Intracellular Electrodes Research Articles

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in <em>Aplysia californica</em>

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool. This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations. To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons. We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation or in vivo. This process can also be applied in other motor pools in Aplysia or in other animal systems.

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse <em>In vivo</em>

The spinal motoneuron has long been a good model system for studying neural function because it is a neuron of the central nervous system with the unique properties of (1) having readily identifiable targets (the muscle fibers) and therefore having a very well-known function (to control muscle contraction); (2) being the convergent target of many spinal and descending networks, hence the name of "final common pathway"; and (3) having a large soma which makes it possible to penetrate them with sharp intracellular electrodes. Furthermore, when studied in vivo, it is possible to record simultaneously the electrical activity of the motoneurons and the force developed by their muscle targets. Performing intracellular recordings of motoneurons in vivo therefore put the experimentalist in the unique position of being able to study, at the same time, all the compartments of the "motor unit" (the name given to the motoneuron, its axon, and the muscle fibers it innervates(1)): the inputs impinging on the motoneuron, the electrophysiological properties of the motoneuron, and the impact of these properties on the physiological function of the motoneurons, i.e. the force produced by its motor unit. However, this approach is very challenging because the preparation cannot be paralyzed and thus the mechanical stability for the intracellular recording is reduced. Thus, this kind of experiments has only been achieved in cats and in rats. However, the study of spinal motor systems could make a formidable leap if it was possible to perform similar experiments in normal and genetically modified mice. For technical reasons, the study of the spinal networks in mice has mostly been limited to neonatal in vitro preparations, where the motoneurons and the spinal networks are immature, the motoneurons are separated from their targets, and when studied in slices, the motoneurons are separated from most of their inputs. Until recently, only a few groups had managed to perform intracellular recordings of motoneurons in vivo(2-4 ), including our team who published a new preparation which allowed us to obtain very stable recordings of motoneurons in vivo in adult mice(5,6). However, these recordings were obtained in paralyzed animals, i.e. without the possibility to record the force output of these motoneurons. Here we present an extension of this original preparation in which we were able to obtain simultaneous recordings of the electrophysiological properties of the motoneurons and of the force developed by their motor unit. This is an important achievement, as it allows us to identify the different types of motoneurons based on their force profile, and thereby revealing their function. Coupled with genetic models disturbing spinal segmental circuitry(7-9), or reproducting human disease(10,11), we expect this technique to be an essential tool for the study of spinal motor system.

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse <em>In vivo</em>

The spinal motoneuron has long been a good model system for studying neural function because it is a neuron of the central nervous system with the unique properties of (1) having readily identifiable targets (the muscle fibers) and therefore having a very well-known function (to control muscle contraction); (2) being the convergent target of many spinal and descending networks, hence the name of "final common pathway"; and (3) having a large soma which makes it possible to penetrate them with sharp intracellular electrodes. Furthermore, when studied in vivo, it is possible to record simultaneously the electrical activity of the motoneurons and the force developed by their muscle targets. Performing intracellular recordings of motoneurons in vivo therefore put the experimentalist in the unique position of being able to study, at the same time, all the compartments of the "motor unit" (the name given to the motoneuron, its axon, and the muscle fibers it innervates1): the inputs impinging on the motoneuron, the electrophysiological properties of the motoneuron, and the impact of these properties on the physiological function of the motoneurons, i.e. the force produced by its motor unit. However, this approach is very challenging because the preparation cannot be paralyzed and thus the mechanical stability for the intracellular recording is reduced. Thus, this kind of experiments has only been achieved in cats and in rats. However, the study of spinal motor systems could make a formidable leap if it was possible to perform similar experiments in normal and genetically modified mice. For technical reasons, the study of the spinal networks in mice has mostly been limited to neonatal in vitro preparations, where the motoneurons and the spinal networks are immature, the motoneurons are separated from their targets, and when studied in slices, the motoneurons are separated from most of their inputs. Until recently, only a few groups had managed to perform intracellular recordings of motoneurons in vivo2-4 , including our team who published a new preparation which allowed us to obtain very stable recordings of motoneurons in vivo in adult mice5,6. However, these recordings were obtained in paralyzed animals, i.e. without the possibility to record the force output of these motoneurons. Here we present an extension of this original preparation in which we were able to obtain simultaneous recordings of the electrophysiological properties of the motoneurons and of the force developed by their motor unit. This is an important achievement, as it allows us to identify the different types of motoneurons based on their force profile, and thereby revealing their function. Coupled with genetic models disturbing spinal segmental circuitry7-9, or reproducting human disease10,11, we expect this technique to be an essential tool for the study of spinal motor system.

P21 Endogenous H2S produced by CSE selectively facilitates acetylcholine release from synaptic terminals of central origin in mouse superior mesenteric ganglion

Abdominal sympathetic prevertebral ganglia in mammals play a major physiological role in regulating motility, absorption and secretion in the gastrointestinal tract and its accessory organs. Hydrogen sulfide (H2S) has emerged as an important messenger molecule in the central nervous system and enteric nervous system. We designed experiments to test the hypotheses that prevertebral ganglia have the enzymatic machinery to generate H2S, that H2S is continuously generated and released in the prevertebral ganglia and that H2S modulates fast cholinergic nicotinic excitatory synaptic input (F-EPSP). Methods The superior mesenteric ganglion (SMG) and attached splanchnic and colonic nerve trunks were dissected from adult SJL/J mice. The use of mouse tissue was approved by the Institutional Animal Care and Use Committee. Sharp glass microelectrodes were used for intracellular recordings and bi-polar stimulating electrodes were used to evoke F-EPSPs. Immunostaining was performed with antibodies for cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS) and with vesicular acetylcholine transporter to identify preganglionic cholinergic terminals. Since the amount of tissue in mouse prevertebral ganglia was less than the amount required for the assay method (gas chromatography), we used guinea pig prevertebral ganglia to measure endogenous H2S generation. Results Immunoreactivity of CSE was found in neurons and glia cells. There was no colocalization of CSE with immunoreactivity of vesicular acetylcholine transporter. Immunoreactivity of CBS was only found in some glia cells. There was no colocalization between immunoreactivity of CBS and immunoreactivity of vesicular acetylcholine transporter. Endogenous H2S was generated and released from homogenized guinea pig sympathetic prevertebral ganglia at a rate of 2.52 ± 0.52 pmol/min/mg tissue weight, suggesting that H2S is generated in intact prevertebral ganglia. To determine whether the F-EPSPs are potentiated by endogenously released H2S in mouse SMG, we tested the effect of inhibiting H2S break-down using stigmatellin, a specific sulfide quinone reductase inhibitor. Stigmatellin (1 μM) significantly (p dl -propargylglycine (PAG) (1 μM) on F-EPSPs evoked by splanchnic and colonic nerve stimulation in the presence of PAG. The amplitude and area of F-EPSPs evoked by splanchnic nerve stimulation were significantly (p 0.05) effect on F-EPSPs evoked by colonic nerve stimulation (30.2 ± 6.1 mV and 808 ± 235 ms · mV with PAG vs. 26.9 ± 7.6 mV and 855 ± 241 ms · mV before PAG; n = 5). Conclusions H2S is enzymatically generated and released in the mouse prevertebral ganglia where it potentiates fast cholinergic synaptic input in a pathway specific manner.

Vertical nanowire electrode arrays as a scalable platform for intracellular interfacing to neuronal circuits

Deciphering the neuronal code--the rules by which neuronal circuits store and process information--is a major scientific challenge. Currently, these efforts are impeded by a lack of experimental tools that are sensitive enough to quantify the strength of individual synaptic connections and also scalable enough to simultaneously measure and control a large number of mammalian neurons with single-cell resolution. Here, we report a scalable intracellular electrode platform based on vertical nanowires that allows parallel electrical interfacing to multiple mammalian neurons. Specifically, we show that our vertical nanowire electrode arrays can intracellularly record and stimulate neuronal activity in dissociated cultures of rat cortical neurons and can also be used to map multiple individual synaptic connections. The scalability of this platform, combined with its compatibility with silicon nanofabrication techniques, provides a clear path towards simultaneous, high-fidelity interfacing with hundreds of individual neurons.

Movement based artifacts may contaminate extracellular electrical recordings from GI muscles

Electrical slow waves drive peristaltic contractions in the stomach and facilitate gastric emptying. In gastroparesis and other disorders associated with altered gastric emptying, motility defects have been related to altered slow wave frequency and disordered propagation. Experimental and clinical measurements of slow waves are made with extracellular or abdominal surface recording. We tested the consequences of muscle contractions and movement on biopotentials recorded from murine gastric muscles with array electrodes and pairs of silver electrodes. Propagating biopotentials were readily recorded from gastric sheets composed of the entire murine stomach. The biopotentials were completely blocked by nifedipine (2 μmol L(-1) ) that blocked contractile movements and peristaltic contractions. Wortmannin, an inhibitor of myosin light chain kinase, also blocked contractions and biopotentials. Stimulation of muscles with carbachol increased the frequency of biopotentials in control conditions but failed to elicit biopotentials with nifedipine or wortmannin present. Intracellular recording with microelectrodes showed that authentic gastric slow waves occur at a faster frequency typically than biopotentials recorded with extracellular electrodes, and electrical slow waves recorded with intracellular electrodes were unaffected by suppression of movement. Electrical transients, equal in amplitude to biopotentials recorded with extracellular electrodes, were induced by movements produced by small transient stretches (<1 mm) of paralyzed or formalin fixed gastric sheets. These data demonstrate significant movement artifacts in extracellular recordings of biopotentials from murine gastric muscles and suggest that movement suppression should be an obligatory control when monitoring electrical activity and characterizing propagation and coordination of electrical events with extracellular recording techniques.

P.1.f.004 Behavioural and neurodevelopmental effects of ACTH and glucocorticoids in neonatal rats

This chapter highlights how studies of the giant dopamine neuron (GDN) located in the left pedal ganglion of the pond snail Planorbis corneus have provided direct demonstration of reverse transport from a neuron in real time. The GDN is advantageous for these studies because it has nomifensine-sensitive plasma membrane dopamine (DA) uptake, reserpine-sensitive vesicular DA uptake, it metabolizes DA to dihydroxyphenylacetic acid, and lacks ascorbic acid, facilitating the unambiguous amperometric detection of DA. Exocytic quantal release events can easily be distinguished from release by reverse transport using carbon fiber electrodes. The large size of the neuron allows the estimation of cytosolic levels of DA with intracellular carbon electrodes. The large size of the cell, the presence of the DA uptake system, and presence of many synaptic vesicles provides an enormous transmitter pool, proving the means to detect reverse transport from a single cell.

Imaging electrical resonance in hair cells

The mechanosensory hair cells of many auditory receptor organs are tuned by an electrical resonance that increases their responses to stimulation over a narrow band of frequencies. The small oscillations of membrane potential characteristic of this phenomenon have previously been detectable only through intracellular electrode measurements, which are laborious and preclude analysis at the level of an entire sensory organ. We used a voltage-sensitive dye to image hair-cell electrical resonance in an intact preparation of the bullfrog's sacculus, a receptor organ sensitive to low-frequency seismic and auditory stimuli. Imaging revealed distinct populations of hair cells whose resonant response varied with the frequency of transepithelial electrical stimulation. Most of the hair cells in the saccular epithelium in vitro were electrically tuned to stimulation at 25-50 Hz. The frequency dependence of the fluorescence signal was sensitive to pharmacological blockade of large-conductance Ca(2+)-sensitive K(+) channels and to enzymatic digestion. At an elevated concentration of Ca(2+), we observed transient fluorescence signals that probably represented action potentials. The stroboscopic imaging and analysis techniques described here present a general approach for studying subthreshold oscillations in electrically excitable cells.

Planar Patch-Clamp Electrodes for Single Cell and Neural Network Studies

The traditional glass pipette patch-clamp technique has contributed greatly to fundamental and pharmacological ion channel studies. The success of this serial technique has driven an effort to create wafer-based patch-clamp platforms using materials with inferior dielectric properties than glass and/or using exotic processing techniques to avoid the difficulties inherent to parallel processing of glass. We have developed a material processing scheme that generates ultrasmooth, high aspect ratio pores in fused quartz wafers. These devices are demonstrated here to be superior planar patch-clamp electrodes achieving gigaohm seals in nearly 80% of trials with a mammalian cell line, with the majority of seals over 10 GΩ and as high as 80 GΩ, competing with the best pipette-based patch-clamp measurements. Our method, amenable to batch fabrication technologies, will enable the acquisition of low noise, ion channel measurements in high throughput.We are currently merging the abovementioned devices with voltage sensitive dye (VSD) imaging and multi-electrode array (MEA) recordings in order to study multisensory integration in the medicinal leech. Initially, the planar pores will function to provide precise placement of neurons in the leech ganglion over the MEA's. The excellent spatial resolution of the VSD's combined with the temporal resolution of MEA's will provide much information of all the neurons that respond to visual stimuli in the ganglion. Further studies may employ the planar pores as intracellular electrodes, allowing voltage control and intracellular recordings of individual neurons in the ganglion.

The projective field of single bipolar cells in the retina

Event Abstract Back to Event The projective field of single bipolar cells in the retina Hiroki Asari1* and Markus Meister2 1 Harvard University, Department of Molecular and Cellular Biology, United States 2 Harvard University, United States The vertebrate retina contains about 10 types of bipolar cells that convey information from photoreceptors to retinal ganglion cells. Previous studies suggest that they form parallel channels, and that each bipolar cell type contributes a specific visual message to select types of ganglion cells. Here we test this hypothesis by determining the full projective field of a single bipolar cell, specifically the responses it elicits in the population of ganglion cells. In the isolated salamander retina, we controlled bipolar cell activity with an intracellular electrode while recording the firing of ganglion cells with a multi-electrode array and manipulating the intervening circuitry pharmacologically. We found that excitation of a single bipolar cell altered the responses of many ganglion cells. The effect was generally excitatory at short distances and inhibitory at longer distances. Electrical synapses among bipolar cells contributed substantially to the lateral spread of excitation, whereas amacrine cells suppressed the excitatory spread and mediated the inhibitory effects. Within the excitatory region, different ganglion cells showed distinct temporal response patterns. A sustained depolarization of the bipolar cell produced a transient burst of spikes in some ganglion cells but sustained firing in others. This range of response kinetics resulted primarily from the interactions of individual bipolar cell terminals with amacrine cells. Our results highlight the diversity of neuronal circuits that distribute signals from a bipolar cell to various ganglion cells, and suggest considerable cross-talk between bipolar cell channels through gap junctions and via amacrine cells. Conference: Computational and Systems Neuroscience 2010, Salt Lake City, UT, United States, 25 Feb - 2 Mar, 2010. Presentation Type: Poster Presentation Topic: Poster session I Citation: Asari H and Meister M (2010). The projective field of single bipolar cells in the retina. Front. Neurosci. Conference Abstract: Computational and Systems Neuroscience 2010. doi: 10.3389/conf.fnins.2010.03.00145 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 02 Mar 2010; Published Online: 02 Mar 2010. * Correspondence: Hiroki Asari, Harvard University, Department of Molecular and Cellular Biology, Paris, United States, asari@fas.harvard.edu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Hiroki Asari Markus Meister Google Hiroki Asari Markus Meister Google Scholar Hiroki Asari Markus Meister PubMed Hiroki Asari Markus Meister Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Enhanced homosynaptic LTD in cerebellar Purkinje cells of the dystrophic MDX mouse

The purpose was to study homosynaptic long-term depression (LTD) at the parallel fiber-Purkinje cell synapse in the mdx mouse, a murine model of the human dystrophinopathy, Duchenne muscular dystrophy (DMD), in order to examine whether the absence of dystrophin affects the induction and extent of this form of synaptic plasticity. Sharp intracellular electrodes were used to record electrically evoked excitatory postsynaptic potentials (EPSPs) from identified Purkinje cells in cerebellar slices. The early phase of homosynaptic LTD, 7-16 min postinduction, was the same in mdx and wildtype Purkinje cells; however, the late phase of LTD, 35-44 min, was significantly enhanced in mdx Purkinje cells. We hypothesize that this enhancement of the late phase of homosynaptic LTD may be due to a disruption of Ca(2+) homeostasis associated with the absence of the protein dystrophin. These findings may explain some of the central nervous system deficiencies reported in DMD boys.

Deletion of P2X3 receptors blunts gastro‐oesophageal sensation in mice

Prior studies have demonstrated P2X receptor expression in the majority of nodose neurons. Immunoreactivity for P2X receptors has also been seen in putative gastric mechanoreceptors, the intraganglionic laminar endings. We therefore hypothesized that deletion of P2X3 receptors will blunt responses to gastric distension in vagal sensory neurons. Using wildtype and P2X3(-/-) mice, we examined responses to purinergic agonists in retrogradely labelled gastric sensory neurons with patch-clamp techniques. Activation of gastro-oesophageal neurons by fluid distension was studied with intracellular electrodes. Distension-evoked ATP release into the gastric lumen was determined with the luciferase assay and intake and gastric emptying of a solid meal was assessed. ATP triggered inward currents in 80% of gastric nodose neurons. In P2X3(-/-) mice, the peak current density was lower compared to controls. Ten of 14 controls but none of 30 neurons from P2X3(-/-) mice responded to alpha,beta-metATP. Gastro-oesophageal sensory neurons of P2X3(-/-) mice showed a blunted response to fluid distension of oesophagus and stomach. This difference was not explained by differences in distension-evoked ATP release, which did not differ between knockout mice and controls. Food intake during a 3-h period was lower in P2X3(-/-) mice. Gastric emptying of a solid meal was slightly faster in knockout mice after 1.5 h, but did not differ between groups at 3 h. Our data support a role of purinergic signalling in gastric vagal afferents. Considering the role of vagal input in sensations of fullness or nausea, P2X receptors may be interesting treatment targets for dyspeptic symptoms.

Electrical stimulation of the mucosa evokes slow EPSPs mediated by NK1 tachykinin receptors and by P2Y1 purinoceptors in different myenteric neurons

Slow excitatory postsynaptic potentials (EPSPs) in enteric neurons arise from diverse sources, but which neurotransmitters mediate specific types of slow EPSPs is unclear. We investigated transmitters and receptors mediating slow EPSPs in myenteric neurons evoked by electrical stimulation of the mucosa in guinea pig small intestine. Segments of ileum or jejunum were dissected to allow access to the myenteric plexus adjacent to intact mucosa, in vitro. AH and S neurons were impaled with conventional intracellular electrodes. Trains of stimuli delivered to the mucosa evoked slow EPSPs in AH neurons that were blocked or depressed by the neurokinin-1 (NK1) tachykinin antagonist SR140333 (100 nM) in 10 of 11 neurons; the NK3 tachykinin receptor antagonist SR142801 (100 nM) had no effect on slow EPSPs in seven of nine AH neurons. Single pulses to the mucosa evoked fast EPSPs and slow depolarizations in S neurons. The depolarizations were divided into intermediate (durations 300-900 ms) or slow (durations 1.3-9 s) EPSPs. The slow EPSPs were blocked by pyridoxal phosphate-6-axophenyl-2-4-disulfonic acid (30 microM, N = 3) or the specific P2Y(1) antagonist MRS 2179 (10 microM, N = 6) and were predominantly in anally projecting S neurons that were immunoreactive for nitric oxide synthase (NOS). In contrast, intermediate EPSPs were predominantly evoked in NOS-negative neurons; these were abolished by MRS 2179 (N = 8). Thus activation of pathways running from the mucosa excites three different types of slow EPSP in myenteric neurons, which are mediated by either a tachykinin (NK1, AH neurons) or a purine nucleotide (P2Y(1), S neurons).

Gap junctions in dorsal root ganglia: Possible contribution to visceral pain

Peripheral injuries can lead to sensitization of neurons in dorsal root ganglia (DRGs), which can contribute to chronic pain. The neurons are sensitized by a combination of physiological and biochemical changes, whose full details are still obscure. Another cellular element in DRGs are satellite glial cells (SGCs), which surround the neurons, but little is known about their role in nociception. We investigated the contribution of SGCs to neuronal sensitization in isolated S1 DRGs from a mouse model of colonic inflammation induced by local application of dinitrosulfonate benzoate (DNBS). Retrograde labeling was used to identify DRG neurons projecting to the colon. Cell-to-cell coupling was determined by intracellular dye injection, and the electrical properties of the neurons were studied with intracellular electrodes. Pain behavior was assessed with von-Frey hairs. The dye injections showed that 10–12 days after DNBS application there was a 6.6-fold increase in gap junction–mediated coupling between SGCs surrounding adjacent neurons, and this occurred preferentially (another 2-fold increase) near neurons that project to the colon. Neuron–neuron coupling incidence increased from 0.7% to 12.1% by colonic inflammation. Inflammation led to an augmented neuronal excitability, and to a reduced pain threshold. Gap junction blockers abolished the inflammation-induced changes in SGCs and neurons, and significantly reversed the pain behavior. We propose that inflammation induces augmented cell coupling in DRGs that contributes to neuronal hyperexcitability, which in turn leads to visceral pain. Gap junction blockers may have potential as analgesic drugs.

Divide et impera: optimizing compartmental models of neurons step by step

Compartmental models of neurons, introduced by Wilfrid Rall in 1964, have become important research tools for both theoretical and experimental neuroscientists to describe electrical (and sometimes also chemical) signalling in neurons. Usually built based on experimental data, they in turn help to interpret experiments, provide a quantitative description of neuronal function in contexts which are not yet directly accessible to experiment, and guide the development of theories of information processing in neurons and neural circuits. Detailed compartmental models, which incorporate anatomical reconstructions of the morphology of a neuron, biophysical descriptions of the kinetics and voltage dependence of its membrane conductances, as well as the membrane capacitance and intracellular resistivity, can predict the evolution in time and space of the membrane potential along the neuronal dendrites, soma and axon in response to arbitrary spatiotemporal patterns of synaptic input or current injection via intracellular electrodes. They can also form the basis for the construction of reduced, or simplified, neuronal models (reviewed by Herz et al. 2006).

Electrophysiological methods for recording synaptic potentials from the NMJ of Drosophila larvae.

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP’s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP’s and minature excitatory junction potentials (mEJP’s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP’s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Separation of single neurons from optical recordings in Tritonia diomedea using ICA

Event Abstract Back to Event Separation of single neurons from optical recordings in Tritonia diomedea using ICA Optical recordings obtained with photodiode arrays and voltage-sensitive dyes allow firing of large numbers of neurons to be recorded during behaviorally-relevant motor programs. Although this technique has tremendous potential for network studies, the resulting datasets can be challenging to interpret; one detector may record multiple neurons and one neuron may appear on many detectors. Earlier studies applied independent component analysis (ICA), a blind source separation technique, to optical traces from Tritonia diomedea's escape swim behavior, to recover components from individual neurons [1] and identify unreported neurons involved in the behavior [2]. Using intracellular and optical methods [3], we evaluate the accuracy of neuronal activity that ICA returns and demonstrate an application of these methods, measuring the change in individual neurons’ activity following stimulus adaptation. To evaluate accuracy, we recorded from neurons in Tritonia with intracellular electrodes while imaging with a 464-element photodiode array using the fast voltage-sensitive absorbance dye RH-155. ICA was run on the optical traces, transforming them into statistically independent components. ICA returned one component for neurons detected by multiple diodes and separated neurons mixed on a single diode. Intracellular recordings confirmed the accuracy of this technique. For each intracellular recording, we found a component that corresponded exactly. Also, the location returned by ICA matched that of the electrode. Additionally, these methods allow us to drive an identified neuron and image its functional connectivity to large numbers of other individual neurons, before and after a treatment of interest. We imaged the responses of several pedal ganglion neurons to a test train of action potentials in CPG neuron C2 driven with an intracellular electrode. Then we applied a sensitizing nerve shock stimulus. Two minutes later we imaged the responses of the same neurons to a second test train. Combining the records and applying ICA, we observed how responses of individual neurons changed after the sensitizing stimulus. Optical recordings in combination with ICA allow quick identification of functional synaptic connections onto previously unknown neurons. Using location maps from ICA, those neurons can be penetrated with intracellular electrodes within the same experiment, enabling new experiments to probe the Tritonia brain over a wide range of conditions and time scales. Given our intracellular validation of ICA's unmixing, these methods promise to be a powerful tool for discovery of unreported neurons and studies of functional connectivity and network plasticity.

Extracellular Charge Adsorption Influences Intracellular Electrochemical Homeostasis in Amphibian Skeletal Muscle

The membrane potential measured by intracellular electrodes, E m, is the sum of the transmembrane potential difference ( E 1) between inner and outer cell membrane surfaces and a smaller potential difference ( E 2) between a volume containing fixed charges on or near the outer membrane surface and the bulk extracellular space. This study investigates the influence of E 2 upon transmembrane ion fluxes, and hence cellular electrochemical homeostasis, using an integrative approach that combines computational and experimental methods. First, analytic equations were developed to calculate the influence of charges constrained within a three-dimensional glycocalyceal matrix enveloping the cell membrane outer surface upon local electrical potentials and ion concentrations. Electron microscopy confirmed predictions of these equations that extracellular charge adsorption influences glycocalyceal volume. Second, the novel analytic glycocalyx formulation was incorporated into the charge-difference cellular model of Fraser and Huang to simulate the influence of extracellular fixed charges upon intracellular ionic homeostasis. Experimental measurements of E m supported the resulting predictions that an increased magnitude of extracellular fixed charge increases net transmembrane ionic leak currents, resulting in either a compensatory increase in Na +/K +-ATPase activity, or, in cells with reduced Na +/K +-ATPase activity, a partial dissipation of transmembrane ionic gradients and depolarization of E m.

Electrophysiological and morphological properties of submucosal neurons in the mouse distal colon

The pathophysiology of intestinal secretion is increasingly studied using transgenic mouse models of colonic inflammation, but little is known about the properties of single submucosal neurons regulating epithelial secretion in the mouse. Therefore, these neurons were characterized in the mouse distal colon submucosal plexus using electrophysiological and morphological techniques. Action potentials and synaptic responses in single neurons were recorded in submucosal preparations in vitro using intracellular electrodes filled with neurobiotin, enabling subsequent morphological examination. Most neurons (approximately 90%) were classified as S-type neurons based on the presence of fast excitatory postsynaptic potentials (EPSPs), absence of prolonged after-hyperpolarizations, and uni-axonal morphology. These neurons rarely fired more than one action potential in response to intracellular depolarization but spontaneous fast EPSPs were observed. Fibre tract stimulation (20 Hz) elicited slow EPSPs in many neurons (37%) and slow inhibitory potentials were also observed in a subset of neurons (18%). None of these cells exhibited electrophysiological features suggestive of AH neurons. However, morphological studies demonstrated that 12% of neurons had two axons, and an immunohistochemical marker of mouse AH neurons, calcitonin gene-related peptide, was co-localized with the pan-neuronal marker HuD in 9% of neurons. Cell counts indicated that mouse distal colon ganglia were much smaller (> 80% contain one to five neurons) compared to guinea-pig distal colon ganglia (approximately 30% contain >10 neurons). This study has shown that mouse distal colon submucosal ganglia are relatively small and, based on combined morphological and electrophysiological features, most neurons exhibit S-type characteristics.

Cell Self Assembly of Intracellular Interface Using Cell Migration

AbstractA cell-driven self-assembly of intracellular nano-device was proposed for bio-hybrid interface. This cells-driven self-assembly employed cell migration force to insert a conductive nanoneedle which would be worked as intracellular electrode. Such a nanoneedle was fabricated in the bottom of a microwell using focused ion beam induce deposition. The microwell structure with a coating of cell adhesion molecules was employed as the scaffold of the cell migration. A glass plate with the microwells had a non cell binding coating of 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer as an anti-biofouling material. Thus a cell adhered only on the wall of a microwell then the cell migrated into the microwell. Adhesion force and migration force induced self-insertion of the nanoneedle into a live cell body using the cell's own migration force. The inserted nanoneedle was made of electrical conductive tungsten, so the intracellular nanoneedle might extract intracellular potential more precisely than extracellular electrode, while inducing much less damage to cells. In the future, the technique of cell-driven self-insertion of nanoneedle may be integrated with multi electrode arrays for developing long-lasting measurements device on cellular network researches, or the risk assessment of the nanomaterials on cellular activities.