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AII amacrine cells and ON-cone bipolar cells in an in vitro retinal slice preparation. A: a pair of electrically coupled AII amacrine cells (cell bodies marked by arrows) in an in vitro slice from rat retina, visualized with infrared gradient contrast videomicroscopy. The retinal layers are indicated by abbreviations (ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer). Scale bar: 10 m. B: composite fluorescence photomicrograph of the pair of AII amacrine cells in A after filling with Lucifer yellow. Photomicrographs were taken after about 80 min of recording, including periods with exposure to 10 and 100 M meclofenamic acid. Due to a slanted orientation of the slice, the flattened projection of the images makes the cells appear shorter than they are. Scale bar: 10 m. C1 and C2: an AII amacrine cell electrically coupled to an ON-cone bipolar cell in an in vitro slice from rat retina, visualized with infrared gradient contrast videomicroscopy. In C1, focus is on cell body of the AII amacrine cell (marked by arrow) and in C2, focus is on cell body of the ON-cone bipolar cell (marked by arrow). Scale bar: 10 m (C1 and C2). D: composite fluorescence photomicrograph of the AII amacrine and ON-cone bipolar cells in C (1, 2) after filling with Lucifer yellow. Arrows point to axon and axon terminal of bipolar cell. Recording pipette of AII amacrine cell is out of focus. Photomicrographs were taken after about 10 min of recording. Scale bar: 10 m.
Source publication
Gap junction channels constitute specialized intercellular contacts that can serve as electrical synapses. In the rod pathway of the retina, electrical synapses between AII amacrine cells express connexin 36 (Cx36) and electrical synapses between AII amacrines and on-cone bipolar cells express Cx36 on the amacrine side and Cx36 or Cx45 on the bipol...
Contexts in source publication
Context 1
... amacrine cells constitute a homogeneous population of cells (Wässle et al. 1993) and were targeted for recording according to the size and location of the cell body in the inner nuclear layer and the thick primary dendrite descending into the inner plexiform layer (Fig. 1A). ON-Cone bipolar cells are a heterogeneous group encompassing four to five cell types ( Euler et al. 1996; Hartveit 1997) and their cell bodies tend to be located distally in the inner nuclear layer, proximally to the row of cell bodies of rod bipolar cells (Euler and Wässle 1995). For recording AII amacrine cell pairs with a high ...
Context 2
... potentially contacting, arboreal dendrites in the proximal part of the inner plexiform layer (n 11 cell pairs). For recording cell pairs of AII amacrine cells and ON-cone bipolar cells with a high probability of physiological coupling, we targeted cells with somata that were as close as possible along a vertical line across the retina ( Fig. 1C; n 4 cell pairs). All cells were filled with Lucifer yellow and, at the end of each recording, fluorescence microscopy allowed visualization of both cells' complete morphology (Fig. 1, B and ...
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Use of null mutant mice is a powerful way to evaluate the role of specific proteins in brain function. Studies performed on knockout mice have revealed some unexpected roles of the gap junction proteins (connexins). Thus, analyses of gene expression in connexin43 (Cx43) null brains indicated that deletion of a single gene (Gja1) induced expression...
Combined confocal microscopy and freeze-fracture replica immunogold labeling (FRIL) were used to examine the connexin identity at electrical synapses in goldfish brain and rat retina, and to test for “co-localization” vs. “close proximity” of connexins to other functionally interacting proteins in synapses of goldfish and mouse brain and rat retina...
Neuronal gap junctions are ubiquitous in the brain, but their precise positions in actual neuronal circuits have been uncertain, and their functional roles remain unclear. In this study, I visualized connexin36-immunoreactive gap junctions and examined the structural features of the interconnected dendrites arising from parvalbumin-positive interne...
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We combined Hodgkin-Huxley equations and gating models of gap junction (GJ) channels to simulate the spread of excitation in two-dimensional networks composed of neurons interconnected by voltage-gated GJs. Each GJ channel contains two fast and slow gates, each exhibiting current-voltage (I-V) rectification and gating properties that depend on tran...
Citations
... Inhibitory neurotransmission was blocked by a mixture of GABA A receptor antagonist bicuculline (Johnston, 2013), GABA B receptor antagonist CGP 55845 (Davies et al., 1993), GABA C receptor antagonist TPMPA (Woodward RM et al., 1996), and glycine receptor antagonist strychnine (Phelan et al., 1989). Meclofenamic acid was used to block the electrical synapse currents (Veruki and Hartveit, 2009). The drug(s), their doses, and the retinal mechanisms they modulate have been summarized (Table 1). ...
... The diffuse, tightly synchronized, low-amplitude rhythm observed under synaptic blockade is consistent with oscillatory dynamics of a gap-junction-coupled syncytium [22]. To test this conjecture, we applied meclofenamic acid (MFA; 60 μM-100 μM), which blocks both gap junction protein connexin 43 (CX43) [23] and connexin 36 (CX36) [24], which are expressed in neurons and glia during mouse development [25] [25]. In these experiments (N = 5), 20X recordings localized to either caudal or rostral regions (Fig. 6A.i) were made under synaptic blockade before and during wash-in of MFA. ...
Breathing is a singularly robust behavior, yet this motor pattern is continuously modulated at slow and fast timescales to maintain blood-gas homeostasis, while intercalating orofacial behaviors. This functional multiplexing goes beyond the rhythmogenic function that is typically ascribed to medullary respiration-modulated networks and may explain lack of progress in identifying the mechanism and constituents of the respiratory rhythm generator. By recording optically along the ventral respiratory column in medulla, we found convergent evidence that rhythmogenic function is distributed over a dispersed and heterogeneous network that is synchronized by electrotonic coupling across a neuronal syncytium. First, high-speed recordings revealed that inspiratory onset occurred synchronously along the column and did not emanate from a rhythmogenic core. Second, following synaptic isolation, synchronized stationary rhythmic activity was detected along the column. This activity was attenuated following gap junction blockade and was silenced by tetrodotoxin. The layering of syncytial and synaptic coupling complicates identification of rhythmogenic mechanism, while enabling functional multiplexing.
... Although in mouse retina, ON and OFF RGCs mainly receive inputs from ON and OFF bipolar cells respectively, signals in each pathway are also influenced by the activities from other pathways via lateral connection. As shown in Fig. 1a, ON RGC mainly receives inputs from ON cone pathway, but the ON cone pathway also interacts with the rod pathway through the gap junction between bipolar cell and AII amacrine cell (Demb and Singer 2012), which could be blocked by meclofenamic acid (MFA) (Veruki and Hartveit 2009). On the other hand, OFF RGC mainly receives inputs from OFF cone pathway, but the outputs of OFF RGC are also modulated by the lateral inhibitory signals from ON pathways (ON cone pathway and rod pathway, Fig. 1b) (Beaudoin et al. 2008;Demb and Singer 2012), and the signals of ON pathways can be blocked by a mGluR6 agonist, L-AP4 (Qiu et al. 2016;Sekaran et al. 2003). ...
Two coordinated dynamic properties (adaptation and sensitization) are observed in retinal ganglion cells (RGCs) under the contrast stimulation. During sustained high-contrast period, adaptation decreases RGCs’ responses while sensitization increases RGCs’ responses. In mouse retina, adaptation and sensitization respectively show OFF- and ON-pathway-dominance. However, the mechanisms which drive the differentiation between adaptation and sensitization remain unclear. In the present study, multi-electrode recordings were conducted on isolated mouse retina under full-field contrast stimulation. Dynamic property was quantified based on the trend of RGC’s firing rate during high-contrast period, light sensitivity was estimated by linear-nonlinear analysis and coding ability was estimated through stimulus reconstruction algorism. γ-Aminobutyric acid (GABA) receptors were pharmacologically blocked to explore the relation between RGCs’ dynamic property and the activity of GABA receptors. It was found that GABAA and GABAC receptors respectively mediated the adaptation and sensitization processes in RGCs’ responses. RGCs’ dynamic property changes occurred after the blockage of GABA receptors were related to the modulation of the cells’ light sensitivity. Further, the blockage of GABAA (GABAC) receptor significantly decreased RGCs’ overall coding ability and eliminated the functional benefits of adaptation (sensitization). Our work suggests that the dynamic property of individual RGC is related to the balance between its GABAA-receptor-mediated inputs and GABAC-receptor-mediated inputs. Blockage of GABA receptors breaks the balance of retinal circuitry for signal processing, and down-regulates the visual information coding ability.
... To determine the relative role of gap junctions and nAChRs on the frequency and area of stage 1 waves in mice, we used two-photon calcium imaging and pharmacology in retinas isolated from E16-18 mice bath loaded with Cal 520. To block gap junctions, we bath applied the gap junction antagonist meclofenamic acid (MFA, 50 μM), which reversibly blocks electrical coupling between retinal interneurons (Veruki and Hartveit, 2009) and developing ipRGCs (Caval-Holme et al., 2019 Application of the gap junction blocker MFA (50 μM) nearly abolished stage 1 waves, causing a significant reduction in frequency of waves and cell participation during waves (Figure 2A-C). We also found a small but significant reduction in wave amplitude, quantified as the average maximum response amplitude of all cells participating in individual waves ( Figure 2D). ...
... In addition to the ACh signaling, gap junctions also play a role in mediating stage 1 retinal waves. Here, we use the gap junction antagonist meclofenamic acid (MFA), which was previously shown to reversibly block junctional conductance (Veruki and Hartveit, 2009), dye coupling (Pan et al., 2007), and spikelets between developing RGCs (Caval-Holme et al., 2019). Application of MFA led to a significant reduction in both the frequency and size of stage 1 waves. ...
Spontaneous activity is a hallmark of developing neural systems. In the retina, spontaneous activity comes in the form of retinal waves, comprised of three stages persisting from embryonic day 16 (E16) to eye opening at postnatal day 14 (P14). Though postnatal retinal waves have been well characterized, little is known about the spatiotemporal properties or the mechanisms mediating embryonic retinal waves, designated Stage 1 waves. Using a custom-built macroscope to record spontaneous calcium transients from whole embryonic retinas, we show that Stage 1 waves are initiated at several locations across the retina and propagate across a broad range of areas. Blocking gap junctions reduced the frequency and size of Stage 1 waves, nearly abolishing them. Global blockade of nAChRs similarly nearly abolished Stage 1 waves. Thus, Stage 1 waves are mediated by a complex circuitry involving subtypes of nAChRs and gap junctions. Stage 1 waves in mice lacking the β2 subunit of the nAChRs (β2-nAChR-KO) persisted with altered propagation properties and were abolished by a gap junction blocker. To assay the impact of Stage 1 waves on retinal development, we compared the spatial distribution of a subtype of retinal ganglion cells, intrinsically photosensitive retinal ganglion cells (ipRGCs), which undergo a significant amount of cell death, in WT and β2-nAChR-KO mice. We found that the developmental decrease of ipRGC density is preserved between WT and β2-nAChR-KO mice, indicating that processes regulating ipRGC distribution are not influenced by spontaneous activity.
... A gap-junction blocker mimics and occludes the effects of the CP-AMPAR antagonist CP-AMPARs are expressed at synapses between RBCs and AII-ACs (Mørkve et al., 2002;Osswald et al., 2007;Singer and Diamond, 2003). AII-ACs, in turn, make excitatory gap-junction connections with On-CBCs ( Figure 1A) (Chun et al., 1993;Kolb and Famiglietti, 1974;Veruki and Hartveit, 2009). Tonic activation of CP-AMPARs on AII-ACs is expected to depolarize the On-CBCs through the gap-junction connections, and thus generate the sustained transmitter release that drives the sustained signals in On-GCs. ...
... Tonic activation of CP-AMPARs on AII-ACs is expected to depolarize the On-CBCs through the gap-junction connections, and thus generate the sustained transmitter release that drives the sustained signals in On-GCs. We tested this hypothesis by applying the gap-junction blocker, meclofenamic acid (MFA, 100 mM), to block the connection between the AII-ACs and the On-CBCs (Veruki and Hartveit, 2009). As expected, MFA reduced background spiking and the sustained component of the light responses in On-midget and On-parasol GCs ( Figure 4C, n = 2 On-midget, n = 1 On-parasol). ...
Midget and parasol ganglion cells (GCs) represent the major output channels from the primate eye to the brain. On-type midget and parasol GCs exhibit a higher background spike rate and thus can respond more linearly to contrast changes than their Off-type counterparts. Here, we show that a calcium-permeable AMPA receptor (CP-AMPAR) antagonist blocks background spiking and sustained light-evoked firing in On-type GCs while preserving transient light responses. These effects are selective for On-GCs and are occluded by a gap-junction blocker suggesting involvement of AII amacrine cells (AII-ACs). Direct recordings from AII-ACs, cobalt uptake experiments, and analyses of transcriptomic data confirm that CP-AMPARs are expressed by primate AII-ACs. Overall, our data demonstrate that under some background light levels, CP-AMPARs at the rod bipolar to AII-AC synapse drive sustained signaling in On-type GCs and thus contribute to the more linear contrast signaling of the primate On- versus Off-pathway.
... Also, MA is a blocker of ATP-sensitive potassium ion channels and neuronal voltage-gated potassium channels, independent of the cyclooxygenase-prostaglandin mechanism [15]. Additionally, it is also known that MA has a high inhibition potential as a gap junction antagonist [16]. In addition to these functions, MA has recently been shown to be an inhibitor of FTO protein, which oxidatively demethylates N6-methyladenosine (m6A) residues on RNA [17]. ...
Prostate cancer is the most common type of cancer among men, and there is still no definitively effective drug treatment. Thus, the search for novel drug agents that may be used for the effective treatment continues. Meclofenamic acid (MA), a non-steroidal anti-inflammatory drug, with anti-tumor effects in various types of cancers was used to investigate its effects on LNCaP cells, a prostate cancer cell line, at the proteome level. The cells were treated with 80 µM MA for 24 h and a comparative proteomic analysis was performed with their untreated control cells. Proteins were extracted from the cells and then were subjected to two-dimensional gel electrophoresis. Protein spots displaying changes in their regulation ratios for more than two-fold were excised from the gels and identified with MALDI-TOF/TOF mass spectrometry. Bioinformatics analysis of the differentially regulated proteins that we identified showed that they were all associated with and took part in related pathways. Glycolytic pathway, cytoskeletal formation, transport activity, protein metabolism, and most notably an mRNA processing pathway were affected by the MA treatment. In addition to presenting a detailed information for what is happening inside the cells upon MA treatment, the proteins affected by MA treatment hold the potential to be novel targets for prostate cancer treatment provided that further in vivo experiments are carried out.
... For recording from HCs, slices were first perfused for 30 minutes in an extracellular solution containing 100 μM of the gap junction blocker meclofenamic acid. 27 Recordings of excitatory postsynaptic currents (EPSCs) were made using a potassium gluconate intracellular solution (in mM/L −1 ): 136.6 K-gluconate, 5 EGTA, 13 TEA-Cl, 15 HEPES, 4 Mg-ATP, and 0.4 GTP (pH 7.2). HCs with a series resistance larger than 30 MΩ were excluded from analysis. ...
... Moreover, we recorded EPSCs after 30 minutes of bath application of 100 μM meclofenamic acid to block gap junctions and thereby horizontal information flow in the retina. 27 All experiments were performed under ambient light conditions and HCs were held at −60 mV. We investigated synaptic development from P6 till adulthood. ...
Purpose:
Cone photoreceptors of the retina use a sophisticated ribbon-containing synapse to convert light-dependent changes in membrane potential into release of synaptic vesicles (SVs). We aimed to study the functional and structural maturation of mouse cone photoreceptor ribbon synapses during postnatal development and to investigate the role of the synaptic ribbon in SV release.
Methods:
We performed patch-clamp recordings from cone photoreceptors and their postsynaptic partners, the horizontal cells during postnatal retinal development to reveal the functional parameters of the synapses. To investigate the occurring structural changes, we applied immunocytochemistry and electron microscopy.
Results:
We found that immature cone photoreceptor terminals were smaller, they had fewer active zones (AZs) and AZ-anchored synaptic ribbons, and they produced a smaller Ca2+ current than mature photoreceptors. The number of postsynaptic horizontal cell contacts to synaptic terminals increased with age. However, tonic and spontaneous SV release at synaptic terminals stayed similar during postnatal development. Multiquantal SV release was present in all age groups, but mature synapses produced larger multiquantal events than immature ones. Remarkably, at single AZs, tonic SV release was attenuated during maturation and showed an inverse relationship with the appearance of anchored synaptic ribbons.
Conclusions:
Our developmental study suggests that the presence of synaptic ribbons at the AZs attenuates tonic SV release and amplifies multiquantal SV release. However, spontaneous SV release may not depend on the presence of synaptic ribbons or voltage-sensitive Ca2+ channels at the AZs.
... S9for more examples). Finally, we sought to determine if Campana cells receive their light-evoked synaptic inputs through gap junctions because Aii-ACs depend on gap junction electrical coupling with ON cone BCs to receive light-evoked synaptic inputs(53)(54)(55)(56). Our results demonstrate that gap junction coupling has a minimal effect on the light-evoked calcium response of Campana cells (SI Appendix,Fig. ...
Significance
Cell classes are the building blocks for the central nervous system. It is widely believed that major neuronal classes have been identified in the retina, although some types in the amacrine cell class have not been fully characterized. Here, we describe a retinal interneuron that does not fit into any existing retinal cell class, and we name them Campana cells. Although Campana cells relay visual signals from photoreceptors to the retinal ganglion cells, they differ from bipolar cells in many morphological, physiological, and molecular features. They are also significantly different from amacrine cells. Therefore, our results open the possibility for an unconventional retinal cell class that plays unique roles in visual processing.
... The concentrations of drugs were as follows (µM; supplier Tocris Bioscience, Bristol, UK; unless otherwise noted): 10 bicuculline methochloride, 1 strychnine (Research Biochemicals Inc., Natick, MA, USA), 10 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 50 (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA), 0.3 or 1 tetrodotoxin (TTX), 50 4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride (ZD7288), and 20 (RS)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP). To block electrical coupling via gap junctions, we added 100 µM 2-[(2,6-dichloro-3-methylphenyl)amino]benzoic acid sodium salt (MFA; Sigma-Aldrich) to the extracellular solution (Veruki and Hartveit 2009). Solutions were either made up freshly for each experiment or were prepared from concentrated aliquots stored at − 20 °C. ...
... Simultaneous, dual recordings of electrically coupled cell pairs with CSEVC amplifiers were performed as described for single cells, but when we recorded currents to estimate a cell's membrane capacitance, the voltage-clamp stimuli were sent simultaneously to both amplifiers to eliminate junctional currents between the two cells. Dual recordings with DSEVC amplifiers were performed as described in earlier studies from our laboratory (Veruki and Hartveit 2009;Veruki et al. 2010). ...
... Using paired recordings from electrically coupled ON-cone bipolar cells and AII amacrine cells, we have previously demonstrated that MFA blocks the electrical synapses between these cells (Veruki and Hartveit 2009). Here, we have re-analyzed data obtained in the previous study to quantify the effect of MFA on input resistance (R N ) and apparent membrane resistance (r m ) and, importantly, to examine how the MFA-evoked changes of these parameters develop in parallel with the gradual reduction of the junctional conductance (G j ). ...
Gap junctions are ubiquitous within the retina, but in general, it remains to be determined whether gap junction coupling between specific cell types is sufficiently strong to mediate functionally relevant coupling via electrical synapses. From ultrastructural, tracer coupling and immunolabeling studies, there is clear evidence for gap junctions between cone bipolar cells, but it is not known if these gap junctions function as electrical synapses. Here, using whole-cell voltage-clamp recording in rat (male and female) retinal slices, we investigated whether the gap junctions of bipolar cells make a measurable contribution to the membrane properties of these cells. We measured the input resistance (RN) of bipolar cells before and after applying meclofenamic acid (MFA) to block gap junctions. In the presence of MFA, RN of ON-cone bipolar cells displayed a clear increase, paralleled by block of the electrical coupling between these cells and AII amacrine cells in recordings of coupled cell pairs. For OFF-cone and rod bipolar cells, RN did not increase in the presence of MFA. The results for rod bipolar cells are consistent with the lack of gap junctions in these cells. However, for OFF-cone bipolar cells, our results suggest that the morphologically identified gap junctions between these cells do not support a junctional conductance that is sufficient to mediate effective electrical coupling. Instead, these junctions might play a role in chemical and/or metabolic coupling between subcellular compartments.
... An approximate 10-fold variation in the size of BC4 dendritic and axonal field sizes were observed in this study, most of which cannot be accounted for by variation in BC4 density across the retina, which is larger than that reported in other studies but likely a consequence of the increased number of cells reconstructed in the current study (Yu et al., 2018). Understanding how this variation in axonal and dendritic arbor size is integrated into retinal output will required physiological studies of BC4 output and will necessarily take into account the role of gap junctions in coupling BCs (Becker, Bonness, & Mobbs, 1998;Hansen, Torborg, Elstrott, & Feller, 2005;Kandler & Katz, 1995;Raviola & Gilula, 1973;Veruki & Hartveit, 2009). ...
Neural circuits in the adult nervous system are characterized by stable, cell type‐specific patterns of synaptic connectivity. In many parts of the nervous system these patterns are established during development through initial over‐innervation by multiple pre‐ or postsynaptic targets, followed by a process of refinement that takes place during development and is in many instances activity dependent. Here we report on an identified synapse in the mouse retina, the cone photoreceptor➔type 4 bipolar cell synapse, and show that its development is distinctly different from the common motif of over‐innervation followed by refinement. Indeed, the majority of cones are contacted by single BC4 s throughout development, but are contacted by multiple BC4 s through ongoing dendritic elaboration between 1 and 6 months of age – well into maturity. We demonstrate that cell density drives contact patterns downstream of single cones in Bax null mice and may serve to maintain constancy in both the dendritic and axonal projective field. This article is protected by copyright. All rights reserved. Bipolar cell to cone contact patterns vary depending on genotype, density and age. a and b, Cones are more likely to be contacted by multiple type 4 bipolar cells (BC4 s) in the Bax−/− retina independent of BC4 density. c and d, Cones are more likely to be sampled by multiple BC4 s as the BC4 density increases. e and f, BC4 number decreases between 5–12 weeks but cones are more likely to be sampled by multiple BC4 s over the same time period.
