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The objective of this study was to test masseter muscle activity and its variability and reliability in terms of repeatable measurements in fully dentate and healthy volunteers during habitual chewing and deliberate unilateral chewing. Three sessions were performed on three consecutive days, each time recording kinematic data and masseter muscle EMG activities through a series of defined jaw exercises, including maximum voluntary contraction, habitual chewing, and left and right unilateral chewing. Asymmetry index (Al) and deliberate chewing index (DCI) scores were used to evaluate the activities of the left and right masseter muscles, which were separately recorded during each of these chewing exercises. DCI scores were side -specific, including two sets of values for left and right unilateral chewing. Reproducibility testing of the values obtained for all parameters revealed good to excellent reproducibility of masseter muscle activity under standardized recording conditions across the consecutive study sessions, with intra-class correlation coefficients (ICCs) ranging from 0.68 to 0.93. Different individuals were found to utilize different strategies of bilateral masseter activation during both habitual chewing and deliberate unilateral chewing.

Myotonia congenita is an autosomal dominantly or recessively inherited muscle disorder causing impaired muscle relaxation and variable degrees of permanent muscle weakness, abnormal currents linked to the chloride channel gene (CLCN1) encoding the chloride channel on skeletal muscle membrane. We describe 12 novel mutations: c.1606G>C (p.Val536Leu), c.2533G>A (p.Gly845Ser), c.2434C>T (p.Gln812X), c.1499T>G (p.E500X), c.1012C>T (p.Arg338X), c.2403+1G>A, c.2840T>A (p.Val947Glu), c.1598C>T (p.Thr533Ile), c.1110delC, c.590T>A (p.Ile197Arg), c.2276insA Fs800X, c.490T>C (p.Trp164Arg) in 22 unrelated Italian patients. To further understand the functional outcome of selected missense mutations (p.Trp164Arg, p.Ile197Arg and p.Gly845Ser, and the previously reported p.Gly190Ser) we characterized the biophysical properties of mutant ion channels in tsA cell model. In the physiological range of muscle membrane potential, all the tested mutations, except p.Gly845Ser, reduced the open probability, increased the fast and slow components of deactivation and affected pore properties. This suggests a decrease in macroscopic chloride currents impairing membrane potential repolarization and causing hyperexcitability in muscle membranes. Detailed clinical features are given of the 8 patients characterized by cell electrophysiology. These data expand the spectrum of CLCN1 mutations and may contribute to genotype-phenotype correlations. Furthermore, we provide insights into the fine protein structure of ClC-1 and its physiological role in the maintenance of membrane resting potential.

Distinguishing excitatory from inhibitory neurons with multielectrode array (MEA) recordings is a serious experimental challenge. The current methods, developed in vitro, mostly rely on spike waveform analysis. These however often display poor resolution and may produce errors caused by the variability of spike amplitudes and neuron shapes. Recent recordings in human brain suggest that the spike waveform features correlate with time-domain statistics such as spiking rate, autocorrelation and coefficient of variation. However, no precise criteria are available to exactly assign identified units to specific neuronal types, either in vivo or in vitro. To solve this problem, we combined MEA recording with fluorescence imaging of neocortical cultures from mice expressing green fluorescent protein (GFP) in GABAergic cells. In this way, we could sort out ‘authentic excitatory neurons’ (AENs) and ‘authentic inhibitory neurons’ (AINs). We thus characterized 1275 units (from 405 electrodes, n=10 experiments), based on autocorrelation, burst length, spike number, spiking rate, squared coefficient of variation and Fano factor (the ratio between spike-count variance and mean). These metrics differed by about one order of magnitude between AINs and AENs. In particular, the Fano factor turned out to provide a firing code which exactly (no overlap) recognizes excitatory and inhibitory units. The difference in Fano factor between all of the identified AEN and AIN groups was highly significant (p < 10-8, ANOVA post-hoc Tukey test). Our results indicate a statistical metric-based approach to distinguish excitatory from inhibitory neurons independently from the spike width.

The dynamics of spontaneous and sensory-evoked up-states have been recently compared, in multi-site recordings in vivo and found to have similarities and differences. Also in vitro, this is evident because we here describe a novel computational method to classify into statistically different states the spontaneous reverberating activity recorded from long-term (12-18 days-in vitro) cultured cortical neurons (from 60-site multi-electrode arrays, MEA). State classification was performed by spike number time histograms (SNTH, or other burst features) of excitatory and inhibitory neuron clusters and revealed that in novel identified states the number of engaged neurons or up-state duration can change. To improve the characterization of each state we also computed the firing spike histograms (FSH) which revealed a new facet of the firing probability of clusters. In exemplary functional experiments we show that: (i) up to 6-7 states can be safely categorized during several hours of recordings without observing spike rate changes, (ii) they disappear after a short pharmacological stimulation being replaced with novel states active and living up to 6-8 h, (iii) antagonists in the nM range can split the activity of a homogeneous network into the chronological coexistence of 2 states, one completely different and one not significantly different from control state. In conclusion, we believe that this novel procedure better characterizes the number of functional states of a network and opens up the possibility of predicting the elementary "vocabulary" used by small networks of neurons.

The dynamics of sensory-evoked and spontaneous up-states have been recently compared, in multi-site recordings in vivo and in vitro, by using various types of statistical analysis such as the spike rate (SR) or the Fano factor (FF). We here describe and validate a novel computational method to classify into statistically different states the spontaneous reverberating activity recorded from long-term (12-18 days-in-vitro) cultured cortical neurons (from 60-site multi-electrode arrays, MEA). State classification was performed by spike number time histograms (SNTH, or other burst features) of excitatory and inhibitory neuron clusters and revealed that also the number of engaged neurons and the FF histograms are linked to the identified firing modes. To improve the characterization of each state we also computed the firing spike histograms (FSH) which revealed a new facet of the firing probability of clusters. Surprisingly, we found that the FF is an enhanced feature as compared to the autocorrelation function (ACF) to clusterize units. In exemplary functional experiments we show that: i) up to 4 different states can be identified in 30’ timesegments, ii) in adjacent time-segments novel states can be recognized, iii) up to 6-7 states can be safely categorized during several hours of recordings, iv) they disappear after a short pharmacological stimulation being replaced with novel states active and living up to 6-8 h. In conclusion, we believe that this novel procedure better characterizes the number of functional states of a network and opens up the possibility of predicting the elementary “vocabulary” used by small networks of neurons.

It has been demonstrated using single-cell and multiunit electrophysiology in layer III entorhinal cortex and disinhibited hippocampal CA3 slices that the balancing of the up-down activity is characterized by both GABAA and GABAB mechanisms. Here we report novel results obtained using multi-electrode array (60 electrodes) simultaneous recordings from reverberating postnatal neocortical networks containing 19.2 ± 1.4% GABAergic neurons, typical of intact tissue. We observed that in each spontaneous active-state the total number of spikes in identifi ed clusters of excitatory and inhibitory neurons is almost equal, thus suggesting a balanced average activity. Interestingly, in the active-state, the early phase is sustained by only 10% of the total spikes and the fi ring rate follows a sigmoidal regenerative mode up to peak at 35 ms with the number of excitatory spikes greater than inhibitory, therefore indicating an early unbalance. Concentrationresponse pharmacology of up- and down-state lifetimes in clusters of excitatory (n = 1067) and inhibitory (n = 305) cells suggests that, besides the GABAA and GABAB mechanisms, others such as GAT-1-mediated uptake, Ih, INaP and IM ion channel activity, robustly govern both upand down-activity. Some drugs resulted to affect up- and/or down-states with different IC50s, providing evidence that various mechanisms are involved. These results should reinforce not only the role of synchrony in CNS networks, but also the recognized analogies between the Hodgkin-Huxley action potential and the population bursts as basic mechanisms for originating membrane excitability and CNS network synchronization, respectively.

Contains fulltext : 87841.pdf (publisher's version ) (Open Access), In a recent paper (Reid et al., 2009) we introduced a method to calculate optimal hierarchies in the visual network that utilizes continuous, rather than discrete, hierarchical levels, and permits a range of acceptable values rather than attempting to fit fixed hierarchical distances. There, to obtain a hierarchy, the sum of deviations from the constraints that define the hierarchy was minimized using linear optimization. In the short time since publication of that paper we noticed that many colleagues misinterpreted the meaning of the term "optimal hierarchy". In particular, a majority of them were under the impression that there was perhaps only one optimal hierarchy, but a substantial difficulty in finding that one. However, there is not only more than one optimal hierarchy but also more than one option for defining optimality. Continuing the line of this work we look at additional options for optimizing the visual hierarchy: minimizing the number of violated constraints and minimizing the maximal size of a constraint violation using linear optimization and mixed integer programming. The implementation of both optimization criteria is explained in detail. In addition, using constraint sets based on the data from Felleman and Van Essen (1991), optimal hierarchies for the visual network are calculated for both optimization methods.

Pompilidotoxins (PMTXs, alpha and beta) are small peptides consisting of 13 amino acids purified from the venom of the solitary wasps Anoplius samariensis (alpha-PMTX) and Batozonellus maculifrons (beta-PMTX). They are known to facilitate synaptic transmission in the lobster neuromuscular junction, and to slow sodium channel inactivation. By using beta-PMTX, alpha-PMTX and four synthetic analogs with amino acid changes, we conducted a thorough study of the effects of PMTXs on sodium current inactivation in seven mammalian voltage-gated sodium channel (VGSC) isoforms and one insect VGSC (DmNa(v)1). By evaluating three components of which the inactivating current is composed (fast, slow and steady-state components), we could distinguish three distinct groups of PMTX effects. The first group concerned the insect and Na(v)1.6 channels, which showed a large increase in the steady-state current component without any increase in the slow component. Moreover, the dose-dependent increase in this steady-state component was correlated with the dose-dependent decrease in the fast component. A second group of effects concerned the Na(v)1.1, Na(v)1.2, Na(v)1.3 and Na(v)1.7 isoforms, which responded with a large increase in the slow component, and showed only a small steady-state component. As with the first group of effects, the slow component was dose-dependent and correlated with the decrease in the fast component. Finally, a third group of effects concerned Na(v)1.4 and Na(v)1.5, which did not show any change in the slow or steady-state component. These data shed light on the complex and intriguing behavior of VGSCs in response to PMTXs, helping us to better understand the molecular determinants explaining isoform-specific effects.

Venom-derived peptide modulators of ion channel gating are regarded as essential tools for understanding the molecular motions that occur during the opening and closing of ion channels. In this study, we present the characterization of five spider toxins on 12 human voltage-gated ion channels, following observations about the target promiscuity of some spider toxins and the ongoing revision of their "canonical" gating-modifying mode of action. The peptides were purified de novo from the venom of Grammostola rosea tarantulas, and their sequences were confirmed by Edman degradation and mass spectrometry analysis. Their effects on seven tetrodotoxin-sensitive Na+ channels, the three human ether-a-go-go (hERG)-related K+ channels, and two human Shaker-related K+ channels were extensively characterized by electrophysiological techniques. All the peptides inhibited ion conduction through all the Na+ channels tested, although with distinctive patterns. The peptides also affected the three pharmaceutically relevant hERG isoforms differently. At higher concentrations, all peptides also modified the gating of the Na+ channels by shifting the activation to more positive potentials, whereas more complex effects were recorded on hERG channels. No effects were evident on the two Shaker-related K+ channels at concentrations well above the IC50 value for the affected channels. Given the sequence diversity of the tested peptides, we propose that tarantula toxins should be considered both as multimode and target-promiscuous ion channel modulators; both features should not be ignored when extracting mechanistic interpretations about ion channel gating. Our observations could also aid in future structure-function studies and might help the development of novel ion channel-specific drugs.

Contains fulltext : 81553.pdf (Publisher’s version ) (Closed access), Although information flow in the neocortex has an apparent hierarchical organization, there is much ambiguity with respect to the definition of such a hierarchy, particularly in higher cortical regions. This ambiguity has been addressed by utilizing observable anatomical criteria, based upon tract tracing experiments, to constrain the definition of hierarchy [Felleman D.J. and van Essen D.C., 1991. Distributed hierarchical processing in the primate. Cereb. Cortex. 1(1), 1-47.]. There are, however, a high number of equally optimal hierarchies that fit these constraints [Hilgetag C.C., O'Neill M.A., Young M.P., 1996. Indeterminate organization of the visual system. Science. 271(5250), 776-777.]. Here, we propose a refined constraint set for optimization which utilizes continuous, rather than discrete, hierarchical levels, and permits a range of acceptable values rather than attempting to fit fixed hierarchical distances. Using linear programming to obtain hierarchies across a number of range sizes, we find a clear hierarchical pattern for both the original and refined versions of the Felleman and Van Essen [Felleman D.J. and van Essen D.C., 1991. Distributed hierarchical processing in the primate. Cereb. Cortex. 1(1), 1-47.] visual network. We also obtain an optimal hierarchy from a refined set of anatomical criteria which allows for the direct specification of hierarchical distance from the laminar distribution of labelled cells (Barone P., Batardiere A., Knoblauch K., Kennedy H., 2000. Laminar distribution of neurons in extrastriate areas projecting to visual areas V1 and V4 correlates with the hierarchical rank and indicates the operation of a distance rule. J. Neurosci. 20(9), 3263-3281.), and discuss the limitations and further possible refinements of such an approach.

Spontaneous bursting activity is present in vivo during CNS development and in vitro in neocortex slices. A prerequisite for understanding the cooperative behavior in neuronal ensembles is large-scale simultaneous extracellular electrophysiology by using either "tetrodes" (4-wire electrode) in awake animals or multi-electrode arrays (MEA) in long-term cultured networks as we did here. We show that from a single low-noise MEA electrode it is possible to identify up to 3-4 types of waveforms whose timestamps show excitatory and inhibitory short-latency (2-4 ms) cross-correlations, indicative of monosynaptic connections. Moreover, the MEA units autocorrelagrams (AC) resulted to have behaviors similar to those demonstrated in vivo by using tetrodes or shanks. Principal component analysis of AC followed by a K-means classification returned 3-4 different clusters whose firing- and burst-related properties were typical of assemblies of putative excitatory and inhibitory neurons. By manipulating the networks with a GABAA antagonist (gabazine), we could detect cell groups selectively responding to blockade of GABA transmission with IC50s of 82 ± 2 and 770 ± 70 nM. These methods, expanded to organotypic co-cultures of CNS regions may be useful to better understand their connecting properties in studies of regenerative medicine. © 2009 Elsevier B.V. All rights reserved.

Contains fulltext : 69620.pdf (publisher's version ) (Closed access), We propose a new technique, called Spatial Objective Relational Transformation (SORT), as an automated approach for derivation of logical relationships between cortical areas in different brain maps registered in the same Euclidean space. Recently, there have been large amounts of voxel-based three-dimensional structural and functional imaging data that provide us with coordinate-based information about the location of differently defined areas in the brain, whereas coordinate-independent, parcellation-based mapping is still commonly used in the majority of animal tracing and mapping studies. Because of the impact of voxel-based imaging methods and the need to attribute their features to coordinate-independent brain entities, this mapping becomes increasingly important. Our motivation here is not to make vague statements where more precise spatial statements would be better, but to find criteria for the identity (or other logical relationships) between areas that were delineated by different methods, in different individuals, or mapped to three-dimensional space using different deformation algorithms. The relevance of this problem becomes immediately obvious as one superimposes and compares different datasets in multimodal databases (e.g. CARET, http://brainmap.wustl.edu/caret), where voxel-based data are registered to surface nodes exploited by the procedure presented here. We describe the SORT algorithm and its implementation in the Java 2 programming language (http://java.sun.com/, which we make available for download. We give an example of practical use of our approach, and validate the SORT approach against a database of the coordinate-independent statements and inferences that have been deduced using alternative techniques.

The transcription factor Sox2 is active in neural stem cells, and Sox2 'knockdown' mice show defects in neural stem/progenitor cells in the hippocampus and eye, and possibly some neurons. In humans, heterozygous Sox2 deficiency is associated with eye abnormalities, hippocampal malformation and epilepsy. To better understand the role of Sox2, we performed in vitro differentiation studies on neural stem cells cultured from embryonic and adult brains of 'knockdown' mutants. Sox2 expression is high in undifferentiated cells, and declines with differentiation, but remains visible in at least some of the mature neurons. In mutant cells, neuronal, but not astroglial, differentiation was profoundly affected. beta-Tubulin-positive cells were abundant, but most failed to progress to more mature neurons, and showed morphological abnormalities. Overexpression of Sox2 in neural cells at early, but not late, stages of differentiation, rescued the neuronal maturation defect. In addition, it suppressed GFAP expression in glial cells. Our results show an in vitro requirement for Sox2 in early differentiating neuronal lineage cells, for maturation and for suppression of alternative lineage markers. Finally, we examined newly generated neurons from Sox2 ;knockdown' newborn and adult mice. GABAergic neurons were greatly diminished in number in newborn mouse cortex and in the adult olfactory bulb, and some showed abnormal morphology and migration properties. GABA deficiency represents a plausible explanation for the epilepsy observed in some of the knockdown mice, as well as in SOX2-deficient individuals.