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Ultrasonic neurostimulation is a potentially potent noninvasive therapy, whose mechanism has yet to be elucidated. We designed a system capable of applying ultrasound with minimal reflections to neuronal cultures. Synaptic transmission was pharmacologically controlled, eliminating network effects, enabling examination of single-cell processes. Short single pulses of low-intensity ultrasound were applied, and time-locked responses were examined using calcium imaging. Low-pressure (0.35 MPa) ultrasound directly stimulated ∼20% of pharmacologically disconnected neurons, regardless of membrane poration. Stimulation was resistant to the blockade of several purinergic receptor and mechanosensitive ion channel types. Stimulation was blocked, however, by suppression of action potentials. Surprisingly, even extremely short (4 μs) pulses were effective, stimulating ∼8% of the neurons. Lower-pressure pulses (0.35 MPa) were less effective than higher-pressure ones (0.65 MPa). Attrition effects dominated, with no indication of compromised viability. Our results detract from theories implicating cavitation, heating, non-transient membrane pores >1.5 nm, pre-synaptic release, or gradual effects. They implicate a post-synaptic mechanism upstream of the action potential, and narrow down the list of possible targets involved., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Although previous research in alcohol dependent populations identified alterations within local structures of the addiction 'reward' circuitry, there is limited research into global features of this network, especially in early recovery. Transcranial magnetic stimulation (TMS) is capable of non-invasively perturbing the brain network while electroencephalography (EEG) measures the network response. The current study is the first to apply a TMS inhibitory paradigm while utilising network science (graph theory) to quantify network anomalies associated with alcohol dependence. Eleven individuals with alcohol-dependence (ALD) in early recovery and 16 healthy controls (HC) were administered 75 single pulses and 75 paired-pulses (inhibitory paradigm) to both the left and right prefrontal cortex (PFC). For each participant, Pearson cross-correlation was applied to the EEG data and correlation matrices constructed. Global network measures (mean degree, clustering coefficient, local efficiency and global efficiency) were extracted for comparison between groups. Following administration of the inhibitory paired-pulse TMS to the left PFC, the ALD group exhibited altered mean degree, clustering coefficient, local efficiency and global efficiency compared to HC. Decreases in local efficiency increased the prediction of being in the ALD group, while all network metrics (following paired-pulse left TMS) were able to adequately discriminate between the groups. In the ALD group, reduced mean degree and global clustering was associated with increased severity of past alcohol use. Our study provides preliminary evidence of altered network topology in patients with alcohol dependence in early recovery. Network anomalies were predictive of high alcohol use and correlated with clinical features of alcohol dependence. Further research using this novel brain mapping technique may identify useful network biomarkers of alcohol dependence and recovery., (© 2022 The Authors. Addiction Biology published by John Wiley & Sons Ltd on behalf of Society for the Study of Addiction.)

Automatic speech recognition (ASR) and natural language processing (NLP) are expected to benefit from an effective, simple, and reliable method to automatically parse conversational speech. The ability to parse conversational speech depends crucially on the ability to identify boundaries between prosodic phrases. This is done naturally by the human ear, yet has proved surprisingly difficult to achieve reliably and simply in an automatic manner. Efforts to date have focused on detecting phrase boundaries using a variety of linguistic and acoustic cues. We propose a method which does not require model training and utilizes two prosodic cues that are based on ASR output. Boundaries are identified using discontinuities in speech rate (pre-boundary lengthening and phrase-initial acceleration) and silent pauses. The resulting phrases preserve syntactic validity, exhibit pitch reset, and compare well with manual tagging of prosodic boundaries. Collectively, our findings support the notion of prosodic phrases that represent coherent patterns across textual and acoustic parameters., Competing Interests: The authors have declared that no competing interests exist.

The interplay between excitation and inhibition is crucial for neuronal circuitry in the brain. Inhibitory cell fractions in the neocortex and hippocampus are typically maintained at 15 to 30%, which is assumed to be important for stable dynamics. We have studied systematically the role of precisely controlled excitatory/inhibitory (E/I) cellular ratios on network activity using mice hippocampal cultures. Surprisingly, networks with varying E/I ratios maintain stable bursting dynamics. Interburst intervals remain constant for most ratios, except in the extremes of 0 to 10% and 90 to 100% inhibitory cells. Single-cell recordings and modeling suggest that networks adapt to chronic alterations of E/I compositions by balancing E/I connectivity. Gradual blockade of inhibition substantiates the agreement between the model and experiment and defines its limits. Combining measurements of population and single-cell activity with theoretical modeling, we provide a clearer picture of how E/I balance is preserved and where it fails in living neuronal networks., Competing Interests: The authors declare no competing interest.

Studies have shown that Methylphenidate (MPH) affects cognitive performance on the neuropsychological tests and clinical symptoms of individuals diagnosed with attention deficit/hyperactivity disorder (ADHD). This study investigated the acute effects of MPH on neuropsychological tests to explore the interaction between MPH and test-retest effects. Twenty youths with ADHD were tested before and after MPH intake in a double-blind placebo-controlled crossover design and compared to twenty matched controls. Participants were tested on a range of standardized tasks including sustained attention to response, N-Back, and Word/Color Stroop. Identical tasks were administered twice each testing day, before and 1 hour after MPH/Placebo administration. Healthy controls were tested similarly with no intervention. Decreases in response time (RT) variability across tasks and in commission errors were found in ADHD after MPH. Conversely, a significant increase in RT variability and increase in omission errors were observed after the placebo. In the control group, RT variability and omission errors increased whereas commission errors decreased, suggesting fatigue and practice effects, respectively. Test-retest reliability was higher in controls than ADHD. It is suggested that cognitive tests are sensitive objective measures for the assessment of responses to MPH in ADHD but are also affected by repetition and fatigue., Competing Interests: Declaration of Conflicting Interests The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. This study was supported by the National Institute for Psychobiology in Israel - Founded by the Charles E. Smith Family., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The electroencephalogram (EEG) of schizophrenia patients is known to exhibit a reduction of signal-to-noise ratio and of phase locking, as well as a facilitation of excitability, in response to a variety of external stimuli. Here, we demonstrate these effects in transcranial magnetic stimulation (TMS)-evoked potentials and in the resting-state EEG. To ensure veracity, we used 3 weekly sessions and analyzed both resting-state and TMS-EEG data. For the TMS responses, our analysis verifies known results. For the resting state, we introduce the methodology of mean-normalized variation to the EEG analysis (quartile-based coefficient of variation), which allows for a comparison of narrow-band EEG amplitude fluctuations to narrow-band Gaussian noise. This reveals that amplitude fluctuations in the delta, alpha, and beta bands of healthy controls are different from those in schizophrenia patients, on time scales of tens of seconds. We conclude that the EEG-measured cortical activity patterns of schizophrenia patients are more similar to noise, both in alpha- and beta-resting state and in TMS responses. Our results suggest that the ability of neuronal populations to form stable, locally, and temporally correlated activity is reduced in schizophrenia, a conclusion, that is, in accord with previous experiments on TMS-EEG and on resting-state EEG., (© The Author(s) 2020. Published by Oxford University Press.)

Background: Transcranial magnetic stimulation (TMS) is a rapidly expanding technology utilized in research and neuropsychiatric treatments. Yet, conventional TMS configurations affect primarily neurons that are aligned parallel to the induced electric field by a fixed coil, making the activation orientation-specific. A novel method termed rotational field TMS (rfTMS), where two orthogonal coils are operated with a 90° phase shift, produces rotation of the electric field vector over almost a complete cycle, and may stimulate larger portion of the neuronal population within a given brain area., Objective: To compare the physiological effects of rfTMS and conventional unidirectional TMS (udTMS) in the motor cortex., Methods: Hand and leg resting motor thresholds (rMT), and motor evoked potential (MEP) amplitudes and latencies (at 120% of rMT), were measured using a dual-coil array based on the H7-coil, in 8 healthy volunteers following stimulation at different orientations of either udTMS or rfTMS., Results: For both target areas rfTMS produced significantly lower rMTs and much higher MEPs than those induced by udTMS, for comparable induced electric field amplitude. Both hand and leg rMTs were orientation-dependent., Conclusions: rfTMS induces stronger physiologic effects in targeted brain regions at significantly lower intensities. Importantly, given the activation of a much larger population of neurons within a certain brain area, repeated application of rfTMS may induce different neuroplastic effects in neural networks, opening novel research and clinical opportunities., Competing Interests: Declaration of competing interest AZ and YR are inventors of deep TMS technology and have financial interest in Brainsway Ltd., a company that develops and commercialize deep TMS devices. GSP, MA, YH and AT are Brainsway employees. All other authors declare no competing interest., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Methylphenidate (MPH) is the leading drug for treatment of attention deficit/hyperactivity disorder (ADHD), yet its underlying neuronal mechanisms are still unclear. Here, we use a dynamical brain networks approach to explore the effects of cognitive effort and MPH on ADHD subjects. Electroencephalography data were recorded from 19 ADHD subjects and 18 controls during a Go/No-Go Task. ADHD subjects completed the task twice a day over 2 days. The second session was administered post-ingestion of placebo/MPH (alternately). Controls performed two tasks in 1 day. The data were divided into 300 ms windows from -300 pre-stimulus until 1200 ms post-stimulus. Brain networks were constructed per subject and window, from which network metrics were extracted and compared across the experimental conditions. We identified an immediate shift of global connectivity and of network segregation after the stimulus for both groups, followed by a gradual return to baseline. Decreased global connectivity was found to be 400-700 ms post-stimulus in ADHD compared with controls, and it was normalized post-MPH. An increase of the networks' segregation occurred post-placebo at 100-400 and 400-700 ms post-stimulus, yet it was inhibited post-MPH. These global alterations resulted mainly from changes in task-relevant frontal and parietal regions. The networks of medicated ADHD subjects and controls exhibited a more significant and lasting change, relative to baseline, compared with those of nonmedicated ADHD. These results suggest impaired network flexibility in ADHD, corrected by MPH.

Methylphenidate (MPH) is a first line drug for attention-deficit/hyperactivity disorder (ADHD), yet the neuronal mechanisms underlying the condition and the treatment are still not fully understood. Previous EEG studies on the effect of MPH in ADHD found changes in evoked response potential (ERP) components that were inconsistent between studies. These inconsistencies highlight the need for a well-designed study which includes multiple baseline sessions and controls for possible fatigue, learning effects and between-days variability. To this end, we employ a double-blind placebo-controlled cross-over study and explore the effect of MPH on the ERP response of subjects with ADHD during a Go/No-Go cognitive task. Our ERP analysis revealed significant differences in ADHD subjects between the placebo and MPH conditions in the frontal-parietal region at 250ms-400ms post stimulus (P3). Additionally, a decrease in the late 650ms-800ms ERP component (LC) is observed in frontal electrodes of ADHD subjects compared to controls. The standard deviation of response time of ADHD subjects was significantly smaller in the MPH condition compared to placebo and correlated with the increased P3 ERP response in the frontoparietal electrodes. We suggest that mental fatigue plays a role in the decrease of the P3 response in the placebo condition compared to pre-placebo, a phenomenon that is significant in ADHD subjects but not in controls, and which is interestingly rectified by MPH., Competing Interests: The authors have declared that no competing interests exist.

The combination of Transcranial Magnetic Stimulation (TMS) with Electroencephalography (EEG) exposes the brain's global response to localized and abrupt stimulations. However, large electric artifacts are induced in the EEG by the TMS, obscuring crucial stages of the brain's response. Artifact removal is commonly performed by data processing techniques. However, an experimentally verified physical model for the origin and structure of the TMS-induced discharge artifacts, by which these methods can be justified or evaluated, is still lacking. We re-examine the known contribution of the skin in creating the artifacts, and outline a detailed model for the relaxation of the charge accumulated at the electrode-gel-skin interface due to the TMS pulse. We then experimentally validate implications set forth by the model. We find that the artifacts decay like a power law in time rather than the commonly assumed exponential. In fact, the skin creates a power-law decay of order 1 at each electrode, which is turned into a power law of order 2 by the reference electrode. We suggest an artifact removal method based on the model which can be applied from times after the pulse as short as 2 milliseconds onwards to expose the full EEG from the brain. The method can separate the capacitive discharge artifacts from those resulting from cranial muscle activation, demonstrating that the capacitive effect dominates at short times. Overall, our insight into the physical process allows us to accurately access TMS-evoked EEG responses that directly follow the TMS pulse, possibly opening new opportunities in TMS-EEG research., Competing Interests: The authors have declared that no competing interests exist.

Background: Alterations in the dynamic coordination of widespread brain networks are proposed to underlie cognitive symptoms of schizophrenia. However, there is limited understanding of the temporal evolution of these networks and how they relate to cognitive impairment. The current study was designed to explore dynamic patterns of network connectivity underlying cognitive features of schizophrenia., Methods: In total, 21 inpatients with schizophrenia and 28 healthy control participants completed a cognitive task while electroencephalography data were simultaneously acquired. For each participant, Pearson cross-correlation was applied to electroencephalography data to construct correlation matrices that represent the static network (averaged over 1200 ms) and dynamic network (1200 ms divided into four windows of 300 ms) in response to cognitive stimuli. Global and regional network measures were extracted for comparison between groups., Results: Dynamic network analysis identified increased global efficiency; decreased clustering (globally and locally); reduced strength (weighted connectivity) around the frontal, parietal, and sensory-motor areas; and increased strength around the occipital lobes (a peripheral hub) in patients with schizophrenia. Regional network measures also correlated with clinical features of schizophrenia. Network differences were prominent 900 ms following the cognitive stimuli before returning to levels comparable to those of healthy control participants., Conclusions: Patients with schizophrenia exhibited altered dynamic patterns of network connectivity across both global and regional measures. These network differences were time sensitive and may reflect abnormalities in the flexibility of the network that underlies aspects of cognitive function. Further research into network dynamics is critical to better understanding cognitive features of schizophrenia and identification of network biomarkers to improve diagnosis and treatment models., (Copyright © 2017 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result of chemical inputs to its synapses. However, neurons can also be excited by an imposed electric field. In particular, recent clinical applications activate neurons by creating an electric field externally. It is therefore of interest to investigate how the neuron responds to the external field and what causes the action potential. Fortunately, precise and controlled application of an external electric field is possible for embryonic neuronal cells that are excised, dissociated and grown in cultures. This allows the investigation of these questions in a highly reproducible system. In this paper some of the techniques used for controlled application of external electric field on neuronal cultures are reviewed. The networks can be either one dimensional, i.e. patterned in linear forms or allowed to grow on the whole plane of the substrate, and thus two dimensional. Furthermore, the excitation can be created by the direct application of electric field via electrodes immersed in the fluid (bath electrodes) or by inducing the electric field using the remote creation of magnetic pulses.

Background: Down syndrome incidence in humans increases dramatically with maternal age. This is mainly the result of increased meiotic errors, but factors such as differences in abortion rate may play a role as well. Since the meiotic error rate increases almost exponentially after a certain age, its contribution to the overall incidence aneuploidy may mask the contribution of other processes., Results: To focus on such selection mechanisms we investigated transmission in trisomic females, using data from mouse models and from Down syndrome humans. In trisomic females the a-priori probability for trisomy is independent of meiotic errors and thus approximately constant in the early embryo. Despite this, the rate of transmission of the extra chromosome decreases with age in females of the Ts65Dn and, as we show, for the Tc1 mouse models for Down syndrome. Evaluating progeny of 73 Tc1 births and 112 Ts65Dn births from females aged 130 days to 250 days old showed that both models exhibit a 3-fold reduction of the probability to transmit the trisomy with increased maternal ageing. This is concurrent with a 2-fold reduction of litter size with maternal ageing. Furthermore, analysis of previously reported 30 births in Down syndrome women shows a similar tendency with an almost three fold reduction in the probability to have a Down syndrome child between a 20 and 30 years old Down syndrome woman., Conclusions: In the two types of mice models for Down syndrome that were used for this study, and in human Down syndrome, older females have significantly lower probability to transmit the trisomy to the offspring. Our findings, taken together with previous reports of decreased supportive environment of the older uterus, add support to the notion that an older uterus negatively selects the less fit trisomic embryos.

Oscillatory activity is widespread in dynamic neuronal networks. The main paradigm for the origin of periodicity consists of specialized pacemaking elements that synchronize and drive the rest of the network; however, other models exist. Here, we studied the spontaneous emergence of synchronized periodic bursting in a network of cultured dissociated neurons from rat hippocampus and cortex. Surprisingly, about 60% of all active neurons were self-sustained oscillators when disconnected, each with its own natural frequency. The individual neuron's tendency to oscillate and the corresponding oscillation frequency are controlled by its excitability. The single neuron intrinsic oscillations were blocked by riluzole, and are thus dependent on persistent sodium leak currents. Upon a gradual retrieval of connectivity, the synchrony evolves: Loose synchrony appears already at weak connectivity, with the oscillators converging to one common oscillation frequency, yet shifted in phase across the population. Further strengthening of the connectivity causes a reduction in the mean phase shifts until zero-lag is achieved, manifested by synchronous periodic network bursts. Interestingly, the frequency of network bursting matches the average of the intrinsic frequencies. Overall, the network behaves like other universal systems, where order emerges spontaneously by entrainment of independent rhythmic units. Although simplified with respect to circuitry in the brain, our results attribute a basic functional role for intrinsic single neuron excitability mechanisms in driving the network's activity and dynamics, contributing to our understanding of developing neural circuits.

Down syndrome (DS) mouse models exhibit cognitive deficits, and are used for studying the neuronal basis of DS pathology. To understand the differences in the physiology of DS model neurons, we used dissociated neuronal cultures from the hippocampi of Ts65Dn and Tc1 DS mice. Imaging of [Ca(2+)]i and whole cell patch clamp recordings were used to analyze network activity and single neuron properties, respectively. We found a decrease of ~ 30% in both fast (A-type) and slow (delayed rectifier) outward potassium currents. Depolarization of Ts65Dn and Tc1 cells produced fewer spikes than diploid cells. Their network bursts were smaller and slower than diploids, displaying a 40% reduction in Δf / f0 of the calcium signals, and a 30% reduction in propagation velocity. Additionally, Ts65Dn and Tc1 neurons exhibited changes in the action potential shape compared to diploid neurons, with an increase in the amplitude of the action potential, a lower threshold for spiking, and a sharp decrease of about 65% in the after-hyperpolarization amplitude. Numerical simulations reproduced the DS measured phenotype by variations in the conductance of the delayed rectifier and A-type, but necessitated also changes in inward rectifying and M-type potassium channels and in the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. We therefore conducted whole cell patch clamp measurements of M-type potassium currents, which showed a ~ 90% decrease in Ts65Dn neurons, while HCN measurements displayed an increase of ~ 65% in Ts65Dn cells. Quantitative real-time PCR analysis indicates overexpression of 40% of KCNJ15, an inward rectifying potassium channel, contributing to the increased inhibition. We thus find that changes in several types of potassium channels dominate the observed DS model phenotype.

Excitation of neurons by an externally induced electric field is a long standing question that has recently attracted attention due to its relevance in novel clinical intervention systems for the brain. Here we use patterned quasi one-dimensional neuronal cultures from rat hippocampus, exploiting the alignment of axons along the linear patterned culture to separate the contribution of dendrites to the excitation of the neuron from that of axons. Network disconnection by channel blockers, along with rotation of the electric field direction, allows the derivation of strength-duration (SD) curves that characterize the statistical ensemble of a population of cells. SD curves with the electric field aligned either parallel or perpendicular to the axons yield the chronaxie and rheobase of axons and dendrites respectively, and these differ considerably. Dendritic chronaxie is measured to be about 1 ms, while that of axons is on the order of 0.1 ms. Axons are thus more excitable at short time scales, but at longer time scales dendrites are more easily excited. We complement these studies with experiments on fully connected cultures. An explanation for the chronaxie of dendrites is found in the numerical simulations of passive, realistically structured dendritic trees under external stimulation. The much shorter chronaxie of axons is not captured in the passive model and may be related to active processes. The lower rheobase of dendrites at longer durations can improve brain stimulation protocols, since in the brain dendrites are less specifically oriented than axonal bundles, and the requirement for precise directional stimulation may be circumvented by using longer duration fields.

In this paper we report the combination of microfluidics, optogenetics and calcium imaging as a cheap and convenient platform to study synaptic communication between neuronal populations in vitro. We first show that Calcium Orange indicator is compatible in vitro with a commonly used Channelrhodopsine-2 (ChR2) variant, as standard calcium imaging conditions did not alter significantly the activity of transduced cultures of rodent primary neurons. A fast, robust and scalable process for micro-chip fabrication was developed in parallel to build micro-compartmented cultures. Coupling optical fibers to each micro-compartment allowed for the independent control of ChR2 activation in the different populations without crosstalk. By analyzing the post-stimuli activity across the different populations, we finally show how this platform can be used to evaluate quantitatively the effective connectivity between connected neuronal populations.

Transcranial Magnetic Stimulation (TMS) is a promising technology for both neurology and psychiatry. Positive treatment outcome has been reported, for instance in double blind, multi-center studies on depression. Nonetheless, the application of TMS towards studying and treating brain disorders is still limited by inter-subject variability and lack of model systems accessible to TMS. The latter are required to obtain a deeper understanding of the biophysical foundations of TMS so that the stimulus protocol can be optimized for maximal brain response, while inter-subject variability hinders precise and reliable delivery of stimuli across subjects. Recent studies showed that both of these limitations are in part due to the angular sensitivity of TMS. Thus, a technique that would eradicate the need for precise angular orientation of the coil would improve both the inter-subject reliability of TMS and its effectiveness in model systems. We show here how rotation of the stimulating field relieves the angular sensitivity of TMS and provides improvements in both issues. Field rotation is attained by superposing the fields of two coils positioned orthogonal to each other and operated with a relative phase shift in time. Rotating field TMS (rfTMS) efficiently stimulates both cultured hippocampal networks and rat motor cortex, two neuronal systems that are notoriously difficult to excite magnetically. This opens the possibility of pharmacological and invasive TMS experiments in these model systems. Application of rfTMS to human subjects overcomes the orientation dependence of standard TMS. Thus, rfTMS yields optimal targeting of brain regions where correct orientation cannot be determined (e.g., via motor feedback) and will enable stimulation in brain regions where a preferred axonal orientation does not exist.

Neurotrophins are known to promote synapse development as well as to regulate the efficacy of mature synapses. We have previously reported that in two-dimensional rat hippocampal cultures, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 significantly increase the number of excitatory input connections. Here we measure the effect of these neurotrophic agents on propagating fronts that arise spontaneously in quasi-one-dimensional rat hippocampal cultures. We observe that chronic treatment with BDNF increased the velocity of the propagation front by about 30%. This change is attributed to an increase in the excitatory input connectivity. We analyze the experiment using the Feinerman-Golomb/Ermentrout-Jacobi/Moses-Osan model for the propagation of fronts in a one-dimensional neuronal network with synaptic delay and introduce the synaptic connection probability between adjacent neurons as a new parameter of the model. We conclude that BDNF increases the number of excitatory connections by favoring the probability to form connections between neurons, but without significantly modifying the range of the connections (connectivity footprint).

We present a theoretical framework using quorum percolation for describing the initiation of activity in a neural culture. The cultures are modeled as random graphs, whose nodes are excitatory neurons with k(in) inputs and k(out) outputs, and whose input degrees k(in) = k obey given distribution functions p(k). We examine the firing activity of the population of neurons according to their input degree (k) classes and calculate for each class its firing probability Φ(k)(t) as a function of t. The probability of a node to fire is found to be determined by its in-degree k, and the first-to-fire neurons are those that have a high k. A small minority of high-k-classes may be called "Leaders," as they form an interconnected sub-network that consistently fires much before the rest of the culture. Once initiated, the activity spreads from the Leaders to the less connected majority of the culture. We then use the distribution of in-degree of the Leaders to study the growth rate of the number of neurons active in a burst, which was experimentally measured to be initially exponential. We find that this kind of growth rate is best described by a population that has an in-degree distribution that is a Gaussian centered around k = 75 with width σ = 31 for the majority of the neurons, but also has a power law tail with exponent -2 for 10% of the population. Neurons in the tail may have as many as k = 4,700 inputs. We explore and discuss the correspondence between the degree distribution and a dynamic neuronal threshold, showing that from the functional point of view, structure and elementary dynamics are interchangeable. We discuss possible geometric origins of this distribution, and comment on the importance of size, or of having a large number of neurons, in the culture.

Objective: The combination of transcranial magnetic stimulation (TMS) and electroencephalography (EEG) has been hampered by the large artifact that the TMS generates in the EEG. Using TMS with EEG necessitates a sophisticated artifact-resistant EEG system that can acquire reliable signals in the crucial several tens of milliseconds immediately following the TMS pulse. Here, we demonstrate the use of a novel artifact removal algorithm together with a 24-bit EEG system to achieve similar recordings as those obtained with the dedicated TMS-compatible EEG system., Methods: This setup was used to compare TMS-evoked responses between a group of healthy controls and a group of patients with schizophrenia, a condition in which effective neural connectivity is thought to be compromised., Results: We observe differences in TMS-evoked responses between the two groups, similar to those recently reported in a study that used a dedicated TMS-compatible EEG system., Conclusions: The standard 24-bit EEG system combined with an artifact removal algorithm produces results similar to the dedicated TMS-compatible system., Significance: This paves the way for more researchers and clinicians to use TMS-evoked responses for research and diagnosis of a wide spectrum of disorders.

The neurotrophic factors brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) have been shown to promote excitatory and inhibitory synapse development. However, a quantitative analysis of their influence on connectivity has proven in general difficult to achieve. In this work we use a novel experimental approach based on percolation concepts that provides a quantification of the average number of connections per neuron. In combination with electrophysiological measurements, we characterize the changes in network connectivity induced by BDNF and NT-3 in rat hippocampal cultures. We show that, on the one hand, BDNF and NT-3 accelerate the maturation of connectivity in the network by about 17 h. On the other hand, BDNF and NT-3 increase the number of excitatory input connections by a factor of about two, but without modifying the number of inhibitory input connections. This scenario of a dominant effect on the excitation is supported by the analysis of spontaneous population bursts in cultures treated with either BDNF or NT-3, which show burst amplitudes that are insensitive to the blockade of inhibition. A leaky integrate-and-fire model reproduces the experimental results well.

We introduce an approach for the quantitative assessment of the connectivity in neuronal cultures, based on the statistical mechanics of percolation on a graph. This allows us to monitor the development of the culture and to see the emergence of connectivity in the network. The culture becomes fully connected at a time equivalent to the expected time of birth. The spontaneous bursting activity that characterizes cultures develops in parallel with the connectivity. The average number of inputs per neuron can be quantitatively determined in units of m(0), the number of activated inputs needed to excite the neuron. For m(0) approximately 15 we find that hippocampal neurons have on average approximately 60-120 inputs, whereas cortical neurons have approximately 75-150, depending on neuronal density. The ratio of excitatory to inhibitory neurons is determined by using the GABA(A) antagonist bicuculine. This ratio changes during development and reaches the final value at day 7-8, coinciding with the expected time of the GABA switch. For hippocampal cultures the inhibitory cells comprise approximately 30% of the neurons in the culture whereas for cortical cultures they are approximately 20%. Such detailed global information on the connectivity of networks in neuronal cultures is at present inaccessible by any electrophysiological or other technique.

Transcranial magnetic stimulation is a remarkable tool for neuroscience research, with a multitude of diagnostic and therapeutic applications. Surprisingly, application of the same magnetic stimulation directly to neurons that are dissected from the brain and grown in vitro was not reported to activate them to date. Here we report that central nervous system neurons patterned on large enough one-dimensional rings can be magnetically stimulated in vitro. In contrast, two-dimensional cultures with comparable size do not respond to excitation. This happens because the one-dimensional pattern enforces an ordering of the axons along the ring, which is designed to follow the lines of the magnetically induced electric field. A small group of sensitive (i.e., initiating) neurons respond even when the network is disconnected, and are presumed to excite the entire network when it is connected. This implies that morphological and electrophysiological properties of single neurons are crucial for magnetic stimulation. We conjecture that the existence of a select group of neurons with higher sensitivity may occur in the brain in vivo as well, with consequences for transcranial magnetic stimulation.

Transcranial Magnetic Stimulation (TMS) is rapidly gaining acceptance as a non-invasive probe into brain functionality. We utilize TMS to study the connectivity of a simple motor network in patients of schizophrenia (N=19), and in healthy control subjects (N=9). TMS was used in an externally paced finger tapping task, perturbing the internal network oscillations invoked by the finger motion as it keeps pace with a metronome. TMS perturbations were synchronized to the metronome and applied to the network at the level of the primary motor cortex (M1). Contrary to initial expectations, TMS did not affect the sensorimotor synchronization of subjects with schizophrenia or their tapping accuracy. TMS did cause extreme deviations in the finger's trajectory, and altered the timing perceptions of subjects with schizophrenia. Additionally, it invoked high-level deficiencies related to attention and volition in the form of lapses, implying that the connectivity between modules in the brain that underlie motor control, sensorimotor synchronization, timing perception and awareness of action, can be disrupted by TMS in subjects with schizophrenia, but not in healthy subjects. The ability to disrupt high level network functions with perturbations to the lower level of M1 supports models describing deficits in connectivity of distributed networks in the brains of schizophrenia patients. It also demonstrates the use of TMS to probe connectivity between components of such networks.

We investigate the propagation of neural activity along one-dimensional rat hippocampal cultures patterned in lines over multielectrode arrays. Activity occurs spontaneously or is evoked by local electrical or chemical stimuli, with different resulting propagation velocities and firing rate amplitudes. A variability of an order of magnitude in velocity and amplitude is observed in spontaneous activity. A linear relation between velocity and amplitude is identified. We define a measure for neuron activation synchrony and find that it correlates with front velocity and is higher for electrically evoked fronts. We present a model that explains the linear relation between amplitude and velocity, which highlights the role of synchrony. The relation to current models for signal propagation in neural media is discussed.

We study the interplay between motor programs and their timing in the brain by using precise pulses of transcranial magnetic stimulation (TMS) applied to the primary motor cortex. The movement of the finger performing a tapping task is periodically perturbed in synchronization with a metronome. TMS perturbation can profoundly affect both the finger trajectory and its kinematics, but the tapping accuracy itself is surprisingly not affected. The motion of the finger during the TMS perturbation can be categorized into two abnormal behaviors that subjects were unaware of: a doubling of the frequency of the tap and a stalling of the finger for half the period. More stalls occurred as the tapping frequency increased. In addition, an enhancement of the velocity of the finger on its way up was observed. We conclude that the timing process involved in controlling the tapping movement is separate from the motor processes in charge of execution of the motor commands. We speculate that the TMS is causing a release of the motor plan ahead of time into activation mode. The observed doubles and stalls are then the result of an indirect interaction in the brain, making use of an existing motor plan to correct the preactivation and obtain the temporal goal of keeping the beat.

Spontaneous activity is typical of in vitro neural networks, often in the form of large population bursts. The origins of this activity are attributed to intrinsically bursting neurons and to noisy backgrounds as well as to recurrent network connections. Spontaneous activity is often observed to emanate from localized sources or initiation zones, propagating from there to excite large populations of neurons. In this study, we use unidimensional cultures to overcome experimental difficulties in identifying initiation zones in vivo and in dissociated two-dimensional cultures. We found that spontaneous activity in these cultures is initiated exclusively in localized zones that are characterized by high neuronal density but also by recurrent and inhibitory network connections. We demonstrate that initiation zones compete in driving network activity in a winner-takes-most scenario.

We study living neural networks by measuring the neurons' response to a global electrical stimulation. Neural connectivity is lowered by reducing the synaptic strength, chemically blocking neurotransmitter receptors. We use a graph-theoretic approach to show that the connectivity undergoes a percolation transition. This occurs as the giant component disintegrates, characterized by a power law with an exponent beta approximately or = 0.65. Beta is independent of the balance between excitatory and inhibitory neurons and indicates that the degree distribution is Gaussian rather than scale free.

The ability of synchronous population activity in layered networks to transmit a rate code is a focus of recent debate. We investigate these issues using a patterned unidimensional hippocampal culture. The network exhibits population bursts that travel its full length, with the advantage that signals propagate along a clearly defined path. The amplitudes of activity are measured using calcium imaging, a good approximate of population rate code, and the distortion of the signal as it travels is analyzed. We demonstrate that propagation along the line is precisely described by information theory as a chain of Gaussian communication channels. The balance of excitatory and inhibitory synapses is crucial for this transmission. However, amplitude information carried along this layered neuronal structure fails within 3 mm, approximately 10 mean axon lengths, and is limited by noise in the synaptic transmission. We conclude that rate codes cannot be reliably transmitted through long layered networks.

Dissociated neurons were cultured on lines of various lengths covered with adhesive material to obtain an experimental model system of linear signal transmission. The neuronal connectivity in the linear culture is characterized, and it is demonstrated that local spiking activity is relayed by synaptic transmission along the line of neurons to develop into a large-scale population burst. Formally, this can be treated as a one-dimensional information channel. Directional propagation of both spontaneous and stimulated bursts along the line, imaged with the calcium indicator Fluo-4, revealed the existence of two different propagation velocities. Initially, a small number of neighboring neurons fire, leading to a slow, small and presumably asynchronous wave of activity. The signal then spontaneously develops to encompass much larger and further populations, and is characterized by fast propagation of high-amplitude activity, which is presumed to be synchronous. These results are well described by an existing theoretical framework for propagation based on an integrate-and-fire model.

We study the dynamic network of e-mail traffic and find that it develops self-organized coherent structures similar to those appearing in many nonlinear dynamic systems. Such structures are uncovered by a general information theoretic approach to dynamic networks based on the analysis of synchronization among trios of users. In the e-mail network, coherent structures arise from temporal correlations when users act in a synchronized manner. These temporally linked structures turn out to be functional, goal-oriented aggregates that must react in real time to changing objectives and challenges (e.g., committees at a university). In contrast, static structures turn out to be related to organizational units (e.g., departments).

A capability for scanning electron microscopy of wet biological specimens is presented. A membrane that is transparent to electrons protects the fully hydrated sample from the vacuum. The result is a hybrid technique combining the ease of use and ability to see into cells of optical microscopy with the higher resolution of electron microscopy. The resolution of low-contrast materials is approximately 100 nm, whereas in high-contrast materials the resolution can reach 10 nm. Standard immunogold techniques and heavy-metal stains can be applied and viewed in the fluid to improve the contrast. Images present a striking combination of whole-cell morphology with a wealth of internal details. A possibility for direct inspection of tissue slices transpires, imaging only the external layer of cells. Simultaneous imaging with photons excited by the electrons incorporates data on material distribution, indicating a potential for multilabeling and specific scintillating markers.

A double pipette system for local, controlled drug infusion is presented. Two concentric pipettes can be manipulated separately and pressurized independently by a designated double holder. The inner pipette is loaded with the desirable solution (drug), and functions as a source, while the outer one is a sink. This gives a flow of the solution between the two pipettes that protrudes only a small distance into the surrounding fluid and does not diffuse away. Time resolution of the infusion is highly controllable, and oscillatory flow can be generated. Three implementations of the double pipette system are demonstrated. We show that local application of neurotransmitters in neuronal networks is an efficient way of stimulating activity in the network. We then present a wet micro lithography technique using topical application of proteins onto the substrate. Finally, we show that we can localize a given drug on a small targeted part of a cell.

A novel method for detection of noble-metal nanoparticles by their nonlinear optical properties is presented and applied for specific labeling of cellular organelles. When illuminated by laser light in resonance with their plasmon frequency these nanoparticles generate an enhanced multiphoton signal. This enhanced signal is measured to obtain a depth-resolved image in a laser scanning microscope setup. Plasmon-resonance images of both live and fixed cells, showing specific labeling of cellular organelles and membranes, either by two-photon autofluorescence or by third-harmonic generation, are presented.

We introduce a quantitative method that utilizes scanning electron microscopy for the analysis of protein chips (SEMPC). SEMPC is based upon counting target-coated gold particles interacting specifically with ligands or proteins arrayed on a derivative microscope glass slide by utilizing backscattering electron detection. As model systems, we quantified the interactions of biotin and streptavidin and of an antibody with its cognate hapten. Our method gives quantitative molecule-counting capabilities with an excellent signal-to-noise ratio and demonstrates a broad dynamic range while retaining easy sample preparation and realistic automation capability. Increased sensitivity and dynamic range are achieved in comparison to currently used array detection methods such as fluorescence, with no signal bleaching, affording high reproducibility and compatibility with miniaturization. Thus, our approach facilitates the determination of the absolute number of molecules bound to the chip rather than their relative amounts, as well as the use of smaller samples.

Beyond the information stored in pages of the World Wide Web, novel types of "meta-information" are created when pages connect to each other. Such meta-information is a collective effect of independent agents writing and linking pages, hidden from the casual user. Accessing it and understanding the interrelation between connectivity and content in the World Wide Web is a challenging problem [Botafogo, R. A. & Shneiderman, B. (1991) in Proceedings of Hypertext (Assoc. Comput. Mach., New York), pp. 63-77 and Albert, R. & Barabási, A.-L. (2002) Rev. Mod. Phys. 74, 47-97]. We demonstrate here how thematic relationships can be located precisely by looking only at the graph of hyperlinks, gleaning content and context from the Web without having to read what is in the pages. We begin by noting that reciprocal links (co-links) between pages signal a mutual recognition of authors and then focus on triangles containing such links, because triangles indicate a transitive relation. The importance of triangles is quantified by the clustering coefficient [Watts, D. J. & Strogatz, S. H. (1999) Nature (London) 393, 440-442], which we interpret as a curvature [Bridson, M. R. & Haefliger, A. (1999) Metric Spaces of Non-Positive Curvature (Springer, Berlin)]. This curvature defines a World Wide Web landscape whose connected regions of high curvature characterize a common topic. We show experimentally that reciprocity and curvature, when combined, accurately capture this meta-information for a wide variety of topics. As an example of future directions we analyze the neural network of Caenorhabditis elegans, using the same methods.