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Glioblastoma is a highly aggressive brain tumor, very invasive and thus difficult to eradicate with standard oncology therapies. Bioelectric treatments based on pulsed electric fields have proven to be a successful method to treat cancerous tissues. However, they rely on stiff electrodes, which cause acute and chronic injuries, especially in soft tissues like the brain. Here we demonstrate the feasibility of delivering pulsed electric fields with flexible electronics using an in ovo vascularized tumor model. We show with fluorescence widefield and multiphoton microscopy that pulsed electric fields induce vasoconstriction of blood vessels and evoke calcium signals in vascularized glioblastoma spheroids stably expressing a genetically encoded fluorescence reporter. Simulations of the electric field delivery are compared with the measured influence of electric field effects on cell membrane integrity in exposed tumor cells. Our results confirm the feasibility of flexible electronics as a means of delivering intense pulsed electric fields to tumors in an intravital 3D vascularized model of human glioblastoma.

Infrared neural stimulation is a promising tool for stimulating the brain because it can be used to excite with high spatial precision without the need of delivering or inserting any exogenous agent into the tissue. Very few studies have explored its use in the brain, as most investigations have focused on sensory or motor nerve stimulation. Using intravital calcium imaging with the genetically encoded calcium indicator GCaMP6f, here we show that the application of infrared neural stimulation induces intracellular calcium signals in Layer 2/3 neurons in mouse cortex in vivo. The number of neurons exhibiting infrared-induced calcium response as well as the amplitude of those signals are shown to be both increasing with the energy density applied. By studying as well the spatial extent of the stimulation, we show that reproducibility of the stimulation is achieved mainly in the central part of the infrared beam path. Stimulating in vivo at such a degree of precision and without any exogenous chromophores enables multiple applications, from mapping the brain’s connectome to applications in systems neuroscience and the development of new therapeutic tools for investigating the pathological brain.

Dendritic hotspots have been shown to play a determining role as local activity foci during sensory information coding and memory formation in cortical neurons in vivo. Here we aim to reveal the characteristics of co-activating more dendritic hotspots themselves within a dynamic time window. Using electrical stimulation and patterned two-photon uncaging for the coincident activation of two neighboring dendritic hotspots in rat hippocampal CA1 interneurons, the evoked local dendritic Ca2+ and the associated excitatory postsynaptic potentials (EPSPs) summed supralinearly. Between active hotspots, supralinearity was present in a relatively broad temporal (∼10 ms) and spatial (∼30 µm) window, mediated by NMDA receptors. Though at larger inter-hotspot distances (>30 µm) supralinear summation of EPSPs was replaced by sublinear integration, the summation of dendritic Ca2+ responses remained supralinear. The inter-hotspot integration represents a novel signal integration state, extending the interaction distance between dendritic inputs, and allowing long-range coincidence detection.

International audience; Functional mapping of the connectivity of the brain is an essential procedure for neurosurgeons aiming for maximal resection of a brain tumor, while minimizing damage to the eloquent cortex. Being able to delineate the location of critical cortical areas in individual patients allows the surgeon to preserve sensorimotor and cognitive functions. Several methods exist for functional mapping, however, none are capable of delivering fast, label free mapping of the cortex with millimeter precision. In this work, we investigate the potential use of infrared neural stimulation (λ=1470nm) as a means of focal stimulation of the mouse cortex in vivo. Here, we show that the application of infrared neural stimulation induces in vivo cortical intracellular calcium signals in Layer II/III mouse neurons, using intravital calcium imaging with the genetically encoded calcium indicator GCaMP6f.

Visual Abstract, Transparent and flexible materials are attractive for a wide range of emerging bioelectronic applications. These include neural interfacing devices for both recording and stimulation, where low electrochemical electrode impedance is valuable. Here the conducting polymer poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) is used to fabricate electrodes that are small enough to allow unencumbered optical access for imaging a large cell population with two-photon (2P) microscopy, yet provide low impedance for simultaneous high quality recordings of neural activity in vivo. To demonstrate this, pathophysiological activity was induced in the mouse cortex using 4-aminopyridine (4AP), and the resulting electrical activity was detected with the PEDOT:PSS-based probe while imaging calcium activity directly below the probe area. The induced calcium activity of the neuronal network as measured by the fluorescence change in the cells correlated well with the electrophysiological recordings from the cortical grid of PEDOT:PSS microelectrodes. Our approach provides a valuable vehicle for complementing classical high temporal resolution electrophysiological analysis with optical imaging.

© 2018 IOP Publishing Ltd. Objective. Neural electrophysiology is often conducted with traditional, rigid depth probes. The mechanical mismatch between these probes and soft brain tissue is unfavorable for tissue interfacing. Making probes compliant can improve biocompatibility, but as a consequence, they become more difficult to insert into the brain. Therefore, new methods for inserting compliant neural probes must be developed. Approach. Here, we present a new bioresorbable shuttle based on the hydrolytically degradable poly(vinyl alcohol) (PVA) and poly(lactic-co-glycolic acid) (PLGA). We show how to fabricate the PVA/PLGA shuttles on flexible and thin parylene probes. The method consists of PDMS molding used to fabricate a PVA shuttle aligned with the probe and to also impart a sharp tip necessary for piercing brain tissue. The PVA shuttle is then dip-coated with PLGA to create a bi-layered shuttle. Main results. While single layered PVA shuttles are able to penetrate agarose brain models, only limited depths were achieved and repositioning was not possible due to the fast degradation. We demonstrate that a bilayered approach incorporating a slower dissolving PLGA layer prolongs degradation and enables facile insertion for at least several millimeters depth. Impedances of electrodes before and after shuttle preparation were characterized and showed that careful deposition of PLGA is required to maintain low impedance. Facilitated by the shuttles, compliant parylene probes were also successfully implanted into anaesthetized mice and enabled the recording of high quality local field potentials. Significance. This work thereby presents a solution towards addressing a key challenge of implanting compliant neural probes using a two polymer system. PVA and PLGA are polymers with properties ideal for translation: commercially available, biocompatible with FDA-approved uses and bioresorbable. By presenting new ways to implant compliant neural probes, we can begin to fully evaluate their chronic biocompatibility and performance compared to traditional, rigid electronics.

Two-photon (TP) uncaging of neurotransmitter molecules is the method of choice to mimic and study the subtleties of neuronal communication either in the intact brain or in slice preparations. However, the currently available caged materials are just at the limit of their usability and have several drawbacks. The local and focal nature of their use may for example be jeopardized by a high spontaneous hydrolysis rate of the commercially available compounds with increased photochemical release rate. Here, using quantum chemical modelling we show the mechanisms of hydrolysis and two-photon activation, and synthesized more effective caged compounds. Furthermore, we have developed a new enzymatic elimination method removing neurotransmitters inadvertently escaping from their compound during experiment. This method, usable both in one and two-photon experiments, allows for the use of materials with an increased rate of photochemical release. The efficiency of the new compound and the enzymatic method and of the new compound are demonstrated in neurophysiological experiments.

An implantable microfluidic ion pump prevents seizures in a mice by delivery of inhibitory neurotransmitters to the seizure source., The persistence of intractable neurological disorders necessitates novel therapeutic solutions. We demonstrate the utility of direct in situ electrophoretic drug delivery to treat neurological disorders. We present a neural probe incorporating a microfluidic ion pump (μFIP) for on-demand drug delivery and electrodes for recording local neural activity. The μFIP works by electrophoretically pumping ions across an ion exchange membrane and thereby delivers only the drug of interest and not the solvent. This “dry” delivery enables precise drug release into the brain region with negligible local pressure increase. The therapeutic potential of the μFIP probe is tested in a rodent model of epilepsy. The μFIP probe can detect pathological activity and then intervene to stop seizures by delivering inhibitory neurotransmitters directly to the seizure source. We anticipate that further tailored engineering of the μFIP platform will enable additional applications in neural interfacing and the treatment of neurological disorders.

The understanding of brain computations requires methods that read out neural activity on different spatial and temporal scales. Following signal propagation and integration across a neuron and recording the concerted activity of hundreds of neurons pose distinct challenges, and the design of imaging systems has been mostly focused on tackling one of the two operations. We developed a high-resolution, acousto-optic two-photon microscope with continuous three-dimensional (3D) trajectory and random-access scanning modes that reaches near-cubic-millimeter scan range and can be adapted to imaging different spatial scales. We performed 3D calcium imaging of action potential backpropagation and dendritic spike forward propagation at sub-millisecond temporal resolution in mouse brain slices. We also performed volumetric random-access scanning calcium imaging of spontaneous and visual stimulation-evoked activity in hundreds of neurons of the mouse visual cortex in vivo. These experiments demonstrate the subcellular and network-scale imaging capabilities of our system.

Inhibitory interneurons are considered to be the controlling units of neural networks, despite their sparse number and unique morphological characteristics compared with excitatory pyramidal cells. Although pyramidal cell dendrites have been shown to display local regenerative events—dendritic spikes (dSpikes)—evoked by artificially patterned stimulation of synaptic inputs, no such studies exist for interneurons or for spontaneous events. In addition, imaging techniques have yet to attain the required spatial and temporal resolution for the detection of spontaneously occurring events that trigger dSpikes. Here we describe a high-resolution 3D two-photon laser scanning method (Roller Coaster Scanning) capable of imaging long dendritic segments resolving individual spines and inputs with a temporal resolution of a few milliseconds. By using this technique, we found that local, NMDA receptor-dependent dSpikes can be observed in hippocampal CA1 stratum radiatum interneurons during spontaneous network activities in vitro. These NMDA spikes appear when approximately 10 spatially clustered inputs arrive synchronously and trigger supralinear integration in dynamic interaction zones. In contrast to the one-to-one relationship between computational subunits and dendritic branches described in pyramidal cells, here we show that interneurons have relatively small (∼14 μm) sliding interaction zones. Our data suggest a unique principle as to how interneurons integrate synaptic information by local dSpikes.

Stratum radiatum interneurons, unlike pyramidal cells, are rich in nicotinic acetylcholine receptors (nAChRs); however, the role of these receptors in plasticity has remained elusive. As opposed to previous physiological studies, we found that functional α7-subunit-containing nAChRs (α7-nAChRs) are abundant on interneuron dendrites of rats. Moreover, dendritic Ca2+ transients induced by activation of α7-nAChRs increase as a function of distance from soma. The activation of these extrasynaptic α7-nAChRs by cholinergic agonists either facilitated or depressed backpropagating action potentials, depending on the timing of α7-nAChR activation. We have previously shown that dendritic α7-nAChRs are involved in the regulation of synaptic transmission, suggesting that α7-nAChRs may play an important role in the regulation of the spike timing-dependent plasticity. Here we provide evidence that long-term potentiation is indeed boosted by stimulation of dendritic α7-nAChRs. Our results suggest a new mechanism for a cholinergic switch in memory encoding and retrieval.

Bradykinin (Bk) is a potent inflammatory mediator that causes hyperalgesia. The action of Bk on the sensory system is well documented but its effects on motoneurons, the final pathway of the motor system, are unknown. By a combination of patch-clamp recordings and two-photon calcium imaging, we found that Bk strongly sensitizes spinal motoneurons. Sensitization was characterized by an increased ability to generate self-sustained spiking in response to excitatory inputs. Our pharmacological study described a dual ionic mechanism to sensitize motoneurons, including inhibition of a barium-sensitive resting K+ conductance and activation of a nonselective cationic conductance primarily mediated by Na+. Examination of the upstream signaling pathways provided evidence for postsynaptic activation of B2 receptors, G protein activation of phospholipase C, InsP3 synthesis, and calmodulin activation. This study questions the influence of motoneurons in the assessment of hyperalgesia since the withdrawal motor reflex is commonly used as a surrogate pain model. DOI: http://dx.doi.org/10.7554/eLife.06195.001, eLife digest When we accidentally place our hand on a hot stove, we normally experience a painful sensation that starts with the sensory nerves under our skin. These nerves respond by transmitting electrical impulses to our brain, where the painful sensation is then processed. At the same time, these impulses are also transmitted to the motor nerves that control the muscles in our hand to trigger an immediate reflex to withdraw the hand from the hot stove. Pain therefore has a useful role as it can reduce how bad an injury is. People with a condition called hyperalgesia have an increased sensitivity to pain. This condition can result from a chemical called bradykinin ‘sensitizing’ the sensory nerves, causing them to transmit more electrical impulses in response to pain than normal. This makes the injury feel much more painful, and can make the pain last for longer than is beneficial. It was less clear whether bradykinin also affects motor nerves and so triggers a withdrawal reflex. By recording the electrical activity of motor nerve cells taken from the spinal cords of newborn rats, Bouhadfane et al. now show that these motor nerves become more active when exposed to bradykinin. Nerve cells generate electrical signals when ions—such as potassium, sodium, and calcium ions—move through channels in the membranes of the cell. Therefore, to investigate how bradykinin influences the electrical activity of motor nerves, Bouhadfane et al. exposed the cells to drugs that inhibit particular ion channels. This revealed that bradykinin sensitizes the motor nerves by blocking a type of potassium ion channel and activating another ion channel that mainly transports sodium ions. Furthermore, Bouhadfane et al. were able to identify the signaling pathways that allow bradykinin to affect the motor nerve cells. The study implies that the neuronal circuitry for pain does not rely exclusively on sensory nerve cells but should also integrate motor nerve cells. A future challenge remains in developing a protocol to resolve the contribution of motor nerve cells to hyperalgesia assessed by reflex withdrawal. DOI: http://dx.doi.org/10.7554/eLife.06195.002

Organic electrochemical transistors are integrated on depth probes to achieve localized electrical stimulation of neurons. The probes feature a mechanical delamination process which leaves only a 4 μm thick film with embedded transistors inside the brain. This considerably reduces probe invasiveness and correspondingly improves future brain-machine interfaces.

Over the last 20 years, neuroscientists have become increasingly interested in two-photon microscopy. One of the reasons for this interest is that two-photon fl uorescence excitation allows counterbalancing the deterioration of the optical signals due to light scattering, and this opens the door for high-resolution imaging at considerable depth in living tissue. Due to progress in fl uorescent marking techniques, to date, two-photon microscopy allows the functional exploration of neuronal activity at multiple scales, from the subprocesses of a single cell (dendrites, single spines, etc.), through single cells or small networks of a few neurons, up to large neuronal populations in the order of a cortical column. Here, we provide some information on the practical aspects of two-photon microscopy applied to imaging neurons in living tissue. We fi rst discuss the advantages, shortcomings, and possible developments of the technique. We provide some practical considerations on the choice of the microscope itself, as well as on its principal elements. Because of recent progress in tackling high-speed imaging of 3D objects, we devote particular attention to the discussion of z -axis scanning techniques. Next, we illustrate some common applications, such as calcium imaging of neuronal activity, in vitro and in vivo. We also brie fl y illustrate how two-photon microscopy can be used for the imaging of erythrocyte fl ow in individual capillaries. Some practical considerations on speci fi c protocols are provided in the form of self-consistent text boxes.

In addition to the classical direct genomic mechanisms of action, oestrogen also exerts poorly understood, nonclassical effects on the signalling system in neurones. In the present study, we investigated whether sex differences exist in gonadectomy- and oestrogen-induced effects on p44/42 mitogen-activated protein kinase (MAPK) phosphorylation in specific brain regions of mice. We demonstrate that MAPK immunoreactivity was not altered by gonadectomy or oestrogen treatment in either sex. However, we show that the level of phosphorylated MAPK (pMAPK) within the anteroventral periventricular nucleus (AVPV) was consistently higher in males than females irrespective of gonadal steroid hormone status. In addition, gonadectomy was found to decrease pMAPK immunoreactivity within the piriform cortex of males. Oestrogen increased pMAPK immunoreactivity in the medial preoptic area and AVPV of females, but failed to have the same effect in male mice. Overall, these results demonstrate a marked sex difference in oestrogen-induced alteration of MAPK phosphorylation in the brain in vivo.

Spontaneous synchronous population activity (SPA) can be detected by electrophysiological methods in cortical slices of epileptic patients, maintained in a physiological medium in vitro. In order to gain additional spatial information about the network mechanisms involved in the SPA generation, we combined electrophysiological studies with two-photon imaging. Neocortical slices prepared from postoperative tissue of epileptic and tumor patients were maintained in a dual perfusion chamber in a physiological incubation medium. SPA was recorded with a 24-channel extracellular linear microelectrode covering all neocortical layers. After identifying the electrophysiologically active regions of the slice, bolus loading of neuronal and glial markers was applied on the tissue. SPA-related [Formula: see text] transients were detected in a large population of neighboring neurons with two-photon microscopy, simultaneous with extracellular SPA and intracellular whole-cell patch-clamp recordings. The intracellularly recorded cells were filled for subsequent anatomy. The cells were reconstructed in three dimensions and examined with light- and transmission electron microscopy. Combining high spatial resolution two-photon [Formula: see text] imaging techniques and high temporal resolution extra- and intracellular electrophysiology with cellular anatomy may permit a deeper understanding of the structural and functional properties of the human neocortex.

Previous studies indicate that somatostatin regulates gonadotropin secretion. We investigated here whether somatostatin has direct effects on GnRH neurons in the adult male and female mice. Dual-labeling immunofluorescence experiments revealed the presence of somatostatin-immunoreactive fibers adjacent to GnRH neurons, and three-dimensional confocal reconstructions demonstrated apparent somatostatin fiber appositions with 50-60% of GnRH neurons located throughout the brain in both male and female mice. Perforated patch-clamp recordings from GnRH-green fluorescent protein neurons revealed that approximately 70% of GnRH neurons responded in a dose-dependent manner to 10-300 nm somatostatin with an acute membrane hyperpolarization and cessation of firing. This effect persisted in the presence of tetrodotoxin and amino acid receptor antagonists, indicating a direct postsynaptic site of action on the GnRH neuron. The identity of the somatostatin receptors underlying this action was assessed using GnRH neuron single-cell RT-PCR. Of the somatostatin receptor subtypes, the sstr2 transcript was the most prevalent and detected in both males and females. The expression of sstr2 by GnRH neurons was confirmed in the sstr2 knockout/LacZ knock-in mouse line. Electrophysiological studies demonstrated that the sstr2-selective agonist seglitide exerted acute hyperpolarizing actions on GnRH neurons identical to those of somatostatin. Together, these studies reveal somatostatin, acting through sstr2, to be one of the most potent inhibitors of electrical excitability of male and female GnRH neurons identified thus far.

We found that by adding Phen to the Eu3+/TTA complexes, the luminescence intensity of Eu3+ can be increased very much. The Phen is bounded to the complexes and can be found not only in the first coordination sphere. It is an efficient shield against the quenching processes and opens a new IMET channel towards the Eu3+. Concerning the ligands, only the S1 excited state plays a role in the IMET.

Epilepsy is a group of neurological disorders which affects millions of people worldwide. Although treatment with medication is helpful in 70% of the cases, serious side effects affect the quality of life of patients. Moreover, a high percentage of epileptic patients are drug resistant; in their case, neurosurgery or neurostimulation are necessary. Therefore, the major goal of epilepsy research is to discover new therapies which are either capable of curing epilepsy without side effects or preventing recurrent seizures in drug-resistant patients. Neuroengineering provides new approaches by using novel strategies and technologies to find better solutions to cure epileptic patients at risk. As a demonstration of a novel experimental protocol in an acute mouse model of epilepsy, a direct in situ electrophoretic drug delivery system is used. Namely, a neural probe incorporating a microfluidic ion pump (µFIP) for on-demand drug delivery and simultaneous recording of local neural activity is implanted and demonstrated to be capable of controlling 4-aminopyridine-induced (4AP-induced) seizure-like event (SLE) activity. The γ-aminobutyric acid (GABA) concentration is kept in the physiological range by the precise control of GABA delivery to reach an antiepileptic effect in the seizure focus but not to cause overinhibition-induced rebound bursts. The method allows both the detection of pathological activity and intervention to stop seizures by delivering inhibitory neurotransmitters directly to the epileptic focus with precise spatiotemporal control. As a result of the developments to the experimental method, SLEs can be induced in a highly localized manner that allows seizure control by the precisely tuned GABA delivery at the seizure onset.

Sharp-wave ripples are transient oscillatory events in the hippocampus that are associated with the reactivation of neuronal ensembles within specific circuits during memory formation. Fast-spiking, parvalbumin-expressing interneurons (FS-PV INs) are thought to provide fast integration in these oscillatory circuits by suppressing regenerative activity in their dendrites. Here, using fast 3D two-photon imaging and a caged glutamate, we challenge this classical view by demonstrating that FS-PV IN dendrites can generate propagating Ca(2+) spikes during sharp-wave ripples. The spikes originate from dendritic hot spots and are mediated dominantly by L-type Ca(2+) channels. Notably, Ca(2+) spikes were associated with intrinsically generated membrane potential oscillations. These oscillations required the activation of voltage-gated Na(+) channels, had the same frequency as the field potential oscillations associated with sharp-wave ripples, and controlled the phase of action potentials. Furthermore, our results demonstrate that the smallest functional unit that can generate ripple-frequency oscillations is a segment of a dendrite.