High Frequency Bursts Research Articles

Intrinsic and Synaptic Contributions to Repetitive Spiking in Dentate Granule Cells.

Repetitive firing of granule cells (GCs) in the dentate gyrus (DG) facilitates synaptic transmission to the CA3 region. This facilitation can gate and amplify the flow of information through the hippocampus. High-frequency bursts in the DG are linked to behavior and plasticity, but GCs do not readily burst. Under normal conditions, a single shock to the perforant path in a hippocampal slice typically drives a GC to fire a single spike, and only occasionally more than one spike is seen. Repetitive spiking in GCs is not robust, and the mechanisms are poorly understood. Here, we used a hybrid genetically encoded voltage sensor to image voltage changes evoked by cortical inputs in many mature GCs simultaneously in hippocampal slices from male and female mice. This enabled us to study relatively infrequent double and triple spikes. We found GCs are relatively homogeneous and their double spiking behavior is cell autonomous. Blockade of GABA type A receptors increased multiple spikes and prolonged the interspike interval, indicating inhibitory interneurons limit repetitive spiking and set the time window for successive spikes. Inhibiting synaptic glutamate release showed that recurrent excitation mediated by hilar mossy cells contributes to, but is not necessary for, multiple spiking. Blockade of T-type Ca2+ channels did not reduce multiple spiking but prolonged interspike intervals. Imaging voltage changes in different GC compartments revealed that second spikes can be initiated in either dendrites or somata. Thus, pharmacological and biophysical experiments reveal roles for both synaptic circuitry and intrinsic excitability in GC repetitive spiking.

Susceptibility of various human cancer cell lines to nanosecond and microsecond range electrochemotherapy: Feasibility of multi-drug cocktails

Electrochemotherapy (ECT) involves combining anticancer drugs with electroporation, which is induced by pulsed electric fields (PEFs), while the effects vary in effectiveness based on the specific parameters of the electrical pulses and susceptibility of the cells to a specific drug. In this work, we utilized conventional microsecond electroporation protocols (0.8 – 1.5 kV/cm × 100 μs × 8, 1 Hz) and the new modality of nanosecond pulses (4 and 8 kV/cm × 500 ns × 100, 1 kHz and 1 MHz), which are compressed into a high frequency burst. Sensitive and resistant lung, breast and ovarian human cancer cell lines were used in the study. In order to overcome drug-resistance, we have investigated the feasibility to use anticancer drug cocktails i.e., bleomycin and cisplatin combinations with metformin, vinorelbine and Dp44mT. The different susceptibility of various human cancer cells lines to electric pulses was determined, the efficacy of ECT was characterized and the type of cell death depending on the combinations of drugs was investigated. The results indicate that synergistic effects of PEFs with drug cocktails may be used to overcome drug-resistance in cancer, while the application of nsPEF provides more flexibility in parametric protocols and modulation of cancer cell death.

In vivo spontaneous activity and coital-evoked inhibition of mouse accessory olfactory bulb output neurons

Little is known about estrous effects on brain microcircuits. We examined the accessory olfactory bulb (AOB) invivo, in anesthetized naturally cycling females, as model microcircuit receiving coital somatosensory information. Whole-cell recordings demonstrate that output neurons are relatively hyperpolarized in estrus and unexpectedly fire high frequency bursts of action potentials. To mimic coitus, a calibrated artificial vagino-cervical stimulation (aVCS) protocol was devised. aVCS evoked stimulus-locked local field responses in the interneuron layer independent of estrous stage. The response is sensitive to α1-adrenergic receptor blockade, as expected since aVCS increases norepinephrine release in AOB. Intriguingly, only in estrus does aVCS inhibit AOB spike output. Estrus-specific output reduction coincides with prolonged aVCS activation of inhibitory interneurons. Accordingly, in estrus the AOB microcircuit sets the stage for coital stimulation to inhibit the output neurons, possibly via high frequency bursting-dependent enhancement of reciprocal synapse efficacy between inter- and output neurons.

Modulation of the thalamus by microburst vagus nerve stimulation: a feasibility study protocol.

Vagus nerve stimulation (VNS) was the first device-based therapy for epilepsy, having launched in 1994 in Europe and 1997 in the United States. Since then, significant advances in the understanding of the mechanism of action of VNS and the central neurocircuitry that VNS modulates have impacted how the therapy is practically implemented. However, there has been little change to VNS stimulation parameters since the late 1990s. Short bursts of high frequency stimulation have been of increasing interest to other neuromodulation targets e.g., the spine, and these high frequency bursts elicit unique effects in the central nervous system, especially when applied to the vagus nerve. In the current study, we describe a protocol design that is aimed to assess the impact of high frequency bursts of stimulation, called "Microburst VNS", in subjects with refractory focal and generalized epilepsies treated with this novel stimulation pattern in addition to standard anti-seizure medications. This protocol also employed an investigational, fMRI-guided titration protocol that permits personalized dosing of Microburst VNS among the treated population depending on the thalamic blood-oxygen-level-dependent signal. The study was registered on clinicaltrials.gov (NCT03446664). The first subject was enrolled in 2018 and the final results are expected in 2023.

Burst firing is required for induction of Hebbian LTP at lateral perforant path to hippocampal granule cell synapses

High frequency burst firing is critical in summation of back-propagating action potentials (APs) in dendrites, which may greatly depolarize dendritic membrane potential. The physiological significance of burst firings of hippocampal dentate GCs in synaptic plasticity remains unknown. We found that GCs with low input resistance could be categorized into regular-spiking (RS) and burst-spiking (BS) cells based on their initial firing frequency (Finit) upon somatic rheobase current injection, and investigated how two types of GCs differ in long-term potentiation (LTP) induced by high-frequency lateral perforant pathway (LPP) inputs. Induction of Hebbian LTP at LPP synapses required at least three postsynaptic APs at Finit higher than 100 Hz, which was met in BS but not in RS cells. The synaptically evoked burst firing was critically dependent on persistent Na+ current, which was larger in BS than RS cells. The Ca2+ source for Hebbian LTP at LPP synapses was primarily provided by L-type calcium channels. In contrast, Hebbian LTP at medial PP synapses was mediated by T-type calcium channels, and could be induced regardless of cell types or Finit of postsynaptic APs. These results suggest that intrinsic firing properties affect synaptically driven firing patterns, and that bursting behavior differentially affects Hebbian LTP mechanisms depending on the synaptic input pathway.

(+)-Borneol enantiomer ameliorates epileptic seizure via decreasing the excitability of glutamatergic transmission.

Epilepsy is one common brain disorder, which is not well controlled by current pharmacotherapy. In this study we characterized the therapeutic potential of borneol, a plant-derived bicyclic monoterpene compound, in the treatment of epilepsy and elucidated the underlying mechanisms. The anti-seizure potency and properties of borneol were assessed in both acute and chronic mouse epilepsy models. Administration of (+)-borneol (10, 30, 100 mg/kg, i.p.) dose-dependently attenuated acute epileptic seizure in maximal-electroshock seizure (MES) and pentylenetetrazol (PTZ)-induced seizure models without obvious side-effect on motor function. Meanwhile, (+)-borneol administration retarded kindling-induced epileptogenesis and relieved fully kindled seizures. Importantly, (+)-borneol administration also showed therapeutic potential in kainic acid-induced chronic spontaneous seizure model, which was considered as a drug-resistant model. We compared the anti-seizure efficacy of 3 borneol enantiomers in the acute seizure models, and found (+)-borneol being the most satisfying one with long-term anti-seizure effect. In electrophysiological study conducted in mouse brain slices containing the subiculum region, we revealed that borneol enantiomers displayed different anti-seizure mechanisms, (+)-borneol (10 μM) markedly suppressed the high frequency burst firing of subicular neurons and decreased glutamatergic synaptic transmission. In vivo calcium fiber photometry analysis further verified that administration of (+)-borneol (100 mg/kg) inhibited the enhanced glutamatergic synaptic transmission in epilepsy mice. We conclude that (+)-borneol displays broad-spectrum anti-seizure potential in different experimental models via decreasing the glutamatergic synaptic transmission without obvious side-effect, suggesting (+)-borneol as a promising anti-seizure compound for pharmacotherapy in epilepsy.

Low frequency neuromuscular electrical stimulation applied to the bulbospongiosus muscle prolongs the ejaculation latency in a rat model.

Premature ejaculation (PE) is common, but its true pathophysiology is not clear, and treatments are limited. We aimed to investigate the effect of neuromuscular electrical stimulation applied in different modes and frequencies to the bulbospongiosus muscle on ejaculation parameters. In this study, 24 male Wistar albino rats were used. The rats were equally divided into three groups: control, high-frequency burst (HFB) and low-frequency (LF) (n = 8 each). Neuromuscular electrical stimulation was applied to the rats for 30 min. In the HFB group, this stimulation was applied in the burst mode at 80 Hz frequency using 200 microseconds (µs) transition time. In the LF group, manual stimulation was applied using a 2 Hz frequency and 200 µs transition time. Following the intraperitoneal administration of para-chloroamphetamine at a dose of 5 mg/kg, ejaculation time, increase in basal seminal vesicle pressure, seminal vesicle maximum pressure, number and interval time of seminal vesicle contractions and bulbospongiosus muscle electromyography activities were evaluated over 30 min. There was a significant difference between the groups in terms of ejaculation time (p = 0.002). The ejaculation times of the LF, HFB and control groups were 1344.71 ± 105.9, 908 ± 62 and 672 ± 149.7 s, respectively. The post hoc analysis revealed that the ejaculation time of the LF group was significantly longer than that of the HFB and control groups (p = 0.033 and p = 0.001, respectively). The remaining parameters did not differ significantly between the groups. The results showed that low-frequency (2 Hz) electrical stimulation applied to the male rats significantly prolonged the ejaculation time. It is thus considered that applying neuromuscular electrical stimulation before planned sexual activity can prevent the rhythmic contractions necessary for completing the ejaculatory process by maintaining subtetanic continuous contraction and prolonging the ejaculation time in patients with premature ejaculation complaints.

An efficient rAAV vector for protein expression in cortical parvalbumin expressing interneurons

Recombinant adeno—associated viruses (rAAV) are extensively used in both research and clinical applications. Despite significant advances, there is a lack of short promoters able to drive the expression of virus delivered genes in specific classes of neurons. We designed an efficient rAAV vector suitable for the rAAV-mediated gene expression in cortical interneurons, mainly in the parvalbumin expressing cells. The vector includes a short parvalbumin promoter and a specialized poly(A) sequence. The degree of conservation of the parvalbumin gene adjoining non-coding regions was used in both the promoter design and the selection of the poly(A) sequence. The specificity was established by co-localizing the fluorescence of the virus delivered eGFP and the antibody for a neuronal marker. rAAV particles were injected in the visual cortex area V1/V2 of adult rats (2–4 months old). Neurons expressing the virus delivered eGFP were mainly positive for interneuronal markers: 66.5 ± 2.8% for parvalbumin, 14.6 ± 2.4% for somatostatin, 7.1 ± 1.2% for vasoactive intestinal peptide, 2.8 ± 0.6% for cholecystokinin. Meanwhile, only 2.1 ± 0.5% were positive for CaMKII, a marker for principal cells in the cortex. The efficiency of the construct was verified by optogenetic experiments: the expression of the virus delivered ChR2 channels was sufficient to evoke by blue light laser high frequency bursts of action potentials in putative fast spiking neurons. We conclude that our promoter allows highly specific expression of the rAAV delivered cDNAs in cortical interneurons with a strong preference for the parvalbumin positive cells.

Spontaneous Activity Predicts Survival of Developing Cortical Neurons.

Spontaneous activity plays a crucial role in brain development by coordinating the integration of immature neurons into emerging cortical networks. High levels and complex patterns of spontaneous activity are generally associated with low rates of apoptosis in the cortex. However, whether spontaneous activity patterns directly encode for survival of individual cortical neurons during development remains an open question. Here, we longitudinally investigated spontaneous activity and apoptosis in developing cortical cultures, combining extracellular electrophysiology with calcium imaging. These experiments demonstrated that the early occurrence of calcium transients was strongly linked to neuronal survival. Silent neurons exhibited a higher probability of cell death, whereas high frequency spiking and burst behavior were almost exclusively detected in surviving neurons. In local neuronal clusters, activity of neighboring neurons exerted a pro-survival effect, whereas on the functional level, networks with a high modular topology were associated with lower cell death rates. Using machine learning algorithms, cell fate of individual neurons was predictable through the integration of spontaneous activity features. Our results indicate that high frequency spiking activity constrains apoptosis in single neurons through sustained calcium rises and thereby consolidates networks in which a high modular topology is reached during early development.

Hemodynamic changes in response to excitatory and inhibitory modulations by transcranial magnetic stimulation at the human sensorimotor cortex

Abstract Transcranial magnetic stimulation (TMS) can non-invasively induce excitatory or inhibitory neural activity depending on the stimulation parameters. However, the neurophysiological basis for both kinds of modulation remains elusive. Here, with a controlled dosage over the 30-s interval, we elicited excitatory and inhibitory modulations over the human primary motor cortex (M1) with TMS bursts of high (10-Hz and 30-Hz; HF) and low frequency (0.5-Hz; LF), respectively, and took functional magnetic resonance images (fMRI) and electromyography (EMG) recordings simultaneously. We used a customized 8-channel head coil array to integrate with an MRI-compatible TMS coil connected to the stimulator on a 3T MRI scanner. Images were prescribed to cover the left M1. Each TMS-fMRI run consisted of five 30-second “TMS on” and 30-second “TMS off” blocks. One of five types of TMS stimuli was administered in the “TMS-on” block: 0.5-Hz, 10-Hz-3-pulses-per-burst (ppb), 10-Hz-5-ppb, 30-Hz-3-ppb, and 30-Hz-5-ppb. In each TR (2s), images were acquired between 0 and 1 s after the start of each volume acquisition, and TMS pulses were delivered during the MRI silent period. EMG responses were calculated the peak-to-peak amplitudes from the time interval between 20 and 40 ms after the TMS onset. Image pre-processing and General Linear Model were performed using SPM12. Excitatory and inhibitory modulations were evidenced by changes in EMG responses to the HF and LF stimulation, respectively. Significantly increased fMRI signal at M1 was only detected under excitatory HF TMS but not during inhibitory LF TMS. The supplementary motor area (SMA) had significant fMRI signal changes after both TMS. The topology of the activated M1 and SMA matched the activated sensorimotor network during voluntary movement. These findings demonstrated the asymmetric hemodynamic responses to excitatory HF and inhibitory LF TMS modulations with region-dependent relationships between the local fMRI signal changes and TMS dosage over different time scales. Keywords: Transcranial magnetic stimulation, Functional magnetic resonance imaging, Primary motor cortex, Frequency

High-frequency burst spiking in layer 5 thick-tufted pyramids of rat primary somatosensory cortex encodes exploratory touch

Diversity of cell-types that collectively shape the cortical microcircuit ensures the necessary computational richness to orchestrate a wide variety of behaviors. The information content embedded in spiking activity of identified cell-types remain unclear to a large extent. Here, we recorded spike responses upon whisker touch of anatomically identified excitatory cell-types in primary somatosensory cortex in naive, untrained rats. We find major differences across layers and cell-types. The temporal structure of spontaneous spiking contains high-frequency bursts (≥100 Hz) in all morphological cell-types but a significant increase upon whisker touch is restricted to layer L5 thick-tufted pyramids (L5tts) and thus provides a distinct neurophysiological signature. We find that whisker touch can also be decoded from L5tt bursting, but not from other cell-types. We observed high-frequency bursts in L5tts projecting to different subcortical regions, including thalamus, midbrain and brainstem. We conclude that bursts in L5tts allow accurate coding and decoding of exploratory whisker touch.

Evaluation of the anticonvulsant effect of nimodipine and amlodipine in mice using maximal electroshock test

Background: Initial stage of seizure is due to high frequency burst of action potential, caused by long lasting depolarization of the neuronal membrane because of large influx of calcium (Ca) ions into cells. As there is role of calcium channels in the initiation of seizure potential, there may be role of calcium channel antagonists in treatment of epilepsy. Therefore, we assessed the anticonvulsant effect of nimodipine and amlodipine in mice and compared it with phenytoin and sodium valproate.Methods: A total 42 mice were randomly divided into 7 groups with 6 mice each. Group 1 vehicle (1% CMC), group 2- phenytoin (25 mg/kg) and group 3- sodium valproate (100 mg/kg) p.o. group 4 and 5- nimodipine (4 mg/kg and 8 mg/kg), group 6 and 7- amlodipine (0.7 mg/kg and 1.3 mg/kg) p.o. in 1% CMC. All animals were tested for convulsions with current strength 50 mA for 0.2 seconds, delivered by ear electrodes of electroconvulsiometer. Parameters assessed were presence or absence of convulsions, onset of (latency to) THLE and duration of THLE in seconds, 24 hours mortality.Results: The difference in percentage of mice being protected from electro convulsions was found to be statistically significant in group 5 (nimodipine 8 mg/kg), group 7 (amlodipine 1.3 mg/kg) as compared to group 1(vehicle control) (p= 0.0152). Onset of (latency to) THLE and duration of THLE, expressed as mean (in seconds) ± standard deviation (SD) and analysed using Kruskal-Wallis non-parametric test showed significant difference in latency to THLE among the groups tested (p value <0.01) and in duration of THLE among the groups tested (p value <0.01), while post- hoc Dunn’s test showed a statistically significant difference between latency to and duration of THLE in the nimodipine 8 mg/kg and amlodipine 1.3 mg/kg groups as compared to vehicle control group (p<0.01). No mortality seen within 24 hours of testing in any group.Conclusions: Nimodipine 8 mg/kg and amlodipine 1.3 mg/kg showed significant anticonvulsant activity (absence of tonic hind limb extension), delayed onset of seizures, reduced duration of seizures comparable to positive control (phenytoin and sodium valproate) and more effective than vehicle control.

Time-frequency super-resolution with superlets

Due to the Heisenberg–Gabor uncertainty principle, finite oscillation transients are difficult to localize simultaneously in both time and frequency. Classical estimators, like the short-time Fourier transform or the continuous-wavelet transform optimize either temporal or frequency resolution, or find a suboptimal tradeoff. Here, we introduce a spectral estimator enabling time-frequency super-resolution, called superlet, that uses sets of wavelets with increasingly constrained bandwidth. These are combined geometrically in order to maintain the good temporal resolution of single wavelets and gain frequency resolution in upper bands. The normalization of wavelets in the set facilitates exploration of data with scale-free, fractal nature, containing oscillation packets that are self-similar across frequencies. Superlets perform well on synthetic data and brain signals recorded in humans and rodents, resolving high frequency bursts with excellent precision. Importantly, they can reveal fast transient oscillation events in single trials that may be hidden in the averaged time-frequency spectrum by other methods.

Firing Differences Between Adult Intralaminar Thalamo-striatal Neurons

Differences in the intrinsic properties of intralaminar thalamo-striatal neurons such as expressing low-threshold-spikes (LTS) or after hyperpolarizing potentials (AHPs) of different duration have been attributed to different maturation stages. However, two morphological types: “diffuse” and “bushy” have been described. Therefore, we explored whether electrophysiological differences persist in adult mice using whole cell recordings. Some recorded neurons were identified by intracellular labeling with biocytin and double labeling with retrograde or anterograde tracings using Cre-mice. We classified these neurons by their AHPs during spontaneous firing. Neurons with long duration AHPs, with fast and slow components, were mostly found in the parafascicular (Pf) nucleus. Neurons with brief AHPs were mainly found in the central lateral (CL) nucleus. However, neurons with both AHPs were found in both nuclei in different proportions. Firing frequency adaptation differed between these neuron classes: those with prolonged AHPs exhibited firing frequency adaptation with fast and slow time constants whereas those with brief AHPs were slow adapters. Neurons with more prolonged AHPs had significant higher input resistances than neurons with brief AHPs. Both cell classes could fire in two modes: trains of single action potentials at depolarized potentials or high frequency bursts on top of LTS at more hyperpolarized potentials. LTS were probably generated by Cav3 calcium channels since they were blocked by the selective antagonist TTA-P2. About 11% of neurons with brief AHPs and 55% of neurons with prolonged AHPs do not show LTS and bursts, even when potassium currents are blocked.

The Marsquake catalogue from InSight, sols 0–478

The InSight (Interior Exploration using Seismic Investigations, Geodesy and Heat Transport) mission began collecting high quality seismic data on Mars in February 2019. This manuscript documents the seismicity observed by SEIS, InSight's seismometer, from this time until the end of March 2020. Within the InSight project, the Marsquake Service (MQS) is responsible for prompt review of all seismic data collected by InSight, detection of events that are likely to be of seismic origin, and curation and release of seismic catalogues. In the first year of data collection, MQS have identified 465 seismic events that we interpret to be from regional and teleseismic marsquakes. Seismic events are grouped into 2 different event families: the low frequency family is dominated by energy at long period below 1 s, and the high frequency family primarily include energy at and above 2.4 Hz. Event magnitudes, from Mars-specific scales, range from 1.3 to 3.7. A third class of events with very short duration but high frequency bursts have been observed 712 times. These are likely associated with a local source driven by thermal stresses. This paper describes the data collected so far in the mission and the procedures under which MQS operates; summarises the content of the current MQS seismic catalogue; and presents the key features of the events we have observed so far, using the largest events as examples.

Electrically-Driven Cell-Type Specific Neuromodulation in the External Globus Pallidus

The identification of distinct cell-types throughout the basal ganglia has been essential in advancing our understanding of network function and improving neurological therapies. In the globus pallidus externa (GPe), interventions targeted to neuronal subpopulations have profound therapeutic potential, but are challenging to implement in clinical settings. We investigated whether electrical stimulation can be tuned to engage cell-type specific responses in the GPe. Although conventional stimulation was non-specific, variance in the synaptic inputs onto Parvalbumin (PV-GPe) and Lim homeobox 6 (Lhx6-GPe) subpopulations enabled us to segregate the responses of these neurons using brief, high frequency bursts of stimulation. The circuit elements responsible for this synaptic variation were found to be asymmetrical inputs from the STN and collaterals of striatonigral neurons. Using a machine-learning approach, we established stimulus parameters that maximize this cell-type specific neuromodulation. These results establish the feasibility of rapidly transforming knowledge about circuit architecture into translatable therapeutic approaches.

Injectable Sensors Based on Passive Rectification of Volume-Conducted Currents.

Sensing implants that can be deployed by catheterization or by injection are preferable over implants requiring invasive surgery. However, present powering methods for active implants and present interrogation methods for passive implants require bulky parts within the implants that hinder the development of such minimally invasive devices. In this article, we propose a novel approach that potentially enables the development of passive sensing systems overcoming the limitations of previous implantable sensing systems in terms of miniaturization. In this approach implants are shaped as thread-like devices suitable for implantation by injection. Their basic structure consists of a thin elongated body with two electrodes at opposite ends and a simple and small circuit made up of a diode, a capacitor and a resistor. The interrogation method to obtain measurements from the implants consists in applying innocuous bursts of high frequency (≥1MHz) alternating current that reach the implants by volume conduction and in capturing and processing the voltage signals that the implants produce after the bursts. As proof-of-concept, and for illustrating how to put in practice this novel approach, here we describe the development and characterization of a system for measuring the conductivity of tissues surrounding the implant. We also describe the implementation and the in vitro validation of a 0.95mm-thick, flexible injectable implant made of off-the-shelf components. For conductivities ranging from about 0.2 to 0.8S/m, when compared to a commercial conductivity meter, the accuracy of the implemented system was about ±10%.

Characterisation of an Oxytocin‐Ires‐Cre Mouse Model for Recording Oxytocin Neuron Bursts in Behaving Mice

The hormone oxytocin initiates uterine contractions required for parturition and mammary duct contraction for milk‐ejection during lactation. Oxytocin is secreted directly into the circulation at the posterior pituitary gland by magnocellular neurons of the hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus. During parturition and lactation, oxytocin neurons fire high frequency bursts that are coordinated across the population, to generate pulses of high circulating oxytocin concentrations required for rhythmic contractions of the uterus and mammary ducts. However, researching oxytocin neuron bursts has stalled because oxytocin neuron bursts are blocked by surgical anaesthesia and are not seen in brain slice preparations. Furthermore, oxytocin neuron bursts have not been studied in freely behaving animals. Hence, new techniques are required to elucidate the mechanisms that generate oxytocin neuron bursts. Fiber photometry uses neuron specific expression of GCaMP, a modified green fluorescent protein (GFP) that senses calcium. Implantation of an optic fiber allows monitoring of changes in GCaMP fluorescence (fluorescence increases during spiking activity) as a proxy measurement of neuronal activity in freely behaving animals. Oxytocin neurons can be transfected using a virus carrying a Cre‐dependant GCaMP. We have crossed oxyotcin‐Ires‐Cre mice (oxytocin neurons express Cre) with tdTomato (red fluorescent protein) mice. The tdTomato expression is Cre‐dependant which allows visual identification of Cre‐positive neurons. We first determined the degree of colocalisation between Cre expression and oxytocin within the PVN. Five oxytocin‐Cre x tdTomato mice were transcardially perfused, their brains removed and sliced coronally at 30 μm. Immunohistochemistry (IHC) for oxytocin was used to determine the colocalisation of oxytocin and endogenous tdTomato (Cre‐positive neurons). Oxytocin positive neurons were found extensively throughout the entire length of the PVN and oxytocin colocalisation with tdTomato was 78.3% (n = 5). We then determined the transfection of Cre‐dependent AAV1.CAG.Flex.GCaMP6s (1.9 x 1013 GC/ml) in oxytocin neurons following an injection. GCaMP6s virus (1 μl) was injected unilaterally immediately dorsal to the PVN using a Hamilton syringe (0.08 cm posterior and 0.022 cm lateral from bregma at a depth of 0.45 cm from the brain surface). Three weeks after the injection, mice were perfused, their brains removed and sliced coronally at 30 μm. Single label IHC for GCaMP6s was used to quantify transfection. Following injections, GCaMP6s is expressed in the top two thirds of the PVN in tdTomato positive neurons (n = 3). To determine the relationship between changes in GCaMP6s fluorescence and spiking activity in oxytocin neurons, we are currently completing in‐vitro from oxytocin neurons while simultaneously recording GCaMP6s fluorescence in brain slices from oxytocin‐Cre mice. Finally, in another group of oxytocin‐Cre mice, an optic fiber will be implanted above the PVN to record oxytocin neurons bursts during lactation in freely behaving mice.

Development of a High-Frequency Burst Pulse Generator for Cancer Cell Treatment and Comparison Between a High-Power Burst Pulse and a Single Pulse

Cancer treatment based on ultrashort pulses of strong electric fields has been developed as a novel biological application. This article focuses on the design of a compact high-power nanosecond pulsed electromagnetic wave generator for realizing cancer treatment via ultrashort pulsed electric fields. For this purpose, we developed a pulsed power generator that continuously outputs multiple pulses via a nonlinear transmission line (NLTL) using magnetic switches. The NLTL comprises a ladder of capacitors and saturable inductors and generates a pulse train by delaying the propagation of each pulse using the magnetic switch of each ladder. In this article, we achieved a higher output frequency compared to the previous generator. The frequency was increased from 13 to 150 MHz by employing a coaxial structure instead of discrete components. In addition, we demonstrate that the application of burst pulses to cancer cells is more effective than single pulses under the same conditions.

Difference of Lethal Effect for Cell Type at Applying High Frequency Burst Pulse for Cancer Treatment

Difference of Lethal Effect for Cell Type at Applying High Frequency Burst Pulse for Cancer Treatment