When Triggers Become Tigers: Taming the Autonomic Nervous System via Sensory Support System Modulation.

Personalized recovery technologies may enable individuals with Substance Use Disorder (SUD) to monitor and manage acute craving and drug use urges in ways that improve drug-seeking decisions in real-time. Direct and indirect regulation of the autonomic nervous system through sensory input monitoring and modulation may enhance control over behavioral decisions and prevent relapse. A personalized sensory support system that monitors neurophysiological reactivity and offers non-pharmacological point-in-time personalized digital interventions may increase awareness of and control over craving reactivity. It is critical to be able to detect these warning signs and intervene early and effectively. The use of wearable technologies that assess point-in-time neurophysiological escalation and shape behavioral response through personalized interventions could be transformative in allowing individuals to better manage their recovery as they transition out of institutions and move back into community settings.

Long-Term Depression Learning in Spinal Cord Networks.

Investigating learning in networks of spinal cord neurons can provide insight into the dynamics of connectivity in human spinal cords. It may also hold implications for developing neural prosthetics and neurocomputers. Culturing neural networks on microelectrode arrays (MEAs) allows for the repeated observation and stimulation of electrophysiological activity in vitro. Here we used MEAs to demonstrate learning in networks of spinal cord neurons. This was done by exposing E17 mouse spinal cord cultures to high frequency artificial spike trains, or tetanization. Unexpectedly, when comparing the networks' responses to low-frequency probing stimulations before and after tetanization, the cultures were found to demonstrate long-term depression (LTD). LTD was most significantly observed between 500-1000 ms after low-frequency probing. These results indicate that periodic high-frequency excitation of spinal cord networks can result in decreased synaptic efficacy.

Zebrafish larvae heartbeat detection from body deformation in low resolution and low frequency video.

Zebrafish (Danio rerio) is a powerful animal model used in many areas of genetics and disease research. Despite its advantages for cardiac research, the heartbeat pattern of zebrafish larvae under different stress conditions is not well documented quantitatively. Several effective automated heartbeat detection methods have been developed to reduce the workload for larva heartbeat analysis. However, most require complex experimental setups and necessitate direct observation of the larva heart. In this paper, we propose the Zebrafish Heart Rate Automatic Method (Z-HRAM), which detects and tracks the heartbeats of immobilized, ventrally positioned zebrafish larvae without direct larva heart observation. Z-HRAM tracks localized larva body deformation that is highly correlated with heart movement. Multiresolution dense optical flow-based motion tracking and principal component analysis are used to identify heartbeats. Here, we present results of Z-HRAM on estimating heart rate from video recordings of seizure-induced larvae, which were of low resolution (1024 × 760) and low frame rate (3 to 4 fps). Heartbeats detected from Z-HRAM were shown to correlate reliably with those determined through corresponding electrocardiogram and manual video inspection. We conclude that Z-HRAM is a robust, computationally efficient, and easily applicable tool for studying larva cardiac function in general laboratory conditions. Graphical abstract Flowchart of the automatic zebrafish heartbeat detection.

Attenuated Glial Reactivity on Topographically Functionalized Poly(3,4-Ethylenedioxythiophene):P-Toluene Sulfonate (PEDOT:PTS) Neuroelectrodes Fabricated by Microimprint Lithography.

Following implantation, neuroelectrode functionality is susceptible to deterioration via reactive host cell response and glial scar-induced encapsulation. Within the neuroengineering community, there is a consensus that the induction of selective adhesion and regulated cellular interaction at the tissue-electrode interface can significantly enhance device interfacing and functionality in vivo. In particular, topographical modification holds promise for the development of functionalized neural interfaces to mediate initial cell adhesion and the subsequent evolution of gliosis, minimizing the onset of a proinflammatory glial phenotype, to provide long-term stability. Herein, a low-temperature microimprint-lithography technique for the development of micro-topographically functionalized neuroelectrode interfaces in electrodeposited poly(3,4-ethylenedioxythiophene):p-toluene sulfonate (PEDOT:PTS) is described and assessed in vitro. Platinum (Pt) microelectrodes are subjected to electrodeposition of a PEDOT:PTS microcoating, which is subsequently topographically functionalized with an ordered array of micropits, inducing a significant reduction in electrode electrical impedance and an increase in charge storage capacity. Furthermore, topographically functionalized electrodes reduce the adhesion of reactive astrocytes in vitro, evident from morphological changes in cell area, focal adhesion formation, and the synthesis of proinflammatory cytokines and chemokine factors. This study contributes to the understanding of gliosis in complex primary mixed cell cultures, and describes the role of micro-topographically modified neural interfaces in the development of stable microelectrode interfaces.

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays.

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

In vivo Characterization of Amorphous Silicon Carbide As a Biomaterial for Chronic Neural Interfaces.

Implantable microelectrode arrays (MEAs) offer clinical promise for prosthetic devices by enabling restoration of communication and control of artificial limbs. While proof-of-concept recordings from MEAs have been promising, work in animal models demonstrates that the obtained signals degrade over time. Both material robustness and tissue response are acknowledged to have a role in device lifetime. Amorphous Silicon carbide (a-SiC), a robust material that is corrosion resistant, has emerged as an alternative encapsulation layer for implantable devices. We systematically examined the impact of a-SiC coating on Si probes by immunohistochemical characterization of key markers implicated in tissue-device response. After implantation, we performed device capture immunohistochemical labeling of neurons, astrocytes, and activated microglia/macrophages after 4 and 8 weeks of implantation. Neuron loss and microglia activation were similar between Si and a-SiC coated probes, while tissue implanted with a-SiC displayed a reduction in astrocytes adjacent to the probe. These results suggest that a-SiC has a similar biocompatibility profile as Si, and may be suitable for implantable MEA applications as a hermetic coating to prevent material degradation.

Synaptic depression depends on charge delivered to network.

In vitro neuronal networks cultured on microelectrode arrays enable the study of network electrophysiology on a fundamental level. Neuronal response to electrical stimulation is an area of interest at the laboratory bench and in the clinic, given its wide application for remedying neurological disorders. Here we investigated the change in cortical network response over time to varied amounts of charge used for stimulation, which may lead to a phenomenon known as selective adaptation. There is a charge threshold that invokes a reverberating network response; when stimulating at 900 mV, five stimulation electrodes were required to elicit a response across the entire network. Stimulating with more charge leads to greater synaptic depression over time when constant periodic stimulation is applied. Stimulating with 5 electrodes led to a decrease in network response to stimulation, whereas stimulating with 12 electrodes led to an extinction of network response. The previously hypothesized selective adaptation mechanism was not observed, implying that our random cortical assemblies have homogeneous excitatory and inhibitory subnetworks.

Chronic intracortical neural recordings using microelectrode arrays coated with PEDOT-TFB.

Microelectrode arrays have been extensively utilized to record extracellular neuronal activity for brain-machine interface applications. Modifying the microelectrodes with conductive polymers such as poly(3,4-ethylenedioxythiophene) (PEDOT) has been reported to be advantageous because it increases the effective surface area of the microelectrodes, thereby decreasing impedance and enhancing charge transfer capacity. However, the long term stability and integrity of such coatings for chronic recordings remains unclear. Previously, our group has demonstrated that use of the smaller counter ion tetrafluoroborate (TFB) during electrodeposition increased the stability of the PEDOT coatings in vitro compared to the commonly used counter ion poly(styrenesulfonate) (PSS). In the current work, we examined the long-term in vivo performance of PEDOT-TFB coated microelectrodes. To do so, we selectively modified half of the microelectrodes on NeuroNexus single shank probes with PEDOT-TFB while the other half of the microelectrodes were modified with gold as a control. The modified probes were then implanted into the primary motor cortex of rats. Single unit recordings were observed on both PEDOT-TFB and gold control microelectrodes for more than 12 weeks. Compared to the gold-coated microelectrodes, the PEDOT-TFB coated microelectrodes exhibited an overall significantly lower impedance and higher number of units per microelectrode specifically for the first four weeks. The majority of PEDOT-TFB microelectrodes with activity had an impedance magnitude lower than 400 kΩ at 1 kHz. Our equivalent circuit modeling of the impedance data suggests stability in the polymer-related parameters for the duration of the study. In addition, when comparing PEDOT-TFB microelectrodes with and without long-term activity, we observed a distinction in certain circuit parameters for these microelectrodes derived from equivalent circuit modeling prior to implantation. This observation may prove useful in qualifying PEDOT-TFB microelectrodes with a greater likelihood of registering long-term activity. Overall, our findings confirm that PEDOT-TFB is a chronically stable coating for microelectrodes to enable neural recording. STATEMENT OF SIGNIFICANCE: Microelectrode arrays have been extensively utilized to record extracellular neuronal activity for brain-machine interface applications. Poly(3,4-ethylenedioxythiophene) (PEDOT) has gained interest because of its unique electrochemical characteristics and its excellent intrinsic electrical conductivity. However, the long-term stability of the PEDOT film, especially for chronic neural applications, is unclear. In this manuscript, we report for the first time the use of highly stable PEDOT doped with tetrafluoroborate (TFB) for long-term neural recordings. We show that PEDOT-TFB coated microelectrodes on average register more units compared to control gold microelectrodes for at least first four weeks post implantation. We collected the in vivo impedance data over a wide frequency spectrum and developed an equivalent circuit model which helped us determine certain parameters to distinguish between PEDOT-TFB microelectrodes with and without long-term activity. Our findings suggest that PEDOT-TFB is a chronically stable coating for neural recording microelectrodes. As such, PEDOT-TFB could facilitate chronic recordings with ultra-small and high-density neural arrays.

Time-Dependent Increase in Network Response to Stimulation.

In vitro neuronal cultures have become a popular method with which to probe network-level neuronal dynamics and phenomena in controlled laboratory settings. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Here we demonstrate the effects of a high frequency electrical stimulation signal in training cultured networks of cortical neurons. Networks receiving this training signal displayed a time-dependent increase in the response to a low frequency probing stimulation, particularly in the time window of 20-50 ms after stimulation. This increase was found to be statistically significant as compared to control networks that did not receive training. The timing of this increase suggests potentiation of synaptic mechanisms. To further investigate this possibility, we leveraged the powerful Cox statistical connectivity method as previously investigated by our group. This method was used to identify and track changes in network connectivity strength.

Amyloid beta modulation of neuronal network activity in vitro.

In vitro assays offer a means of screening potential therapeutics and accelerating the drug development process. Here, we utilized neuronal cultures on planar microelectrode arrays (MEA) as a functional assay to assess the neurotoxicity of amyloid-ß 1-42 (Aß42), a biomolecule implicated in the Alzheimer׳s disease (AD). In this approach, neurons harvested from embryonic mice were seeded on the substrate-integrated microelectrode arrays. The cultured neurons form a spontaneously active network, and the spiking activity as a functional endpoint could be detected via the MEA. Aß42 oligomer, but not monomer, significantly reduced network spike rate. In addition, we demonstrated that the ionotropic glutamate receptors, NMDA and AMPA/kainate, play a role in the effects of Aß42 on neuronal activity in vitro. To examine the utility of the MEA-based assay for AD drug discovery, we tested two model therapeutics for AD, methylene blue (MB) and memantine. Our results show an almost full recovery in the activity within 24h after administration of Aß42 in the cultures pre-treated with either MB or memantine. Our findings suggest that cultured neuronal networks may be a useful platform in screening potential therapeutics for Aß induced changes in neurological function.

Access resistance of stimulation electrodes as a function of electrode proximity to the retina.

OBJECTIVE: Epiretinal prostheses seek to effectively stimulate the retina by positioning electrode arrays close to its surface so current pulses generate narrow retinal electric fields. Our objective was to evaluate the use of the electrical impedance of insulated platinum electrodes as a measure of the proximity of insulated platinum electrodes to the inner surface of the retina. APPROACH: We examined the impedance of platinum disk electrodes, 0.25 mm in diameter, insulated with two widths (0.8 and 1.6 mm outer diameter) of transparent fluoropolymer in a rabbit retinal eyecup preparation. Optical coherence tomography measured the electrode's proximity to the retinal surface which was correlated with changes in the voltage waveform at the electrode. Electrode impedance changes during retinal deformation were also studied. MAIN RESULTS: When the 1.6 mm diameter insulated electrodes advanced towards the retinal surface from 1000 µm, their voltage step at current pulse onset increased, reflecting an access resistance increase of 3880 ± 630 Ω, with the 50% midpoint averaging 30 µm, while thin 0.8 mm insulated electrode advancement showed an access resistance increase 50% midpoint averaging 16 µm. Using impedance spectroscopy, electrode-retina proximity differences were seen in the 1.6 mm insulated electrode impedance modulus between 1 and 100 kHz and the waveform phase angle at 0.3-10 kHz, while thin 0.8 mm insulated electrode advancement produced smaller impedance modulus changes with retinal proximity between 3 and 100 kHz. These impedance changes with retinal proximity may reflect different sized zones of eye wall being coupled in series with the insulated platinum electrode. SIGNIFICANCE: The proximity of stimulus electrodes to neural tissue in fluid-filled spaces can be estimated from access resistance changes in the stimulus pulse waveform. Because many prosthetic devices allow back telemetry communication of the stimulus electrode waveform, it is possible these series resistance increases observed with retinal proximity could be used as a metric of stimulus electrode placement.

A importância da placa estabilizadora no tratamento do bruxismo e cefaleia matinal - relato de caso / The importance of the stabilization splint in the treatment of bruxism and morning headaches - case report

Este estudo teve como objetivo relatar um caso de bruxismo e cefaleia matinal, o qual foi tratado com placa estabilizadora para impedir o desgaste dentário e reduzir a dor orofacial de origem muscular. A paciente procurou a disciplina de Clínica Integrada com queixa de dores de cabeça e na face frequentes logo ao acordar. Após anamnese, exame físico intraoral e palpação muscular, a paciente foi diagnosticada com cefaleia matutina e dor facial de origem muscular relacionadas com bruxismo noturno (atividade parafuncional). Como tratamento, foi proposta a confecção de uma placa estabilizadora para uso noturno. No primeiro retorno após uma semana, a paciente relatou melhora na cefaleia matutina, e após um mês, os sintomas praticamente desapareceram. A mesma foi instruída a continuar usando a placa para dormir com o objetivo de proteger seus dentes de um desgaste maior, mesmo que as dores cessassem ompletamente. Recomendou-se retorno após 3 meses para acompanhamento dos sinais e sintomas. Apesar da inexistência de tratamento curativo, o tratamento com placa estabilizadora apresenta-se como uma importante opção terapêutica no controle da dor muscular e prevenção dos desgastes dentários.

This study aimed to report a clinical case of bruxism and morning headaches in which the patient was treated with a stabilization splint to prevent tooth wear and reduce muscular orofacial pain. The patient complained of frequent headaches and facial pain upon waking. After anamnesis, intraoral examination, and muscle palpation, the patient was diagnosed with morning headaches and muscular orofacial pain caused by nocturnal bruxism (parafunctional activity). The patient was treated with a stabilization splint for nocturnal use. One week after the insertion of the splint, the patient reported relief in morning headache, and after a month the symptoms had almost completely disappeared. The patient was instructed to keep using the splint to sleep in order to prevent tooth wear, even if the pain ceased completely. An appointment at 3 months post-insertion was scheduled for follow-up of signs and symptoms. Despite the lack of curative treatment for sleeping bruxism, the stabilization splint therapy is an important therapeutic option for pain control and prevention of tooth wear

Improving the performance of poly(3,4-ethylenedioxythiophene) for brain-machine interface applications.

Conducting polymers, especially poly(3,4-ethylenedioxythiophene) (PEDOT) based materials, are important for developing highly sensitive and microscale neural probes. In the present work, we show that the conductivity and stability of PEDOT can be significantly increased by switching the widely used counter anion poly(styrenesulfonate) (PSS) to the smaller tetrafluoroborate (TFB) anion during the electrodeposition of the polymer. Time-dependent impedance measurements of polymer modified implantable microwires were conducted in physiological buffer solutions under accelerated aging conditions and the relative stability of PEDOT:PSS and PEDOT:TFB modified microwires was compared over time. This study was also extended to carbon nanotube (CNT) incorporated PEDOT:PSS which, according to some reports, is claimed to enhance the stability and electrical performance of the polymer. However, no noticeable difference was observed between PEDOT:PSS and CNT:PEDOT:PSS in our measurements. At the biologically relevant frequency of 1kHz, PEDOT:TFB modified microwires exhibit approximately one order of magnitude higher conductivity and demonstrate enhanced stability over both PEDOT:PSS and CNT:PEDOT:PSS modified microwires. In addition, PEDOT:TFB is not neurotoxic and we show the proof-of-concept for both in vitro and in vivo neuronal recordings using PEDOT:TFB modified microelectrode arrays and chronic electrodes, respectively. Our findings suggest that PEDOT:TFB is a promising conductive polymer coating for the recording of neural activities.

Use of cortical neuronal networks for in vitro material biocompatibility testing.

Neural interfaces aim to restore neurological function lost during disease or injury. Novel implantable neural interfaces increasingly capitalize on novel materials to achieve microscale coupling with the nervous system. Like any biomedical device, neural interfaces should consist of materials that exhibit biocompatibility in accordance with the international standard ISO10993-5, which describes in vitro testing involving fibroblasts where cytotoxicity serves as the main endpoint. In the present study, we examine the utility of living neuronal networks as functional assays for in vitro material biocompatibility, particularly for materials that comprise implantable neural interfaces. Embryonic mouse cortical tissue was cultured to form functional networks where spontaneous action potentials, or spikes, can be monitored non-invasively using a substrate-integrated microelectrode array. Taking advantage of such a platform, we exposed established positive and negative control materials to the neuronal networks in a consistent method with ISO 10993-5 guidance. Exposure to the negative controls, gold and polyethylene, did not significantly change the neuronal activity whereas the positive controls, copper and polyvinyl chloride (PVC), resulted in reduction of network spike rate. We also compared the functional assay with an established cytotoxicity measure using L929 fibroblast cells. Our findings indicate that neuronal networks exhibit enhanced sensitivity to positive control materials. In addition, we assessed functional neurotoxicity of tungsten, a common microelectrode material, and two conducting polymer formulations that have been used to modify microelectrode properties for in vivo recording and stimulation. These data suggest that cultured neuronal networks are a useful platform for evaluating the functional toxicity of materials intended for implantation in the nervous system.

Lifetime assessment of atomic-layer-deposited Al2O3-Parylene C bilayer coating for neural interfaces using accelerated age testing and electrochemical characterization.

The lifetime and stability of insulation are critical features for the reliable operation of an implantable neural interface device. A critical factor for an implanted insulation's performance is its barrier properties that limit access of biological fluids to the underlying device or metal electrode. Parylene C is a material that has been used in FDA-approved implantable devices. Considered a biocompatible polymer with barrier properties, it has been used as a substrate, insulation or an encapsulation for neural implant technology. Recently, it has been suggested that a bilayer coating of Parylene C on top of atomic-layer-deposited Al2O3 would provide enhanced barrier properties. Here we report a comprehensive study to examine the mean time to failure of Parylene C and Al2O3-Parylene C coated devices using accelerated lifetime testing. Samples were tested at 60°C for up to 3 months while performing electrochemical measurements to characterize the integrity of the insulation. The mean time to failure for Al2O3-Parylene C was 4.6 times longer than Parylene C coated samples. In addition, based on modeling of the data using electrical circuit equivalents, we show here that there are two main modes of failure. Our results suggest that failure of the insulating layer is due to pore formation or blistering as well as thinning of the coating over time. The enhanced barrier properties of the bilayer Al2O3-Parylene C over Parylene C makes it a promising candidate as an encapsulating neural interface.

Dynamic steering of in vitro cortical neurons using field stimulation.

Neurological disorders are often characterized by abnormal neuronal activity. In the case of epilepsy, this can manifest itself in the form of uncontrolled synchronous activity often in the form of bursting. Pattern steering is the ability to apply stimulation to a network that effectively changes its dynamical firing pattern. In an epileptic network, the stimulation would be used to move the seizing network from its abnormal state to a normal state. This idea is explored here in cultured networks of cortical neurons plated on microelectrode arrays. Stimulation was applied to the bath resulting in an electric field generated throughout the network. This field was verified as sub-threshold in strength using a finite element model simulation. Stimulated networks showed a significant suppression in the number of bursts and increase in the interburst interval as compared to control networks. This observed burst suppression suggests that the sub-threshold stimulating field moved networks from a state of high frequency bursting to a state of low frequency bursting.

Real-time tracking of neuronal network structure using data assimilation.

A nonlinear data assimilation technique is applied to determine and track effective connections between ensembles of cultured spinal cord neurons measured with multielectrode arrays. The method is statistical, depending only on confidence intervals, and requiring no form of arbitrary thresholding. In addition, the method updates connection strengths sequentially, enabling real-time tracking of nonstationary networks. The ensemble Kalman filter is used with a generic spiking neuron model to estimate connection strengths as well as other system parameters to deal with model mismatch. The method is validated on noisy synthetic data from Hodgkin-Huxley model neurons before being used to find network connections in the neural culture recordings.

Voice controlled wheelchairs: fine control by humming.

People without disabilities seamlessly control devices with their hands. Interestingly, their hands can perform coarse and fine control. Implementing smooth control for computerized systems is not straightforward and most of the time it is not intuitive either. Here we offer a solution to that problem: smooth control through humming. Voice commands have become ubiquitous in modern technology. Speech-to-text applications abound. Smooth control, on the other hand, has not been tackled yet. Here we design and implement a humming control technique, and demonstrate a hardware implementation with a powered wheelchair. Once actuated, the speed with which the chair moves will depend on the subtle variation on the fundamental frequency of the user's humming, acquired through an accelerometer measuring vocal cord vibration. We also discuss two signal processing techniques that handle commonly encountered issues when trying to resolve frequencies in real time data. The hardware implementation shows performance of 80% and higher in speech recognition for signal-to-noise ratio (SNR) higher than 8dB and 100% in smooth control and frequency detection for all tested SNRs. We also discuss potential applications of smooth humming control to other assistive technology.

Differential responses to ω-agatoxin IVA in murine frontal cortex and spinal cord derived neuronal networks.

ω-Agatoxin-IVA is a well known P/Q-type Ca(2+) channel blocker and has been shown to affect presynaptic Ca(2+) currents as well postsynaptic potentials. P/Q-type voltage gated Ca(2+) channels play a vital role in presynaptic neurotransmitter release and thus play a role in action potential generation. Monitoring spontaneous activity of neuronal networks on microelectrode arrays (MEAs) provides an important tool for examining this neurotoxin. Changes in extracellular action potentials are readily observed and are dependent on synaptic function. Given the efficacy of murine frontal cortex and spinal cord networks to detect neuroactive substances, we investigated the effects of ω-agatoxin on spontaneous action potential firing within these networks. We found that networks derived from spinal cord are more sensitive to the toxin than those from frontal cortex; a concentration of only 10nM produced statistically significant effects on activity from spinal cord networks whereas 50 nM was required to alter activity in frontal cortex networks. Furthermore, the effects of the toxin on frontal cortex are more complex as unit specific responses were observed. These manifested as either a decrease or increase in action potential firing rate which could be statistically separated as unique clusters. Administration of bicuculline, a GABAA inhibitor, isolated a single response to ω-agatoxin, which was characterized by a reduction in network activity. These data support the notion that the two clusters detected with ω-agatoxin exposure represent differential responses from excitatory and inhibitory neuronal populations.

Detecting connectivity changes in neuronal networks.

We develop a method from semiparametric statistics (Cox, 1972) for the purpose of tracking links and connection strengths over time in a neuronal network from spike train data. We consider application of the method as implemented in Masud and Borisyuk (2011), and evaluate its use on data generated independently of the Cox model hypothesis, in particular from a spiking model of Izhikevich in four different dynamical regimes. Then, we show how the Cox method can be used to determine statistically significant changes in network connectivity over time. Our methodology is demonstrated using spike trains from multi-electrode array measurements of networks of cultured mammalian spinal cord cells.