Double Patch Clamp Research Articles

Single or Double Patch-Clamp Recordings In Ex Vivo Slice Preparation: Functional Connectivity, Synapse Dynamics, and Optogenetics.

Patch-clamp recordings are the method of choice to define cell-type specific electrophysiological properties of single neurons and the synaptic connectivity between pairs of connected neurons in brain slices. In combination with optogenetic tools, patch-clamp recordings allow for the investigation of long-range afferent connectivity from identified distant brain areas. Here we describe the necessary equipment to carry out patch clamp recordings, surgical methods for dissection and preparation of horizontal brain slices containing the hippocampus, and a step-by-step guide for establishing patch clamp recordings in the whole-cell configuration. We provide protocols for single neuron stimulation via the patch pipette and for photostimulation experiments that activate axon terminals expressing light sensitive ion channels.

Mutual inhibition circuit as underlying mechanism for bi-stable perception and non-linear responses in vision

It has been hypothesized that mutual inhibition circuit underlies bi-stable perception. It is possible that this circuit functions to resolve ambiguity of input image by quickly shifting the balance of competing signals in response to conflicting features. Furthermore, it is possible that feedback signals influence this "biased competition" to reflect global properties of the image. I developed a system to record from two pyramidal cells in primary visual cortex in vitro by double patch clamp recording technique combined with so-called "dynamic clamp" system. In this system, two neurons can be connected by modeled synapses. When a connection between the two neurons are established by mutually inhibiting model synapses, simultaneous activation of the two neurons caused bi-stable activities: dominance of neural activities alternated between the two neurons in about 1~2 seconds interval. Next, I ran paradigms analogous to various experimental paradigms used for behavioral studies of bi-stable perception. Levelt's 4th law states that increasing the strength of input contrast causes increase of switching frequency. Similarly, increasing the depolarizing current injected to both neurons showed increase of switching frequencies at the higher range of injected current. Flash suppression paradigm was modeled by injecting depolarizing current to one neuron for few seconds followed by simultaneous injections of the current to the two neurons. The simultaneous injection evoked bi-stability that mostly started with the dominant activity of the non-adapted neuron. Intermittent presentation of bi-stable figure is known to prolong the percept duration. When this paradigm was modeled by injecting depolarizing current intermittently, the prolongation effect was not observed. Finally, by placing a stimulus electrode to V2, feedback excitation was evoked. The stimulation often caused the switch of dominance. Hence, feedback signals can influence the outcome of the competition. With this system, the details of neural processes that undergo bi-stable activities can be investigated. Meeting abstract presented at VSS 2017

Unidirectional signal propagation in primary neurons micropatterned at a single-cell resolution.

The structure and connectivity of cultured neuronal networks can be controlled by using micropatterned surfaces. Here, we demonstrate that the direction of signal propagation can be precisely controlled at a single-cell resolution by growing primary neurons on micropatterns. To achieve this, we first examined the process by which axons develop and how synapses form in micropatterned primary neurons using immunocytochemistry. By aligning asymmetric micropatterns with a marginal gap, it was possible to pattern primary neurons with a directed polarization axis at the single-cell level. We then examined how synapses develop on micropatterned hippocampal neurons. Three types of micropatterns with different numbers of short paths for dendrite growth were compared. A normal development in synapse density was observed when micropatterns with three or more short paths were used. Finally, we performed double patch clamp recordings on micropatterned neurons to confirm that these synapses are indeed functional, and that the neuronal signal is transmitted unidirectionally in the intended orientation. This work provides a practical guideline for patterning single neurons to design functional neuronal networks in vitro with the direction of signal propagation being controlled.

Slow chemical transmission between dorsal root ganglion neuron somata

Somatic sensory neuron somata are located within the dorsal root ganglia (DRG) and are mostly ensheathed by individual satellite glial cell sheets. It has been noted, however, that a subpopulation of these DRG somata are intimately associated, separated only by a single thin satellite glial cell membrane septum. We set out to test whether such neuron-glial cell-neuron trimers (NGlNs) are also linked functionally. The presence of NGlNs in chick DRGs was confirmed by electron microscopy. Selective satellite glial cell immunostains were identified and were used to image the inter-neuron septa in DRG frozen sections. We used a gentle, dispase-based enzymatic method to isolate chick and rat NGlNs in vitro for double patch clamp recordings. In the majority of pairs tested, an action potential-like stimulus train delivered to one soma resulted in a delayed, noisy and long-duration response in its idle partner. The response to a second stimulus train given minutes later was markedly facilitated. Both bidirectional and unidirectional transmission was observed between the paired neurons. Transmission was chemical and block by the general purinergic blocker suramin implicated ATP as a neurotransmitter. We conclude that the two neuronal somata in the NGlN can communicate by chemical transmission, which may involve a transglial, bi-synaptic pathway. This novel soma-to-soma transmission reflects a novel form of processing that may play a role in sensory disorders in the DRG and interneuron communication in the central nervous system.

Molecular Mechanisms Governing Cx26/Cx32 Heterotypic Docking and Functional Gap Junction Channel Formation

Based on the crystal structure of Cx26 gap junction channel, we generated homology models of Cx26/Cx32 heterotypic channels and examined docking compatibility of wildtype and mutant Cx26/Cx32 hemichannel pairs. According to the model, 60 hydrogen bonds (HBs) were indentified between a pair of Cx32/Cx26 hemichannels. The HB-forming residues between E2 domains are completely conserved in a group of heterotypically compatible connexins. Asparagine175 in E2 domain of Cx32 was a pivotal residue forming 3 HBs with K168, T177 and D179 of inter-docked Cx26. Cx32 mutations, N175Y or N175H, destroy 3/3 or 2/3 HBs, respectively, at the E2-E2 docking interface. Our models predict that the Cx32 N175Y and N175H hemichannels are unlikely to dock with Cx26 hemichannels properly due to steric hindrance and/or electrostatic repulsion, resulting in loss of HBs at the docking interface.Experimentally, we tagged RFP and GFP to the carboxyl terminals of wild-type and mutant Cx26, Cx32 respectively. Cx32 N175Y/H-GFPs showed impaired trafficking to cell-cell interfaces. Double patch-clamp analyses demonstrated that these Cx32 mutants did not form functional homotypic channels with themselves indicating docking defects. When Cx32-GFP expressing cells were co-cultured with Cx26-RFP expressing cells, these two wild-type connexins were frequently co-localized at the cell-cell interfaces and formed functional Cx32/Cx26 heterotypic channels with asymmetrical voltage gating profile. No co-localization was found at the cell-cell interfaces between Cx26 and Cx32 N175Y or N175H expressing cells; also no functional N175Y/Cx26 and N175H/Cx26 heterotypic channels were recorded. Interestingly, when a Cx26 mutant K168V was paired with Cx32 N175H, heterotypic docking was restored as evidenced by co-localization at cell-cell junctions and asymmetrical electrical coupling.Our results demonstrate that HBs and spatial compatibility surrounding N175 of Cx32 are key factors for heterotypic docking between Cx32 and Cx26 hemichannels.

Asparagine 175 of Connexin32 Is a Critical Residue for Docking and Forming Functional Heterotypic Gap Junction Channels with Connexin26

The gap junction channel is formed by proper docking of two hemichannels. Depending on the connexin(s) in the hemichannels, homotypic and heterotypic gap junction channels can be formed. Previous studies suggest that the extracellular loop 2 (E2) is an important molecular domain for heterotypic compatibility. Based on the crystal structure of the Cx26 gap junction channel and homology models of heterotypic channels, we analyzed docking selectivity for several hemichannel pairs and found that the hydrogen bonds between E2 domains are conserved in a group of heterotypically compatible hemichannels, including Cx26 and Cx32 hemichannels. According to our model analysis, Cx32N175Y mutant destroys three hydrogen bonds in the E2-E2 interactions due to steric hindrance at the heterotypic docking interface, which makes it unlikely to dock with the Cx26 hemichannel properly. Our experimental data showed that Cx26-red fluorescent protein (RFP) and Cx32-GFP were able to traffic to cell-cell interfaces forming gap junction plaques and functional channels in transfected HeLa/N2A cells. However, Cx32N175Y-GFP exhibited mostly intracellular distribution and was occasionally observed in cell-cell junctions. Double patch clamp analysis demonstrated that Cx32N175Y did not form functional homotypic channels, and dye uptake assay indicated that Cx32N175Y could form hemichannels on the cell surface similar to wild-type Cx32. When Cx32N175Y-GFP- and Cx26-RFP-transfected cells were co-cultured, no colocalization was found at the cell-cell junctions between Cx32N175Y-GFP- and Cx26-RFP-expressing cells; also, no functional Cx32N175Y-GFP/Cx26-RFP heterotypic channels were identified. Both our modeling and experimental data suggest that Asn(175) of Cx32 is a critical residue for heterotypic docking and functional gap junction channel formation between the Cx32 and Cx26 hemichannels.

Asparagine175 of Cx32 is a Critical Residue for Docking and Forming Functional Heterotypic Gap Junction Channels with Cx26

Gap junctions result from the docking of two hemichannels. Depend on the connexin(s) in the hemichannels, homotypic and heterotypic gap junction channels can be formed. Previous chimera studies with domain exchange between different connexins indicated that the extracellular loop2 (E2) is important molecular domain to heterotypic compatibility. Based on the high resolution structure of Cx26 gap junction channel and homology models of heterotypic channels, we analyzed docking selectivity for several hemichannel pairs, and found that the hydrogen bonds between E2 domains are conserved in several heterotypically compatible hemichannels, e.g. between Cx26 and Cx32. According to our model analysis, Cx32 mutation, Cx32N175Y, destroys three hydrogen bonds in the E2-E2 interactions at the heterotypic docking interface. Our model predicts that the Cx32N175Y hemichannel is unlikely to dock with Cx26 hemichannel properly due to steric hindrance at the docking interface. Experimentally, we tagged GFP and RFP to the carboxyl terminals of Cx32 and Cx26 to generate Cx32GFP (Cx32N175YGFP) and Cx26RFP, respectively. Our data showed that tagged Cx26 and Cx32 were able to traffic to cell interfaces forming gap junction plaque-like structures in transfected HeLa/N2A cells. However, Cx32N175YGFP exhibited mostly intracellular distribution and rarely seen in cell-cell interfaces. Double patch clamp analysis demonstrated that this Cx32 mutant did not form functional homotypic channels. When wild-type or mutant Cx32GFP expressing cells were cocultured with Cx26RFP expressing cells, Cx32GFP and Cx26RFP were frequently colocalized at the cell-cell interfaces and formed functional Cx32/Cx26 heterotypic channels. No colocalization was found at the cell-cell interfaces between Cx32N175Y and Cx26 expressing cells, also no functional Cx32N175Y/Cx26 heterotypic channels were identified. Our modeling and experimental data indicate that N175 of Cx32 is a critical residue for heterotypic docking between Cx32 and Cx26 hemichannels.

A new experimental model of focal seizures in the entorhinal cortex

Despite intensive studies, our understanding of the cellular and molecular mechanisms underlying epileptogenesis remains largely unsatisfactory. Our defective knowledge derives in part from the lack of adequate experimental models of the distinct phases that characterize the epileptic event, that is, initiation, propagation, and cessation. The aim of our study is the development of a new brain slice model in which a focal seizure can be repetitively evoked at a precise and predictable site. Epileptiform activities were studied by fast Ca²(+) imaging coupled with simultaneous single and double patch-clamp or extracellular recordings from neurons of entorhinal cortex (EC) slices from Wistar rats and C57BL6J mice at postnatal days 13-17. In the presence of 4-aminopyridine (4-AP) and low Mg²(+) , activation of layer V-VI neurons by local N-methyl-d-aspartate (NMDA) applications evolved into an ictal discharge (ID) that propagated to the entire EC. NMDA-evoked IDs were similar to spontaneous events. IDs with similar pattern and duration could be repetitively triggered from the same site by successive NMDA stimulations. The high ID reproducibility is an important feature of the model that allowed testing of the effects of currently used antiepileptic drugs (AEDs) on initiation, propagation, and cessation of focal ictal events. By offering the unique opportunity to repetitively evoke an ID from the same restricted site, this model represents a powerful approach to study the cellular and molecular events at the basis of initiation, propagation, and cessation of focal seizures.

Phosphorylation of connexin43 on serine 306 regulates electrical coupling

Phosphorylation is a key regulatory event in controlling the function of the cardiac gap junction protein connexin43 (Cx43). Three new phosphorylation sites (S296, S297, S306) have been identified on Cx43; two of these sites (S297 and S306) are dephosphorylated during ischemia. The functional significance of these new sites is currently unknown. The purpose of this study was to examine the role of S296, S297, and S306 in the regulation of electrical intercellular communication. To mimic constitutive dephosphorylation, serine was mutated to alanine at the three sites and expressed in HeLa cells. Electrical coupling and single channel measurements were performed by double patch clamp. Protein expression levels were assayed by western blotting, localization of Cx43, and phosphorylation of S306 by immunolabeling. Free hemichannels were assessed by biotinylation. Macroscopic conductance in cells expressing S306A was reduced to 57% compared to wild type (WT), whereas coupling was not significantly changed in cells expressing either S296A or S297A. S306A-expressing cells displayed similar protein and free hemichannel abundance compared to WT Cx43, whereas the fractional area of plaques in cell-to-cell interfaces was increased. However, single channel measurements showed a WT Cx43 main state conductance of 119 pS, whereas the main state conductance of S306A channels was reduced to 95 pS. Furthermore, channel gating was affected in S306A channels. Lack of phosphorylation at serine 306 results in reduced coupling, which can be explained by reduced single channel conductance. We suggest that dephosphorylation of S306 partly explains the electrical uncoupling seen in myocardial ischemia.

Exocytosis of Post-Golgi Vesicles Is Regulated by Components of the Endocytic Machinery

Post-Golgi vesicles target and deliver most biosynthetic cargoes to the cell surface. However, the molecules and mechanisms involved in fusion of these vesicles are not well understood. We have employed a system to simultaneously monitor release of luminal and membrane biosynthetic cargoes from individual post-Golgi vesicles. Exocytosis of these vesicles is not calcium triggered. Release of luminal cargo can be accompanied by complete, partial, or no release of membrane cargo. Partial and no release of membrane cargo of a fusing vesicle are fates associated with kiss-and-run exocytosis, and we find that these are the predominant mode of post-Golgi vesicle exocytosis. Partial cargo release by post-Golgi vesicles occurs because of premature closure of the fusion pore and is modulated by the activity of clathrin, actin, and dynamin. Our results demonstrate that these components of the endocytic machinery modulate the nature and extent of biosynthetic cargo delivery by post-Golgi vesicles at the cell membrane.

Coordinated Synchronization in the Electrically Coupled Network of Terminal Nerve Gonadotropin-Releasing Hormone Neurons as Demonstrated by Double Patch-Clamp Study

The peptidergic neurons play important roles such as neuromodulatory and neuroendocrine functions in the central nervous system. However, our knowledge about the organization and the function of the peptidergic neuromodulator systems is still very poor. The terminal nerve GnRH peptidergic neurons of a teleost, the dwarf gourami (Colisa lalia), serve as an excellent model system for such study. The cell bodies are large and make up a tight cell cluster, and the easy access to the cell bodies on the ventral surface of the brain makes the electrophysiological measurements in a precisely controlled manner. Here we show direct evidence to demonstrate the electrical coupling and the synchronization of the neural firing activity among the terminal nerve GnRH neurons by using the double patch-clamp recording technique. The electrical coupling coefficient was strong enough (ranged from 0.083 to 0.370) to synchronize spontaneous firings of GnRH neurons in the cluster. A model, in which the firings in the cluster occur within a small time window (dozens of milliseconds), was verified by using the serial loose-seal extracellular patch-clamp recordings and the cross-correlogram analysis. The present findings provide several insights for understanding the physiological mechanisms and functional significance of synchronized activities in the peptidergic and/or aminergic neuromodulator system as well as in the peptidergic neuroendocrine cells.

Nitric oxide specifically reduces the permeability of Cx37-containing gap junctions to small molecules.

Gap junction intercellular communication (GJIC) plays a significant role in the vascular system. Regulation of GJIC is a dynamic process, with alterations in connexin (Cx) protein expression and post-translational modification as contributing mechanisms. We hypothesized that the endothelial autacoid nitric oxide (NO) would reduce dye coupling in human umbilical vein endothelial cells (HUVECs). In our subsequent experiments, we sought to isolate the specific Cx isoform(s) targeted by NO or NO-activated signaling pathways. Since HUVEC cells variably express three Cx (Cx37, Cx40, and Cx43), this latter aim required the use of transfected HeLa cells (HeLaCx37, HeLaCx43), which do not express Cx proteins in their wild type form. Dye coupling was measured by injecting fluorescent dye (e.g., Alexa Fluor 488) into a single cell and determining the number of stained adjacent cells. Application of the NO donor SNAP (2 microM, 20 min) reduced dye coupling in HUVEC by 30%. In HeLa cells, SNAP did not reduce dye transfer of cells expressing Cx43, but decreased the dye transfer from Cx37-expressing cells to Cx43-expressing cells by 76%. The effect of SNAP on dye coupling was not mediated via cGMP. In contrast to its effect on dye coupling, SNAP had no effect on electrical coupling, measured by a double patch clamp in whole cell mode. Our results demonstrate that NO inhibits the intercellular transfer of small molecules by a specific influence on Cx37, suggesting a potential role of NO in controlling certain aspects of vascular GJIC.

Impact of micropatterned surfaces on neuronal polarity

Experimental control over cellular polarity in a neuronal network is a promising tool to study synapse formation and network behavior. We aimed to exploit a mechanism described by Stenger et al. [J. Neurosci. Methods 82 (1998) 167] to manipulate the direction of axonal versus dendritic outgrowth on a micropattern. The group had used laser ablation to create patterns of aminated silanes for cell attachment on a background of repellent fluorinated silanes. The pattern offered continuous adhesive pathways for axonal and interrupted pathways for dendritic outgrowth. By microcontact printing, we created similar patterns containing continuous and interrupted pathways consisting of extracellular matrix proteins on a background of polystyrene. Neuronal polarity was determined on the functional level through double patch clamp measurements, detecting synapses and their orientation. Although our pattern reproduced the properties that were assumed to be critical for the described effect, namely contrasting pathways of different adhesiveness, we failed to reproduce the above results. It is indicated that other qualities of alternative pathways than mere differences in adhesiveness are required to orient neuronal polarity in vitro. We suggest that the effect observed by Stenger et al. has to be attributed to less universal characteristics of the micropattern, e.g. to the specific chemical groups that were utilized.

Role of cyclic nucleotide phosphodiesterase isoforms in cAMP compartmentation following beta2-adrenergic stimulation of ICa,L in frog ventricular myocytes.

The role of cyclic nucleotide phosphodiesterase (PDE) isoforms in the beta2-adrenergic stimulation of the L-type Ca2+ current (ICa,L) was investigated in frog ventricular myocytes using double patch-clamp and double-barrelled microperfusion techniques. Isoprenaline (ISO, 1 nM to 10 microM) was applied on one half of the cell, either alone or in the presence of PDE inhibitors, and the local and distant responses of ICa,L were used to determine the gradient of local vs. distant cAMP concentration (alpha). IBMX (100 microM), a non-selective PDE inhibitor, reduced alpha from 40 to 4.4 indicating a 9-fold reduction in intracellular cAMP compartmentation when all PDE activity was blocked. While PDE1 and PDE2 inhibition had no effect, PDE3 inhibition by milrinone (3 microM) or PDE4 inhibition by Ro 20-1724 (3 microM) reduced alpha by 6- and 4-fold, respectively. A simultaneous application of milrinone and Ro 20-1724 produced a similar effect to IBMX, showing that PDE3 and PDE4 were the major PDEs accounting for cAMP compartmentation. Okadaic acid (3 microM), a non-selective phosphatase inhibitor, or H89 (1 microM), an inhibitor of cAMP-dependent protein kinase (PKA), had no effect on the distant response of ICa,L to ISO indicating that PDE activation by PKA played a minor role in cAMP compartmentation. Our results demonstrate that PDE activity determines the degree of cAMP compartmentation in frog ventricular cells upon beta2-adrenergic stimulation. PDE3 and PDE4 subtypes play a major role in this process, and contribute equally to ensure a functional coupling of beta2-adrenergic receptors with nearby Ca2+ channels via local elevations of cAMP.

Role of cyclic nucleotide phosphodiesterase isoforms in cAMP compartmentation following beta2-adrenergic stimulation of ICa,L in frog ventricular myocytes

The role of cyclic nucleotide phosphodiesterase (PDE) isoforms in the β2-adrenergic stimulation of the L-type Ca2+ current (ICa,L) was investigated in frog ventricular myocytes using double patch-clamp and double-barrelled microperfusion techniques. Isoprenaline (ISO, 1 nM to 10 μM) was applied on one half of the cell, either alone or in the presence of PDE inhibitors, and the local and distant responses of ICa,L were used to determine the gradient of local vs. distant cAMP concentration (α). IBMX (100 μM), a non-selective PDE inhibitor, reduced α from 40 to 4.4 indicating a 9-fold reduction in intracellular cAMP compartmentation when all PDE activity was blocked. While PDE1 and PDE2 inhibition had no effect, PDE3 inhibition by milrinone (3 μM) or PDE4 inhibition by Ro 20-1724 (3 μM) reduced α by 6- and 4-fold, respectively. A simultaneous application of milrinone and Ro 20-1724 produced a similar effect to IBMX, showing that PDE3 and PDE4 were the major PDEs accounting for cAMP compartmentation. Okadaic acid (3 μM), a non-selective phosphatase inhibitor, or H89 (1 μM), an inhibitor of cAMP-dependent protein kinase (PKA), had no effect on the distant response of ICa,L to ISO indicating that PDE activation by PKA played a minor role in cAMP compartmentation. Our results demonstrate that PDE activity determines the degree of cAMP compartmentation in frog ventricular cells upon β2-adrenergic stimulation. PDE3 and PDE4 subtypes play a major role in this process, and contribute equally to ensure a functional coupling of β2-adrenergic receptors with nearby Ca2+ channels via local elevations of cAMP.

Effects of n‐alcohols on junctional coupling and amylase secretion of pancreatic acinar cells

We have tested the effects of alcohols differing by their alkyl chain length on the membrane channels and amylase secretion of rat pancreatic acinar cells. In intact acini, alcohols with a chain of seven, eight, or nine carbons (C-7, C-8, and C-9) induced dye uncoupling and increased basal amylase release. These effects were readily reversible after alcohol removal. By contrast, an alcohol with a chain of 15 carbons (C-15) and several alcohols with chains of fewer than six carbons (C-2, C-4, and C-6) did not uncouple acinar cells and had no effects of amylase secretion. Neither did alkanes and oxidized derivatives of C-7 and C-8 alcohols did not affect dye coupling. Double patch-clamp experiments on pairs of acinar cells, under conditions of strong cytosolic Ca2+ and pH buffering, showed that C-7, C-8, and C-9 alcohols blocked completely and reversibly the electrical conductance of junctional channels. Furthermore, studies of single voltage-clamped acinar cells revealed that the uncoupling alcohols did not affect the resting nonjunctional membrane conductances. Thus the alcohols that did not affect acinar cells coupling did not affect amylase secretion, whereas the alcohols that caused uncoupling increased secretion. The latter effect was not mediated by changes in the conductance of nonjunctional membrane, cytosolic Ca2+, and pH and, as revealed by an immunological hemolytic plaque assay for amylase, had a time course consistent with the rapid (within 1 min) inhibition of coupling. These data provide new support for the view that the regulation of cell-to-cell communications is correlated with that of digestive enzyme secretion.