Behavioral, neural and ultrastructural alterations in a graded-dose 6-OHDA mouse model of early-stage Parkinson's disease.

Studying animal models furthers our understanding of Parkinson's disease (PD) pathophysiology by providing tools to investigate detailed molecular, cellular and circuit functions. Different versions of the neurotoxin-based 6-hydroxydopamine (6-OHDA) model of PD have been widely used in rats. However, these models typically assess the result of extensive and definitive dopaminergic lesions that reflect a late stage of PD, leading to a paucity of studies and a consequential gap of knowledge regarding initial stages, in which early interventions would be possible. Additionally, the better availability of genetic tools increasingly shifts the focus of research from rats to mice, but few mouse PD models are available yet. To address these, we characterize here the behavioral, neuronal and ultrastructural features of a graded-dose unilateral, single-injection, striatal 6-OHDA model in mice, focusing on early-stage changes within the first two weeks of lesion induction. We observed early onset, dose-dependent impairments of overall locomotion without substantial deterioration of motor coordination. In accordance, histological evaluation demonstrated a partial, dose-dependent loss of dopaminergic neurons of substantia nigra pars compacta (SNc). Furthermore, electron microscopic analysis revealed degenerative ultrastructural changes in SNc dopaminergic neurons. Our results show that mild ultrastructural and cellular degradation of dopaminergic neurons of the SNc can lead to certain motor deficits shortly after unilateral striatal lesions, suggesting that a unilateral dose-dependent intrastriatal 6-OHDA lesion protocol can serve as a successful model of the early stages of Parkinson's disease in mice.

Fabrication and in vivo 2-photon microscopy validation of transparent PEDOT:PSS microelectrode arrays.

Transparent microelectrode arrays enable simultaneous electrical recording and optical imaging of neuronal networks in the brain. Electrodes made of the conducting polymer poly(3,4-ethylenedioxythiophene) doped with polystyrene sulfonate (PEDOT:PSS) are transparent; however, device fabrication necessitates specific processes to avoid deterioration of the organic material. Here, we present an innovative fabrication scheme for a neural probe that consists of transparent PEDOT:PSS electrodes and demonstrate its compatibility with 2-photon microscopy. The electrodes show suitable impedance to record local field potentials from the cortex of mice and sufficient transparency to visualize GCaMP6f-expressing neurons underneath the PEDOT:PSS features. The results validate the performance of the neural probe, which paves the way to study the complex dynamics of in vivo neuronal activity with both a high spatial and temporal resolution to better understand the brain.

Flexible Organic Electronic Devices for Pulsed Electric Field Therapy of Glioblastoma.

Glioblastoma is difficult to eradicate with standard oncology therapies due to its high degree of invasiveness. Bioelectric treatments based on pulsed electric fields (PEFs) are promising for the improvement of treatment efficiency. However, they rely on rigid electrodes that cause acute and chronic damage, especially in soft tissues such as the brain. In this work, flexible electronics were used to deliver PEFs to tumors and the biological response was evaluated with fluorescent microscopy. Interdigitated gold electrodes on a thin, transparent parylene-C substrate were coated with the conducting polymer PEDOT:PSS, resulting in a conformable and biocompatible device. The effects of PEFs on tumors and their microenvironment were examined using various biological models. First, monolayers of glioblastoma cells were cultured on top of the electrodes to investigate phenomena in vitro. As an intermediate step, an in ovo model was developed where engineered tumor spheroids were grafted in the embryonic membrane of a quail. Due to the absence of an immune system, this led to highly vascularized tumors. At this early stage of development, embryos have no immune system, and tumors are not recognized as foreign bodies. Thus, they can develop fast while developing their own vessels from the existing embryo vascular system, which represents a valuable 3D cancer model. Finally, flexible electrode delivery of PEFs was evaluated in a complete organism with a functional immune system, using a syngenic, orthograft (intracranial) mouse model. Tumor spheroids were grafted into the brain of transgenic multi-fluorescent mice prior to the implantation of flexible organic electrode devices. A sealed cranial window enabled multiphoton imaging of the tumor and its microenvironment during treatment with PEFs over a period of several weeks.

Two-photon GCaMP6f imaging of infrared neural stimulation evoked calcium signals in mouse cortical neurons in vivo.

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.

CLARITY analysis of the Cl/pH sensor expression in the brain of transgenic mice.

Genetically encoded biosensors are widely used in cell biology for the non-invasive imaging of concentrations of ions or the activity of enzymes, to evaluate the distribution of small molecules, proteins and organelles, and to image protein interactions in living cells. These fluorescent molecules can be used either by transient expression in cultured cells or in entire organisms or through stable expression by producing transgenic animals characterized by genetically encoded and heritable biosensors. Using the mouse Thy1 mini-promoter, we generated a line of transgenic mice expressing a genetically encoded sensor for the simultaneous measurements of intracellular Cl- and pH. This construct, called ClopHensor, consists of a H+- and Cl--sensitive variant of the enhanced green fluorescent protein (E2GFP) fused with a red fluorescent protein (DsRedm). Stimulation of hippocampal Schaffer collaterals proved that the sensor is functionally active. To reveal the expression pattern of ClopHensor across the brain of Thy1::ClopHensor mice, we obtained transparent brain samples using the CLARITY method and imaged them with confocal and light-sheet microscopy. We then developed a semi-quantitative approach to identify brain structures with high intrinsic sensor fluorescence. This approach allowed us to assess cell morphology and track axonal projection, as well as to confirm E2GFP and DsRedm fluorescence colocalization. This analysis also provides a map of the brain areas suitable for non-invasive monitoring of intracellular Cl-/pH in normal and pathological conditions. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

Electrophoretic Delivery of γ-aminobutyric Acid (GABA) into Epileptic Focus Prevents Seizures in Mice.

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.

Electrophoretic drug delivery for seizure control.

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.

A bilayered PVA/PLGA-bioresorbable shuttle to improve the implantation of flexible neural probes.

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.

High efficiency two-photon uncaging coupled by the correction of spontaneous hydrolysis.

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.

Multimodal Characterization of Neural Networks Using Highly Transparent Electrode Arrays.

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.

Accurate spike estimation from noisy calcium signals for ultrafast three-dimensional imaging of large neuronal populations in vivo.

Extracting neuronal spiking activity from large-scale two-photon recordings remains challenging, especially in mammals in vivo, where large noises often contaminate the signals. We propose a method, MLspike, which returns the most likely spike train underlying the measured calcium fluorescence. It relies on a physiological model including baseline fluctuations and distinct nonlinearities for synthetic and genetically encoded indicators. Model parameters can be either provided by the user or estimated from the data themselves. MLspike is computationally efficient thanks to its original discretization of probability representations; moreover, it can also return spike probabilities or samples. Benchmarked on extensive simulations and real data from seven different preparations, it outperformed state-of-the-art algorithms. Combined with the finding obtained from systematic data investigation (noise level, spiking rate and so on) that photonic noise is not necessarily the main limiting factor, our method allows spike extraction from large-scale recordings, as demonstrated on acousto-optical three-dimensional recordings of over 1,000 neurons in vivo.

Localized Neuron Stimulation with Organic Electrochemical Transistors on Delaminating Depth Probes.

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.

Sensitization of neonatal rat lumbar motoneuron by the inflammatory pain mediator bradykinin.

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.

Dendritic spikes induce ripples in parvalbumin interneurons during hippocampal sharp waves.

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.

Combined two-photon imaging, electrophysiological, and anatomical investigation of the human neocortex in vitro.

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.

Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes.

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.

Roller Coaster Scanning reveals spontaneous triggering of dendritic spikes in CA1 interneurons.

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.

Enhanced dendritic action potential backpropagation in parvalbumin-positive basket cells during sharp wave activity.

In this study two-photon imaging and single cell electrophysiological measurements were carried out in PV+ hippocampal interneurons to compare the dendritic calcium dynamics of somatically evoked backpropagating action potentials (BAPs) and in vitro sharp wave oscillation (SPW) activated BAPs at different distances from the soma. In the case of 300 µm thick, non-oscillating slices, the BAP-evoked Ca(2+) (BAP-Ca(2+)) influx propagated along the dendritic tree in a non-uniform manner and its amplitude gradually reduced when measured at more distal regions. In contrast to the evoked BAP-Ca(2+)s, the spontaneous SPW-induced Ca(2+) influx had only a small distance-dependent decrement. Our results suggest that similarly to nicotinic acetylcholine receptor activation, synaptic activity during hippocampal SPWs increases AP backpropagation into distant dendritic segments. Bath application of Nimodipine, a specific Ca(2+) channel blocker and tetrodotoxine decreased the amplitude of the somatically evoked Ca(2+) influx, which suggests that L-type Ca(2+) channels play an important role both during somatically evoked and SPW-induced BAPs.

Somatostatin inhibition of gonadotropin-releasing hormone neurons in female and male mice.

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.

Dendritic nicotinic receptors modulate backpropagating action potentials and long-term plasticity of interneurons.

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 alpha7-subunit-containing nAChRs (alpha7-nAChRs) are abundant on interneuron dendrites of rats. Moreover, dendritic Ca2+ transients induced by activation of alpha7-nAChRs increase as a function of distance from soma. The activation of these extrasynaptic alpha7-nAChRs by cholinergic agonists either facilitated or depressed backpropagating action potentials, depending on the timing of alpha7-nAChR activation. We have previously shown that dendritic alpha7-nAChRs are involved in the regulation of synaptic transmission, suggesting that alpha7-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 alpha7-nAChRs. Our results suggest a new mechanism for a cholinergic switch in memory encoding and retrieval.