increase of the density of dendritic spines and enhancement of synaptic transmission through ionotropic glutamate receptors are important events, leading to synaptic plasticity and eventually hippocampus-dependent spatial learning and memory formation. Here we have undertaken an innovative approach to upregulate hippocampal plasticity. RNS60 is a 0.9% saline solution containing charge-stabilized nanobubbles that are generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. RNS60, but not NS (normal saline), PNS60 (saline containing a comparable level of oxygen without the TCP modification), or RNS10.3 (TCP-modified normal saline without excess oxygen), stimulated morphological plasticity and synaptic transmission via NMDA- and AMPA-sensitive calcium influx in cultured mouse hippocampal neurons. Using mRNA-based targeted gene array, real-time PCR, immunoblot, and immunofluorescence analyses, we further demonstrate that RNS60 stimulated the expression of many plasticity-associated genes in cultured hippocampal neurons. Activation of type IA, but not type IB, phosphatidylinositol-3 (PI-3) kinase by RNS60 together with abrogation of RNS60-mediated upregulation of plasticity-related proteins (NR2A and GluR1) and increase in spine density, neuronal size, and calcium influx by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a specific inhibitor of PI-3 kinase, suggest that RNS60 upregulates hippocampal plasticity via activation of PI-3 kinase. Finally, in the 5XFAD transgenic model of Alzheimer’s disease (AD), RNS60 treatment upregulated expression of plasticity-related proteins PSD95 and NR2A and increased AMPA- and NMDA-dependent hippocampal calcium influx. These results describe a novel property of RNS60 in stimulating hippocampal plasticity, which may help AD and other dementias.     

Degradation of Postsynaptic Scaffold GKAP and Regulation of Dendritic Spine Morphology by the TRIM3 Ubiquitin Ligase in Rat Hippocampal Neurons

Changes in neuronal activity modify the structure of dendritic spines and alter the function and protein composition of synapses. Regulated degradation of postsynaptic density (PSD) proteins by the ubiquitin-proteasome system is believed to play an important role in activity-dependent synaptic remodeling. Stimulating neuronal activity in vitro and in vivo induces the ubiquitination and degradation of GKAP/SAPAP and Shank, major scaffold proteins of the PSD. However, the specific ubiquitin ligases that regulate postsynaptic protein composition have not been identified. Here we identify the RING finger-containing protein TRIM3 as a specific E3 ubiquitin ligase for the PSD scaffold GKAP/SAPAP1. Present in PSD fractions from rat brain, TRIM3 stimulates ubiquitination and proteasome-dependent degradation of GKAP, and induces the loss of GKAP and associated scaffold Shank1 from postsynaptic sites. Suppression of endogenous TRIM3 by RNA interference (RNAi) results in increased accumulation of GKAP and Shank1 at synapses, as well as enlargement of dendritic spine heads. RNAi of TRIM3 also prevented the loss of GKAP induced by synaptic activity. Thus, TRIM3 is a novel E3 ligase that mediates activity-dependent turnover of PSD scaffold proteins and is a negative regulator of dendritic spine morphology.     

Biofilm Formation and Detachment in Gram-Negative Pathogens Is Modulated by Select Bile Acids

Biofilms are a ubiquitous feature of microbial community structure in both natural and host environments; they enhance transmission and infectivity of pathogens and provide protection from human defense mechanisms and antibiotics. However, few natural products are known that impact biofilm formation or persistence for either environmental or pathogenic bacteria. Using the combination of a novel natural products library from the fish microbiome and an image-based screen for biofilm inhibition, we describe the identification of taurine-conjugated bile acids as inhibitors of biofilm formation against both Vibrio cholerae and Pseudomonas aeruginosa. Taurocholic acid (1) was isolated from the fermentation broth of the fish microbiome-derived strain of Rhodococcus erythropolis and identified using standard NMR and MS methods. Screening of the twelve predominant human steroidal bile acid components revealed that a subset of these compounds can inhibit biofilm formation, induce detachment of preformed biofilms under static conditions, and that these compounds display distinct structure-activity relationships against V. cholerae and P. aeruginosa. Our findings highlight the significance of distinct bile acid components in the regulation of biofilm formation and dispersion in two different clinically relevant bacterial pathogens, and suggest that the bile acids, which are endogenous mammalian metabolites used to solubilize dietary fats, may also play a role in maintaining host health against bacterial infection.     

Consolidation of an Olfactory Memory Trace in the Olfactory Bulb Is Required for Learning-Induced Survival of Adult-Born Neurons and Long-Term Memory

Background

It has recently been proposed that adult-born neurons in the olfactory bulb, whose survival is modulated by learning, support long-term olfactory memory. However, the mechanism used to select which adult-born neurons following learning will participate in the long-term retention of olfactory information is unknown. We addressed this question by investigating the effect of bulbar consolidation of olfactory learning on memory and neurogenesis.

Methodology/Principal Findings

Initially, we used a behavioral ecological approach using adult mice to assess the impact of consolidation on neurogenesis. Using learning paradigms in which consolidation time was varied, we showed that a spaced (across days), but not a massed (within day), learning paradigm increased survival of adult-born neurons and allowed long-term retention of the task. Subsequently, we used a pharmacological approach to block consolidation in the olfactory bulb, consisting in intrabulbar infusion of the protein synthesis inhibitor anisomycin, and found impaired learning and no increase in neurogenesis, while basic olfactory processing and the basal rate of adult-born neuron survival remained unaffected. Taken together these data indicate that survival of adult-born neurons during learning depends on consolidation processes taking place in the olfactory bulb.

Conclusion/Significance

We can thus propose a model in which consolidation processes in the olfactory bulb determine both survival of adult-born neurons and long-term olfactory memory. The finding that adult-born neuron survival during olfactory learning is governed by consolidation in the olfactory bulb strongly argues in favor of a role for bulbar adult-born neurons in supporting olfactory memory.     

Frizzled-5 Receptor Is Involved in Neuronal Polarity and Morphogenesis of Hippocampal Neurons

The Wnt signaling pathway plays important roles during different stages of neuronal development, including neuronal polarization and dendritic and axonal outgrowth. However, little is known about the identity of the Frizzled receptors mediating these processes. In the present study, we investigated the role of Frizzled-5 (Fzd5) on neuronal development in cultured Sprague-Dawley rat hippocampal neurons. We found that Fzd5 is expressed early in cultured neurons on actin-rich structures localized at minor neurites and axonal growth cones. At 4 DIV, Fzd5 polarizes towards the axon, where its expression is detected mainly at the peripheral zone of axonal growth cones, with no obvious staining at dendrites; suggesting a role of Fzd5 in neuronal polarization. Overexpression of Fzd5 during the acquisition of neuronal polarity induces mislocalization of the receptor and a loss of polarized axonal markers. Fzd5 knock-down leads to loss of axonal proteins, suggesting an impaired neuronal polarity. In contrast, overexpression of Fzd5 in neurons that are already polarized did not alter polarity, but decreased the total length of axons and increased total dendrite length and arborization. Fzd5 activated JNK in HEK293 cells and the effects triggered by Fzd5 overexpression in neurons were partially prevented by inhibition of JNK, suggesting that a non-canonical Wnt signaling mechanism might be involved. Our results suggest that, Fzd5 has a role in the establishment of neuronal polarity, and in the morphogenesis of neuronal processes, in part through the activation of the non-canonical Wnt mechanism involving JNK.     

Regulation of Dendrite and Spine Morphogenesis and Plasticity by Catenins

The appropriate regulation of dendrite, spine, and synapse morphogenesis in neurons both during and after development is critical for the formation and maintenance of neural circuits. It is becomingly increasingly clear that the cadherin–catenin cell adhesion complex, a complex that has been widely studied in epithelia, regulates neuronal morphogenesis. More interestingly, the catenins, cytosolic proteins that bind to cadherins, regulate multiple aspects of neuronal morphogenesis including dendrite, spine, and synapse morphogenesis and plasticity, both independent of and dependent on their ability to bind cadherins. In this review, we examine some of the more recent and exciting studies that implicate individual catenins in various aspects of neuronal morphogenesis and plasticity.     

Prolonged Decay of Evoked Inhibitory Postsynaptic Currents in Hippocampal Neurons Is Not Shaped by Asynchronous Release

During evoked release, several quanta of neurotransmitter are synchronously released in several GABA-ergic synapses. Assuming that not more than one vesicle is released at each release site, the decay of miniature and evoked IPSC (mIPSC and eIPSC, respectively) should coincide. In this study, we found that in a considerable part of the cultured hippocampal neurons eIPSC decayed more slowly than mIPSC did. We investigated the mechanisms underlying this difference using conventional electrophysiological approaches, deconvolution, simulations, and nonstationary noise analysis. Our results indicate that asynchronous release of synaptic vesicles cannot explain the prolonged decay of the GABA-ergic IPSC. We suggest that some interaction between the quanta at the pre- and/or post-synaptic level should result in a slower decay of the eIPSC in comparison with that of mIPSC.     

The Downregulation of Truncated TrkB Receptors Modulated by MicroRNA-185 Activates Full-Length TrkB Signaling and Suppresses the Epileptiform Discharges in Cultured Hippocampal Neurons

Neurochemical Research - Epilepsy is a common neurological disorder characterised by occurrence of spontaneous recurrent epileptiform discharges (SREDs) in neurons. The cellular mechanisms that...     

The Interaction of Heparin Tetrasaccharides with Chemokine CCL5 Is Modulated by Sulfation Pattern and pH

Interactions between chemokines such as CCL5 and glycosaminoglycans (GAGs) are essential for creating haptotactic gradients to guide the migration of leukocytes into inflammatory sites, and the GAGs that interact with CCL5 with the highest affinity are heparan sulfates/heparin. The interaction between CCL5 and its receptor on monocytes, CCR1, is mediated through residues Arg-17 and -47 in CCL5, which overlap with the GAG-binding ⁴⁴RKNR⁴⁷ “BBXB” motifs. Here we report that heparin and tetrasaccharide fragments of heparin are able to inhibit CCL5-CCR1 binding, with IC₅₀ values showing strong dependence on the pattern and extent of sulfation. Modeling of the CCL5-tetrasaccharide complexes suggested that interactions between specific sulfate and carboxylate groups of heparin and residues Arg-17 and -47 of the protein are essential for strong inhibition; tetrasaccharides lacking the specific sulfation pattern were found to preferentially bind CCL5 in positions less favorable for inhibition of the interaction with CCR1. Simulations of a 12-mer heparin fragment bound to CCL5 indicated that the oligosaccharide preferred to interact simultaneously with both ⁴⁴RKNR⁴⁷ motifs in the CCL5 homodimer and engaged residues Arg-47 and -17 from both chains. Direct engagement of these residues by the longer heparin oligosaccharide provides a rationalization for its effectiveness as an inhibitor of CCL5-CCR1 interaction. In this mode, histidine (His-23) may contribute to CCL5-GAG interactions when the pH drops just below neutral, as occurs during inflammation. Additionally, an examination of the contribution of pH to modulating CCL5-heparin interactions suggested a need for careful interpretation of experimental results when experiments are performed under non-physiological conditions.     

Refinement and Pattern Formation in Neural Circuits by the Interaction of Traveling Waves with Spike-Timing Dependent Plasticity

Traveling waves in the developing brain are a prominent source of highly correlated spiking activity that may instruct the refinement of neural circuits. A candidate mechanism for mediating such refinement is spike-timing dependent plasticity (STDP), which translates correlated activity patterns into changes in synaptic strength. To assess the potential of these phenomena to build useful structure in developing neural circuits, we examined the interaction of wave activity with STDP rules in simple, biologically plausible models of spiking neurons. We derive an expression for the synaptic strength dynamics showing that, by mapping the time dependence of STDP into spatial interactions, traveling waves can build periodic synaptic connectivity patterns into feedforward circuits with a broad class of experimentally observed STDP rules. The spatial scale of the connectivity patterns increases with wave speed and STDP time constants. We verify these results with simulations and demonstrate their robustness to likely sources of noise. We show how this pattern formation ability, which is analogous to solutions of reaction-diffusion systems that have been widely applied to biological pattern formation, can be harnessed to instruct the refinement of postsynaptic receptive fields. Our results hold for rich, complex wave patterns in two dimensions and over several orders of magnitude in wave speeds and STDP time constants, and they provide predictions that can be tested under existing experimental paradigms. Our model generalizes across brain areas and STDP rules, allowing broad application to the ubiquitous occurrence of traveling waves and to wave-like activity patterns induced by moving stimuli.     

Notch Is Required in Adult Drosophila Sensory Neurons for Morphological and Functional Plasticity of the Olfactory Circuit

Olfactory receptor neurons (ORNs) convey odor information to the central brain, but like other sensory neurons were thought to play a passive role in memory formation and storage. Here we show that Notch, part of an evolutionarily conserved intercellular signaling pathway, is required in adult Drosophila ORNs for the structural and functional plasticity of olfactory glomeruli that is induced by chronic odor exposure. Specifically, we show that Notch activity in ORNs is necessary for the odor specific increase in the volume of glomeruli that occurs as a consequence of prolonged odor exposure. Calcium imaging experiments indicate that Notch in ORNs is also required for the chronic odor induced changes in the physiology of ORNs and the ensuing changes in the physiological response of their second order projection neurons (PNs). We further show that Notch in ORNs acts by both canonical cleavage-dependent and non-canonical cleavage-independent pathways. The Notch ligand Delta (Dl) in PNs switches the balance between the pathways. These data define a circuit whereby, in conjunction with odor, N activity in the periphery regulates the activity of neurons in the central brain and Dl in the central brain regulates N activity in the periphery. Our work highlights the importance of experience dependent plasticity at the first olfactory synapse.     

Physiological,Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static^1,2 or record extracellular neuronal activity during a movement.^3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.Download video file.^{(43M, mov)}     

Phosphorylation of Gephyrin in Hippocampal Neurons by Cyclin-dependent Kinase CDK5 at Ser-270 Is Dependent on Collybistin

Gephyrin is a scaffold protein essential for the postsynaptic clustering of inhibitory glycine and different subtypes of GABA(A) receptors. The cellular and molecular mechanisms involved in gephyrin-mediated receptor clustering are still not well understood. Here we provide evidence that the gephyrin-binding protein collybistin is involved in regulating the phosphorylation of gephyrin. We demonstrate that the widely used monoclonal antibody mAb7a is a phospho-specific antibody that allows the cellular and biochemical analysis of gephyrin phosphorylation at Ser-270. In addition, another neighbored epitope determinant was identified at position Thr-276. Analysis of the double mutant gephyrin(T276A,S277A) revealed significant reduction in gephyrin cluster formation and altered oligomerization behavior of gephyrin. Moreover, pharmacological inhibition of cyclin-dependent kinases in hippocampal neurons reduced postsynaptic gephyrin mAb7a immunoreactivities. In vitro phosphorylation assays and phosphopeptide competition experiments revealed a phosphorylation at Ser-270 depending on enzyme activities of cyclin-dependent kinases CDK1, -2, or -5. These data indicate that collybistin and cyclin-dependent kinases are involved in regulating the phosphorylation of gephyrin at postsynaptic membrane specializations.     

The Biological Activity of the Prototypic Cyclotide Kalata B1 Is Modulated by the Formation of Multimeric Pores

The cyclotides are a large family of circular mini-proteins containing a cystine knot motif. They are expressed in plants as defense-related proteins, with insecticidal activity. Here we investigate their role in membrane interaction and disruption. Kalata B1, a prototypic cyclotide, was found to induce leakage of the self-quenching fluorophore, carboxyfluorescein, from phospholipid vesicles. Alanine-scanning mutagenesis of kalata B1 showed that residues essential for lytic activity are clustered, forming a bioactive face. Kalata B1 was sequestered at the membrane surface and showed slow dissociation from vesicles. Electrophysiological experiments showed that conductive pores were induced in liposome patches on incubation with kalata B1. The conductance calculated from the current-voltage relationship indicated that the diameter of the pores formed in the bilayer patches is 41–47 Å. Collectively, the findings provide a mechanistic explanation for the diversity of biological functions ascribed to this fascinating family of ultrastable macrocyclic peptides.The cyclotides are a family of topologically unique macrocyclic peptides abundant in plants of the Rubiaceae (coffee) and Violaceae (violet) families. They possess unusual structural and biophysical properties and are composed of a head-to-tail cyclic backbone and a cystine knot (1). The cystine knot is formed by a disulfide bond that penetrates a ring made by two other disulfide bonds and their connecting backbone segments. The structure of kalata B1, the prototypic cyclotide, is illustrated in Fig. 1 (2). The cyclic cystine knot at the core of three-dimensional structure contributes to the exceptional chemical and biological stability of cyclotides (3) and underpins their exciting potential for pharmaceutical and agricultural applications (4).Open in a separate windowFIGURE 1.Structural representation of the kalata B1 sequence showing the cystine knot topology and head-to-tail cyclized backbone (PDB ID 1nb1). The six cysteine residues are bolded and labeled with Roman numerals. The three disulfide bonds are shown as cylinders connecting the cysteine residues (I–IV, II–V, and III–VI). The backbone segments between successive cysteine residues are referred to as loops. Loops 2, 3, 5, and 6 are marked, with the small arrows indicating the direction of the peptide chain from amino to carboxyl ends. Loops 1 and 4 form part of the embedded ring of cystine knot. The broad arrows indicate β-strands in the peptide backbone that are typically associated with cystine knot motifs (47).Cyclotides display a diverse range of biological activities, including anti-human immunodeficiency virus (5–8), neurotensin antagonism (9), hemolytic (10), antimicrobial (11), anti-fouling (12), and pesticidal activities (13–19). Cyclotides have been postulated to be defense-related proteins on the basis of their pesticidal activity and the suite of natural isoforms present in individual plants (20). Little is known about their mechanism of action, but their observed activities potentially might be associated with membrane interactions. Studies utilizing analytical ultracentrifugation (21) have shown that the cyclotide kalata B2 forms specific oligomers in solution, which could potentially have a role in the formation of membrane-spanning pores. A membrane-based mechanism of action is supported by a recent surface plasmon resonance study, which demonstrated that several kalata-like cyclotides bind to phosphatidylethanolamine-containing membranes (22). More recently, the cyclotide cycloviolacin O2 was shown to be cytotoxic to a human lymphoma cell line and induce leakage of calcein-loaded HeLa cells (23). NMR studies showed that the binding of kalata B1, and other analogues, to dodecylphosphocholine micelles is modulated by both electrostatic and hydrophobic interactions (24, 25). Although these findings indicate that cyclotides interact with a wide range of membranes, with incorporation of the cyclotide structure into monolayers (micelles), and result in leakage of cellular contents from bilayers (HeLa cells), a functional study of cyclotide-membrane interactions is to date lacking.In the present study, the membrane-binding ability of prototypic cyclotide kalata B1 is delineated, and the mechanism of action is defined. For the first time we show that cyclotides form pores with channel-like activities in membranes. Dye leakage experiments indicate that kalata B1 induces membrane permeability, and electrophysiological measurements provide unequivocal evidence of pore formation, probably involving the insertion of oligomers of kalata B1 into the lipid bilayers. The results indicate that the diverse range of biological activities reported for cyclotides can be accounted for by membrane permeabilization associated with transmembrane pores with channel-like activity.