Non-stationary Noise Analysis Research Articles

Spontaneous IPSCs and glycine receptors with slow kinetics in wide‐field amacrine cells in the mature rat retina

The functional properties of glycine receptors were analysed in different types of wide-field amacrine cells, narrowly stratifying cells considered to play a role in larger-scale integration across the retina. The patch-clamp technique was used to record spontaneous IPSCs (spIPSCs) and glycine-evoked patch responses from mature rat retinal slices (4-7 weeks postnatal). Glycinergic spIPSCs were blocked reversibly by strychnine (300 nM). Compared to previously described spIPSCs in AII amacrine cells, the spIPSCs in wide-field amacrine cells displayed a very slow decay time course (tau(fast) approximately 15 ms; tau(slow) approximately 57 ms). The kinetic properties of spIPSCs in whole-cell recordings were paralleled by even slower deactivation kinetics of responses evoked by brief pulses of glycine (3 mm) to outside-out patches from wide-field amacrine cells (tau(fast) approximately 45 ms; tau(slow) approximately 350 ms). Non-stationary noise analysis of patch responses and spIPSCs yielded similar average single-channel conductances (approximately 31 and approximately 34 pS, respectively). Similar, as well as both lower- and higher-conductance levels could be identified from directly observed single-channel gating during the decay phase of spIPSCs and patch responses. These results suggest that the slow glycinergic spIPSCs in wide-field amacrine cells involve alpha2beta heteromeric receptors. Taken together with previous work, the kinetic properties of glycine receptors in different types of amacrine cells display a considerable range that is probably a direct consequence of differential expression of receptor subunits. Unique kinetic properties are likely to differentially shape the glycinergic input to different types of amacrine cells and thereby contribute to distinct integrative properties among these cells.

Structure and Mechanism of Kainate Receptor Modulation by Anions

L-glutamate, the major excitatory neurotransmitter in the human brain, activates a family of ligand-gated ion channels, the major subtypes of which are named AMPA, kainate, and NMDA receptors. In common with many signal transduction proteins, glutamate receptors are modulated by ions and small molecules, including Ca(2+), Mg(2+), Zn(2+), protons, polyamines, and steroids. Strikingly, the activation of kainate receptors by glutamate requires the presence of both Na(+) and Cl(-) in the extracellular solution, and in the absence of these ions, receptor activity is abolished. Here, we identify the site and mechanism of action of anions. Surprisingly, we find that Cl(-) ions are essential structural components of kainate receptors. Cl(-) ions bind in a cavity formed at the interface between subunits in a dimer pair. In the absence of Cl(-), dimer stability is reduced, the rate of desensitization increases, and the fraction of receptors competent for activation by glutamate drops precipitously.

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.

Diversity of GABAA receptor synaptic currents on individual pyramidal cortical neurons

Miniature GABA(A) receptor-mediated inhibitory postsynaptic currents (mIPSCs) in cortical pyramidal neurons have previously been categorized into two types: small amplitude mIPSCs with a mono-exponential deactivation (mono-mIPSCs) and relatively larger mIPSCs with bi-exponential deactivation (bi-mIPSCs). The aim of this study was to determine if the GABA(A) channels that underlie these mIPSCSs are molecularly distinct. We found, using non-stationary noise analysis, that the difference in their amplitude could be not accounted for by their single channel conductance (both were 40 pS). Next, using alpha subunit selective GABA(A) receptor modulators, we examined the identity of the alpha subunits that may be expressed in the synapses that give rise to these mIPSCs. Zolpidem (100 and 500 nM, alpha1 selective) affected the deactivation of a subset of the mono-mIPSCs, indicating that alpha1 subunits are not highly expressed in these synapses. However, zolpidem (100 nM) prolonged the deactivation of all bi-mIPSCs, indicating a high abundance of alpha1 subunits in these synapses. SB-205384 (alpha3 selective) had no effect on the mono-mIPSCs but the bi-mIPSCs were prolonged. Furosemide (alpha4 selective) reduced the amplitude of only the mono-mIPSCs. L655,708 (alpha5 selective) reduced the amplitude of both populations and shortened the duration of the mono-mIPSCs. Finally, we found that the neuroactive steroid pregesterone sulphate reduced the amplitude of both mIPSC types. These results provide pharmacological evidence that synapses on cortical pyramidal neurons are molecularly distinct. The purpose of these different types of synapses may be to provide different inhibitory timing patterns on these cells.

Self-phosphorylation modulates the gating of rat liver gap junction channels: A nonstationary noise analysis

The effect of phosphorylation on the gating of rat liver gap junction hemichannels (Cx 32) has been investigated. It has been shown that self-phosphorylation of rat liver Cx 32 protein reduces the permeability of proteoliposomes as well the current flowing through multichannels in lipid bilayer membrane (BLM). The low frequency power spectral density analyses of nonstationary noise evolved due to the gating of Cx 32 multichannels demonstrated that self-phosphorylation modulated the channel functioning. A clear change in the power spectrum slopes ( α) of the nonstationary noise profiles confirmed the modulation of the channel dynamics due to self-regulation.

Muscle Chloride Channel Dysfunction in Two Mouse Models of Myotonic Dystrophy

Muscle degeneration and myotonia are clinical hallmarks of myotonic dystrophy type 1 (DM1), a multisystemic disorder caused by a CTG repeat expansion in the 3′ untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. Transgenic mice engineered to express mRNA with expanded (CUG)250 repeats (HSA LR mice) exhibit prominent myotonia and altered splicing of muscle chloride channel gene (Clcn1) transcripts. We used whole-cell patch clamp recordings and nonstationary noise analysis to compare and biophysically characterize the magnitude, kinetics, voltage dependence, and single channel properties of the skeletal muscle chloride channel (ClC-1) in individual flexor digitorum brevis (FDB) muscle fibers isolated from 1–3-wk-old wild-type and HSA LR mice. The results indicate that peak ClC-1 current density at −140 mV is reduced >70% (−48.5 ± 3.6 and −14.0 ± 1.6 pA/pF, respectively) and the kinetics of channel deactivation increased in FDB fibers obtained from 18–20- d-old HSA LR mice. Nonstationary noise analysis revealed that the reduction in ClC-1 current density in HSA LR FDB fibers results from a large reduction in ClC-1 channel density (170 ± 21 and 58 ± 11 channels/pF in control and HSA LR fibers, respectively) and a modest decrease in maximal channel open probability(0.91 ± 0.01 and 0.75 ± 0.03, respectively). Qualitatively similar results were observed for ClC-1 channel activity in knockout mice for muscleblind-like 1 (Mbnl1 ΔE3/ΔE3), a second murine model of DM1 that exhibits prominent myotonia and altered Clcn1 splicing (Kanadia et al., 2003). These results support a molecular mechanism for myotonia in DM1 in which a reduction in both the number of functional sarcolemmal ClC-1 and maximal channel open probability, as well as an acceleration in the kinetics of channel deactivation, results from CUG repeat–containing mRNA molecules sequestering Mbnl1 proteins required for proper CLCN1 pre-mRNA splicing and chloride channel function.

Functional segregation of synaptic GABAA and GABAC receptors in goldfish bipolar cell terminals

The transmission of light responses to retinal ganglion cells is regulated by inhibitory input from amacrine cells to bipolar cell (BC) synaptic terminals. GABA(A) and GABA(C) receptors in BC terminals mediate currents with different kinetics and are likely to have distinct functions in limiting BC output; however, the synaptic properties and localization of the receptors are currently poorly understood. By recording endogenous GABA receptor currents directly from BC terminals in goldfish retinal slices, I show that spontaneous GABA release activates rapid GABA(A) receptor miniature inhibitory postsynaptic currents (mIPSCs) (predominant decay time constant (tau(decay)), 1.0 ms) in addition to a tonic GABA(C) receptor current. The GABA(C) receptor antagonist (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) has no effect on the amplitude or kinetics of the rapid GABA(A) mIPSCs. In addition, inhibition of the GAT-1 GABA transporter, which strongly regulates GABA(C) receptor currents in BC terminals, fails to reveal a GABA(C) component in the mIPSCs. These data suggest that GABA(A) and GABA(C) receptors are highly unlikely to be synaptically colocalized. Using non-stationary noise analysis of the mIPSCs, I estimate that GABA(A) receptors in BC terminals have a single-channel conductance (gamma) of 17 pS and that an average of just seven receptors mediates a quantal event. From noise analysis of the tonic current, GABA(C) receptor gamma is estimated to be 4 pS. Identified GABA(C) receptor mIPSCs exhibit a slow decay (tau(decay), 54 ms) and are mediated by approximately 42 receptors. The distinct properties and localization of synaptic GABA(A) and GABA(C) receptors in BC terminals are likely to facilitate their specific roles in regulating the transmission of light responses in the retina.

Glucocorticoids Specifically Enhance L-Type Calcium Current Amplitude and Affect Calcium Channel Subunit Expression in the Mouse Hippocampus

Previous studies have shown that corticosterone enhances whole cell calcium currents in CA1 pyramidal neurons, through a pathway involving binding of glucocorticoid receptor homodimers to the DNA. We examined whether glucocorticoids show selectivity for L- over N-type of calcium currents. Moreover, we addressed the putative gene targets that eventually lead to the enhanced calcium currents. Electrophysiological recordings were performed in nucleated patches that allow excellent voltage control. Calcium currents in these patches almost exclusively involve N- and L-type channels. We found that L- but not N-type calcium currents were largely enhanced after treatment with a high dose of corticosterone sufficient to activate glucocorticoid receptors. Voltage dependency and kinetic properties of the currents were unaffected by the hormone. Nonstationary noise analysis suggests that the increased current is not caused by a larger unitary conductance, but rather to a doubling of the number of functional channels. Quantitative real-time PCR revealed that transcripts of the Ca(v)1 subunits encoding for the N- or L-type calcium channels are not upregulated in the mouse CA1 area; instead, a strong, direct, and consistent upregulation of the beta4 subunit was observed. This indicates that the corticosteroid-induced increase in number of L-type calcium channels is not caused by a simple transcriptional regulation of the pore-forming subunit of the channels.

Two types of GABAergic miniature inhibitory postsynaptic currents in mouse substantia gelatinosa neurons

The physiological and pharmacological properties of γ-aminobutyric acid (GABA)ergic miniature inhibitory postsynaptic currents (mIPSCs) were investigated in substantia gelatinosa neurons of mouse spinal cord using whole-cell patch clamp recordings. Two cell populations were pharmacologically identified based on the effect of propofol (10 μM) on the mIPSC decay kinetics: those exhibiting propofol-sensitive mIPSCs, with a slow decay kinetic (mIPSC SLOW ), and those exhibiting propofol-resistant mIPSCs, with a fast decay kinetic (mIPSC FAST ) (decay time constants of 14.2 ± 0.7 and 7.4 ± 0.8 ms, respectively). The frequency and amplitude of both types of mIPSCs were not affected by propofol. Miniature IPSC FAST showed midazolam insensitivity, while midazolam prolonged the decay phase of mIPSC SLOW without modulation of the frequency and amplitude. Exogenous GABA-evoked responses in the neurons with mIPSC SLOW were potentiated by propofol, while those in neurons with mIPSC FAST were unaffected by propofol. Furthermore, non-stationary noise analysis of the two kinetically and pharmacologically distinct mIPSCs revealed different conductance of GABA A receptor channels underlying the synaptic events. Pharmacological responses to propofol and midazolam suggested that mIPSC FAST and mIPSC SLOW in substantia gelatinosa neurons can be mediated by GABA A receptors with different subunit compositions.

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 approximately 500 mus, and a decay phase best fitted by a double-exponential function with tau(fast) approximately 4.8 ms (97.5% amplitude contribution) and tau(slow) approximately 33 ms. Decay kinetics were voltage dependent. Ultrafast application of brief ( approximately 2-5 ms) pulses of glycine (3 mm) to patches, evoked responses with fast deactivation kinetics best fitted with a double-exponential function with tau(fast) approximately 4.6 ms (85% amplitude contribution) and tau(slow) approximately 17 ms. Double-pulse experiments indicated recovery from desensitization after a 100-ms pulse of glycine with a double-exponential time course (tau(fast) approximately 71 ms and tau(slow) approximately 1713 ms). Non-stationary noise analysis of spIPSCs and patch responses, and directly observed channel gating yielded similar single-channel conductances ( approximately 41 to approximately 47 pS). In addition, single-channel gating occurred at approximately 83 pS. These results suggest that the fast glycinergic spIPSCs in AII cells are probably mediated by alpha1beta heteromeric receptors with a contribution from alpha1 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.

Kindling‐induced alterations in GABAA receptor‐mediated inhibition and neurosteroid activity in the rat piriform cortex

The piriform cortex makes strong interconnections with limbic structures (amygdala, entorhinal cortex and hippocampus) that are involved in memory processing. These connections have also been implicated in the development of temporal lobe epilepsy. However, little is known about how neurones in this region may change during seizure genesis. Here we tested the hypothesis that in the kindling model of temporal lobe epilepsy GABAA receptor-mediated inhibition is altered in the piriform cortex. To do this we performed whole-cell patch-clamp recordings in piriform cortex brain slices obtained from non-kindled and amygdala-kindled adult rats. We found that kindling coincided with an increase in the amplitude and duration of miniature inhibitory post-synaptic currents (mIPSCs) recorded from non-pyramidal neurones, whereas the mIPSCs occurring on pyramidal (excitatory) cells did not change. Non-stationary noise analysis of mIPSCs occurring on the non-pyramidal neurones showed that inferred unitary conductance of synaptic channels were the same before and after kindling, implying that the channel number increased significantly. Immunocytochemical analysis of the inhibitory innervation showed that it was also unaltered by seizure induction. We also found that the effect of the positive modulator tetrahydrodeoxycorticosterone was reduced on the pyramidal neurones after kindling. In contrast, the potentiating effects of tetrahydrodeoxycorticosterone on non-pyramidal cells were about the same after kindling as in control (sham) rats. These data indicate that amygdala kindling causes a shift in the inhibition 'balance' between the pyramidal and non-pyramidal cells, perhaps leading to the disinhibition of pyramidal cells.

Studying properties of neurotransmitter receptors by non‐stationary noise analysis of spontaneous synaptic currents

The properties of neurotransmitter receptor channels are important for determining synaptic transmission in the nervous system. The presence of quantal variability complicates the use of conventional non-stationary noise analysis for determining the unitary conductance and number of channels involved in synaptic currents. Peak-scaled non-stationary noise analysis has been used to compensate for quantal variability, but there is evidence that the resulting variance versus mean relationships can be transformed from parabolic to skewed. We have used computer modelling based on experimentally derived kinetic schemes to investigate such relationships and demonstrate that their shape is a consequence of the temporal structure of the fluctuations during synaptic responses. Covariance analysis showed that peak-scaling generates a skewed relationship when the covariance function decays rapidly (compared to the average response waveform), corresponding to a low correlation between fluctuations at the peak and in neighbouring regions of the decay phase. A parabolic relationship is obtained when the covariance function decays more slowly, corresponding to a higher correlation. Irrespective of a skewed or parabolic relationship, we demonstrate that the unitary current can be reliably estimated, with a coefficient of variation (CV) as low as 0.05 and bias as low as +/-2% under ideal conditions. While the shape of the variance versus mean curve after peak-scaled non-stationary noise analysis is ultimately a consequence of the kinetic properties of the channels, inadequate alignment of individual waveforms can transform the relationship from parabolic to skewed, and low-pass filtering can transform the relationship from skewed to parabolic. These findings have important implications for analysis of experimental data.

Empirical considerations regarding the use of ensemble-variance analysis of macroscopic currents

Two fundamental properties of an ion channel are its single channel current ( i) and its open probability ( P o), which are most directly defined by single channel recordings. However, under some circumstances either practical limitations (e.g., extreme voltages) or unique channel properties may preclude the use of single channel recordings for definition of such properties. As an alternative, the variance in macroscopic currents (non-stationary noise analysis or variance–mean analysis) has been exploited to define fundamental elementary properties of the underlying channels. Although some limitations of the variance–mean approach have been considered in previous work by others, here simulation methods were used to define the conditions under which variance analysis can be suitable for providing estimates of i and P o. Of particular interest is the extent to which reasonably reliable estimates of P o can be obtained, even under conditions of P o less than 0.5. Empirically, the analysis indicates that, with sufficient numbers of sweeps and with constraints on initial estimates of i, reasonably reliable estimates of P o can be made down to 0.2. The impact of a number of other factors on the utility of variance–mean analysis are also considered, including effects of inactivation, filtering, and the consequences of particular gating schemes.

Optimizing fluctuation analysis of GABA Aergic IPSCs for accurate unitary currents

Nonstationary fluctuation analysis of synaptic currents requires division of currents into bins of time, with little agreement on how to select an optimal bin width. We used simulated inhibitory postsynaptic currents (simIPSCs) in an empirical approach to establish the optimal bin width needed for estimation of the unitary current, i e. We found acceptable accuracy (≤5%) at bin widths shorter than the length of the stationary segment of simIPSCs that persisted when Gaussian noise was added to the simulated currents. We also studied evoked and spontaneous IPSCs mediated by receptors for γ-aminobutyrate (GABA) in thalamic neurons. Similar to simIPSCs, analysis of the IPSCs yielded saturating relationships between bin width and accuracy of unitary current estimate. Whereas standard error decreased, the accuracy of i e estimates increased with decreasing bin width, forming a plateau at bins below 2–3 ms in duration. Using this approach, one can reliably determine the optimal bin width for nonstationary noise analysis.

Functional characterization of P2X3receptors fused with fluorescent proteins

P2X receptor function in the CNS is poorly understood, and currently available data are partly inconsistent. In the presented study, we investigated P2X3 receptors stably expressed in HEK293 cells. Non-stationary noise analysis of whole cell currents and rapid ATP application through flash photolysis allowed for assessing the single channel conductance (6.6 pS) and the fast activation kinetics of the receptor (20 ms). The characteristics of channel desensitization and pharmacological properties matched previous findings. The properties of wild type receptors were compared with P2X3 constructs carrying a fluorescent tag (ECFP or DsRed2) at the C-terminus. These fluorescently labeled subunits formed functional receptors, with neither the affinity of the ligand binding site nor channel properties (ion selectivity, gating kinetics, single channel conductance) differing from wild type. We conclude that both fusion proteins tested here are suitable for generating transgenic mice, which can be expected to promote understanding of the physiological role of P2X3 receptors in CNS signaling.

Heterogeneity of Postsynaptic Receptor Occupancy Fluctuations among Glycinergic Inhibitory Synapses in the Zebrafish Hindbrain

The amplitude of glycinergic miniature inhibitory postsynaptic currents (mIPSCs) varies considerably in neurons recorded in the isolated hindbrain of 50-h-old zebrafish larvae. At this age, glycinergic synapses are functionally mature. In order to measure the occupancy level of postsynaptic glycine receptors (GlyRs) and to determine the pre- and/or postsynaptic origin of its variability, we analysed mIPSCs within bursts evoked by alpha-latrotoxin (0.1-1 nM). Two types of burst were observed according to their mIPSC frequencies: 'slow' bursts with clearly spaced mIPSCs and 'fast' bursts characterised by superimposed events. Non-stationary noise analysis of mIPSCs in some 'slow' bursts recorded in the presence or in the absence of Ca2+ denoted that mIPSC amplitude variance did not depend on the quantity of neurotransmitters released (presynaptic origin), but rather on intrinsic stochastic behaviour of the same group of GlyRs (postsynaptic origin). In these bursts, the open probability measured at the peak of the mIPSCs was close to 0.5 while the maximum open probability is close to 0.9 for the synaptic isoform of GlyRs (heteromeric alpha1/beta GlyRs). In 'fast' bursts with superimposed events, a correlation was found between the amplitude of mIPSCs and the basal current level measured at their onset, which could suggest that the same group of GlyRs is activated during such bursts. Altogether, our results indicate that glycine synapses can display different release modes in the presence of alpha-latrotoxin. They also indicate that, in our model, postsynaptic GlyRs cannot be saturated by the release of a single vesicle.

Counting channels: a tutorial guide on ion channel fluctuation analysis.

Ion channels open and close in a stochastic fashion, following the laws of probability. However, distinct from tossing a coin or a die, the probability of finding the channel closed or open is not a fixed number but can be modified (i.e., we can cheat) by some external stimulus, such as the voltage. Single-channel records can be obtained using the appropriate electrophysiological technique (e.g., patch clamp), and from these records the open probability and the channel conductance can be calculated. Gathering these parameters from a membrane containing many channels is not straightforward, as the macroscopic current I = iNP(o), where i is the single-channel current, N the number of channels, and P(o) the probability of finding the channel open, cannot be split into its individual components. In this tutorial, using the probabilistic nature of ion channels, we discuss in detail how i, N, and P(o max) (the maximum open probability) can be obtained using fluctuation (nonstationary noise) analysis (Sigworth FJ. G Gen Physiol 307: 97-129, 1980). We also analyze the sources of possible artifacts in the determination of i and N, such as channel rundown, inadequate filtering, and limited resolution of digital data acquisition by use of a simulation computer program (available at www.cecs.cl).

Kinetic modulation of HERG potassium channels by the volatile anesthetic halothane.

(human ether-a-gogo related gene) encodes the cardiac rapidly activating delayed rectifier potassium currents (I(kr)), which play an important role in cardiac action potential repolarization. General anesthetics, like halothane, can prolong Q-T interval, suggesting that they act on myocellular repolarization, possibly involving HERG channels. Evidence for direct modulation of HERG channels by halothane is still lacking. To gain insight on HERG channel modulation by halothane the authors recorded macroscopic currents expressed in Xenopus oocytes and conducted non-stationary noise analysis to evaluate single channel parameters modified by the anesthetic. Macroscopic currents were recorded in 120 mM K(+) internal-5 mM K(+) external solutions with the cut open oocyte technique. Macropatch recordings for non-stationary noise analysis of HERG tail currents were made in symmetrical 120 mM K(+) solutions. Pulse protocols designed for HERG current recording were elicited from a holding potential of -80 mV. Halothane was delivered via gravity-fed perfusion. Halothane (0.7%, 1.5%, and 3%) decreased macroscopic currents in a concentration-dependent manner (average reduction by 14%, 22%, and 35% in the range of -40 mV to 40 mV) irrespective of potential. HERG currents had slower activation and accelerated deactivation and inactivation. Non-stationary noise analysis revealed that halothane, 1.5%, decreased channel P(o) by 27%, whereas single-channel current amplitudes and number of channels in the patch remained unchanged. Halothane inhibits HERG currents expressed in oocytes in a concentration-dependent manner. It slowed down activation and accelerated deactivation and inactivation of HERG channels. The authors' results demonstrate that halothane decreased HERG currents by modulating kinetic properties of HERG channels, decreasing their open probability. Partial block of I(kr) currents could contribute to delayed myocellular repolarization and altered cardiac electrophysiology.

Glycinergic synaptic transmission to bullfrog retinal bipolar cells is input-specific

Glycinergic inhibitory postsynaptic currents (IPSCs) focally elicited at the dendrites and axon terminals were recorded from bipolar cells in the bullfrog retinal slice, using the whole-cell clamp technique. IPSCs driven by input from interplexiform cells at bipolar cell dendrites ( ipc-IPSCs) had a much slower decay time constant (25.2±7.8 ms) than IPSCs driven by input from amacrine cells at bipolar cell axon terminals ( ac-IPSCs) (14.7±5.5 ms). Furthermore, peak-scaled non-stationary noise analysis revealed that the weighted mean single-channel conductance of the glycine receptors underlying bipolar cell dendritic ipc-IPSCs (20.8±6.6 pS) was significantly larger than that of those underlying bipolar cell axon terminal ac-IPSCs (12.9±2.9 pS). These results demonstrate that glycinergic synaptic transmission with different properties at bipolar cell dendrites and axon terminals differentially mediates intraretinal centrofugal signal transfer from the inner retina to the outer retina provided by interplexiform cells and lateral inhibition offered by amacrine cells in the inner retina.

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 microM for AMPA and 169 microM for kainate). Application of AMPA to patches evoked desensitizing responses with an EC50 of 217 and 88 microM for the peak and steady-state responses, respectively. Kainate-evoked responses of patches displayed no desensitization (EC50 = 162 microM). 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 microM). 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 (P(Ca)/P(Cs) = 1.9-2.1). Stationary noise analysis indicated that kainate activated channels with an apparent chord conductance of approximately 9 pS. Non-stationary noise analysis indicated that AMPA and glutamate activated channels with apparent chord conductances of approximately 9, approximately 15, approximately 23 and approximately 38 pS. Discrete single-channel gating corresponding to chord conductances of approximately 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.