Margaret Lin Veruki

Animal cells connected by nanotubes can be electrically coupled through interposed gap-junction channels

Tunneling nanotubes (TNTs) are recently discovered conduits for a previously unrecognized form of cell-to-cell communication. These nanoscale, F-actin–containing membrane tubes connect cells over long distances and facilitate the intercellular exchange of small molecules and organelles. Using optical membrane-potential measurements combined with mechanical stimulation and whole-cell patch-clamp recording, we demonstrate that TNTs mediate the bidirectional spread of electrical signals between TNT-connected normal rat kidney cells over distances of 10 to 70 μm. Similar results were obtained for other cell types, suggesting that electrical coupling via TNTs may be a widespread characteristic of animal cells. Strength of electrical coupling depended on the length and number of TNT connections. Several lines of evidence implicate a role for gap junctions in this long-distance electrical coupling: punctate connexin 43 immunoreactivity was frequently detected at one end of TNTs, and electrical coupling was voltage-sensitive and inhibited by meclofenamic acid, a gap-junction blocker. Cell types lacking gap junctions did not show TNT-dependent electrical coupling, which suggests that TNT-mediated electrical signals are transmitted through gap junctions at a membrane interface between the TNT and one cell of the connected pair. Measurements of the fluorescent calcium indicator X-rhod-1 revealed that TNT-mediated depolarization elicited threshold-dependent, transient calcium signals in HEK293 cells. These signals were inhibited by the voltage-gated Ca2+ channel blocker mibefradil, suggesting they were generated via influx of calcium through low voltage-gated Ca2+ channels. Taken together, our data suggest a unique role for TNTs, whereby electrical synchronization between distant cells leads to activation of downstream target signaling.

Activation of a presynaptic glutamate transporter regulates synaptic transmission through electrical signaling.

Whereas glutamate transporters in glial cells and postsynaptic neurons contribute significantly to re-uptake of synaptically released transmitter, the functional role of presynaptic glutamate transporters is poorly understood. Here, we used electrophysiological recording to examine the functional properties of a presynaptic glutamate transporter in rat retinal rod bipolar cells and its role in regulating glutamatergic synaptic transmission between rod bipolar cells and amacrine cells. Release of glutamate activated the presynaptic transporter with a time course that suggested a perisynaptic localization. The transporter was also activated by spillover of glutamate from neighboring rod bipolar cells. By recording from pairs of rod bipolar cells and AII amacrine cells, we demonstrate that activation of the transporter-associated anion current hyperpolarizes the presynaptic terminal and thereby inhibits synaptic transmission by suppressing transmitter release. Given the evidence for presynaptic glutamate transporters, similar mechanisms could be of general importance for transmission in the nervous system.

AII (Rod) Amacrine Cells Form a Network of Electrically Coupled Interneurons in the Mammalian Retina

AII (rod) amacrine cells in the mammalian retina are reciprocally connected via gap junctions, but there is no physiological evidence that demonstrates a proposed function as electrical synapses. In whole-cell recordings from pairs of AII amacrine cells in a slice preparation of the rat retina, bidirectional, nonrectifying electrical coupling was observed in all pairs with overlapping dendritic trees (average conductance approximately 700 pS). Coupling displayed characteristics of a low-pass filter, with no evidence for amplification of spike-evoked electrical postsynaptic potentials by active conductances. Coincidence detection, as well as precise temporal synchronization of subthreshold membrane potential oscillations and TTX-sensitive spiking, was commonly observed. These results indicate a unique mode of operation and integrative capability of the network of AII amacrine cells.

Cellular and subcellular expression of monocarboxylate transporters in the pigment epithelium and retina of the rat

The cellular and subcellular expression of the monocarboxylate transporters MCT1, MCT2 and MCT4 [corresponding to MCT3 of Price N. T. et al. (1998) Biochem. J. 329, 321-328] were investigated in the pigment epithelium and outer retina of rats. Immunofluorescence and postembedding immunogold analyses revealed strong MCT1 labelling in the apical membrane of the pigment epithelial and no detectable signal in the basolateral membrane. In contrast, antibodies to the glucose transporter GLUT1 produced intense labelling in both membranes. Neither MCT1 nor GLUT1 was enriched in intracellular compartments. The monocarboxylate transporter MCT4 was very weakly expressed in the retinal pigment epithelium of adult animals, but occurred at higher concentrations at this site in 14-day-old rats. However, even at the latter stage, the immunolabelling of MCT4 was weak compared to that of MCT1. In the neural retina, the data were consistent with a predominant glial localization of MCT1. Specifically, immunogold particles signalling MCT1 occurred in Müller cell microvilli and in the velate processes between the photoreceptors. No labelling was obtained with antibodies to MCT2. Taken together with previous biochemical analyses, the present findings indicate that MCT1 is involved in the outward transport of lactate through the retinal pigment epithelial cells, and in the transfer of lactate between Müller cells and photoreceptors.

Functional properties of spontaneous EPSCs and non-NMDA receptors in rod amacrine (AII) cells in the rat retina

The functional properties of spontaneous, glutamatergic EPSCs and non‐NMDA receptors in AII amacrine cells were studied in whole cells and patches from slices of the rat retina using single and dual electrode voltage clamp recording. Pharmacological analysis verified that the EPSCs (Erev∼0 mV) were mediated exclusively by AMPA‐type receptors. EPSCs displayed a wide range of waveforms, ranging from simple monophasic events to more complex multiphasic events. Amplitude distributions of EPSCs were moderately skewed towards larger amplitudes (modal peak 23 pA). Interevent interval histograms were best fitted with a double exponential function. Monophasic, monotonically rising EPSCs displayed very fast kinetics with an average 10–90 % rise time of ∼340 μs and a decay phase well fitted by a single exponential (τdecay∼760 μs). The specific AMPA receptor modulator cyclothiazide markedly slowed the decay phase of spontaneous EPSCs (τdecay∼3 ms). An increase in temperature decreased both 10–90 % rise time and τdecay with Q10 values of 1.3 and 1.5, respectively. The decay kinetics were slower at positive membrane potentials compared to negative membrane potentials (205 mV/e‐fold change in τdecay). Step depolarization of individual presynaptic rod bipolar cells or OFF‐cone bipolar cells evoked transient, CNQX‐sensitive responses in AII amacrine cells with average peak amplitudes of ∼330 pA. Ultrafast application of brief (∼1 ms) or long (∼500 ms) pulses of glutamate to outside‐out patches evoked strongly desensitizing responses with very fast deactivation and desensitization kinetics, well fitted by single (τdecay∼1.1 ms) and double exponential (τ1∼3.5 ms; τ2∼21 ms) functions, respectively. Double‐pulse experiments indicated fast recovery from desensitization (τ∼12.4 ms). Our results indicate that spontaneous, AMPA receptor‐mediated EPSCs in AII amacrine cells have very fast, voltage‐dependent kinetics that can be well accounted for by the kinetic properties of the AMPA receptors themselves.

Functional characteristics of non-NMDA-type ionotropic glutamate receptor channels in AII amacrine cells in rat retina.

The properties of non‐NMDA glutamate receptor channels in AII amacrine cells were studied by patch‐clamp recording from rat retinal slices. Application of AMPA or kainate to intact cells evoked currents with no apparent desensitization (EC50 of 118 μM for AMPA and 169 μM for kainate). Application of AMPA to patches evoked desensitizing responses with an EC50 of 217 and 88 μM for the peak and steady‐state responses, respectively. Kainate‐evoked responses of patches displayed no desensitization (EC50= 162 μM). Cyclothiazide strongly potentiated AMPA‐evoked responses and the AMPA‐receptor antagonist GYKI 53655 inhibited both AMPA‐ and kainate‐evoked responses (IC50= 0.5–1.7 μM). Pre‐equilibration with GYKI 53655 completely blocked the response to kainate and pretreatment with concanavalin A did not unmask a response mediated by kainate receptors. AMPA‐ and kainate‐evoked currents reversed close to 0 mV. AMPA‐evoked peak and steady‐state response components in patches displayed linear and outwardly rectifying I–V relations with an RI (ratio of the slope conductances at +40 mV and ‐60 mV) of 0.96 ± 0.11 and 5.6 ± 1.3, respectively. AMPA‐evoked currents displayed a voltage‐dependent relaxation after steps to positive or negative membrane potentials, indicating that the outward rectification of the steady‐state response is caused by a voltage‐dependent kinetic parameter of channel gating. Under bi‐ionic conditions ([Ca2+]out= 30 mm, [Cs+]in= 171 mm), the reversal potentials of AMPA‐ and kainate‐evoked currents indicated channels with significant Ca2+ permeability (PCa/PCs= 1.9–2.1). Stationary noise analysis indicated that kainate activated channels with an apparent chord conductance of ∼9 pS. Non‐stationary noise analysis indicated that AMPA and glutamate activated channels with apparent chord conductances of ∼9, ∼15, ∼23 and ∼38 pS. Discrete single‐channel gating corresponding to chord conductances of ∼23 pS could be directly observed in some responses. Thus, our results indicate expression of high‐affinity, voltage‐sensitive AMPA receptors with significant Ca2+ permeability and relatively large single‐channel chord conductances in AII amacrine cells.

AII amacrine cells express functional NMDA receptors

NMDA and non-NMDA receptors mediate the majority of fast excitatory synaptic transmission in the CNS. AII amacrine cells in the mammalian retina receive glutamatergic input from rod bipolar cells and are known to express non-NMDA receptors. We have investigated the expression of NMDA receptors in these cells by recording responses to exogenously applied NMDA in whole-cell recordings in slices of rat retina. Most cells displayed clear responses to NMDA. The responses could be blocked by a specific NMDA receptor antagonist and were characterized by voltage-dependent block with outward rectification. These results suggest that NMDA receptors could play a role in mediating excitatory synaptic input to AII amacrine cells.

Studying properties of neurotransmitter receptors by non-stationary noise analysis of spontaneous postsynaptic currents and agonist-evoked responses in outside-out patches

Chemical synaptic transmission depends on neurotransmitter-gated ion channels concentrated in the postsynaptic membrane of specialized synaptic contacts. The functional characteristics of these neurotransmitter receptor channels are important for determining the properties of synaptic transmission. Whole-cell recording of postsynaptic currents (PSCs) and outside-out patch recording of transmitter-evoked currents are important tools for estimating the single-channel conductance and the number of receptors contributing to the PSC activated by a single transmitter quantum. When single-channel activity cannot be directly resolved, non-stationary noise analysis is a valuable tool for determining these parameters. Peak-scaled non-stationary noise analysis can be used to compensate for quantal variability in synaptic currents. Here, we present detailed protocols for conventional and peak-scaled non-stationary noise analysis of spontaneous PSCs and responses in outside-out patches. In addition, we include examples of computer code for individual functions used in the different stages of non-stationary noise analysis. These analysis procedures require 3–8 h.

Functional properties of spontaneous IPSCs and glycine receptors in rod amacrine (AII) cells in the rat retina

AII amacrine cells play a crucial role in retinal signal transmission under scotopic conditions. We have used rat retinal slices to investigate the functional properties of inhibitory glycine receptors on AII cells by recording spontaneous IPSCs (spIPSCs) in whole cells and glycine‐evoked responses in outside‐out patches. Glycinergic spIPSCs displayed fast kinetics with an average 10–90% rise time of ∼500 μs, and a decay phase best fitted by a double‐exponential function with τfast∼ 4.8 ms (97.5% amplitude contribution) and τslow∼ 33 ms. Decay kinetics were voltage dependent. Ultrafast application of brief (∼2–5 ms) pulses of glycine (3 mm) to patches, evoked responses with fast deactivation kinetics best fitted with a double‐exponential function with τfast∼ 4.6 ms (85% amplitude contribution) and τslow∼ 17 ms. Double‐pulse experiments indicated recovery from desensitization after a 100‐ms pulse of glycine with a double‐exponential time course (τfast∼ 71 ms and τslow∼ 1713 ms). Non‐stationary noise analysis of spIPSCs and patch responses, and directly observed channel gating yielded similar single‐channel conductances (∼41 to ∼47 pS). In addition, single‐channel gating occurred at ∼83 pS. These results suggest that the fast glycinergic spIPSCs in AII cells are probably mediated by α1β heteromeric receptors with a contribution from α1 homomeric receptors. We hypothesize that glycinergic synaptic input may target the arboreal dendrites of AII cells, and could serve to shunt excitatory input from rod bipolar cells and transiently uncouple the transcellular current through electrical synapses between AII cells and between AII cells and ON‐cone bipolar cells.

Electrical synapses between AII amacrine cells in the retina: Function and modulation.

Adaptation enables the visual system to operate across a large range of background light intensities. There is evidence that one component of this adaptation is mediated by modulation of gap junctions functioning as electrical synapses, thereby tuning and functionally optimizing specific retinal microcircuits and pathways. The AII amacrine cell is an interneuron found in most mammalian retinas and plays a crucial role for processing visual signals in starlight, twilight and daylight. AII amacrine cells are connected to each other by gap junctions, potentially serving as a substrate for signal averaging and noise reduction, and there is evidence that the strength of electrical coupling is modulated by the level of background light. Whereas there is extensive knowledge concerning the retinal microcircuits that involve the AII amacrine cell, it is less clear which signaling pathways and intracellular transduction mechanisms are involved in modulating the junctional conductance between electrically coupled AII amacrine cells. Here we review the current state of knowledge, with a focus on the recent evidence that suggests that the modulatory control involves activity-dependent changes in the phosphorylation of the gap junction channels between AII amacrine cells, potentially linked to their intracellular Ca(2+) dynamics. This article is part of a Special Issue entitled Electrical Synapses.