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NEUROSCIENCE |
1 University of Bergen, Department of Biomedicine, Bergen, Norway
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Abstract |
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fast
15 ms; slow
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 (fast
45 ms; slow
350 ms). Non-stationary noise analysis of patch responses and spIPSCs yielded similar average single-channel conductances (31 and 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 
2
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.
(Received 26 December 2006;
accepted after revision 27 February 2007;
first published online 1 March 2007)
Corresponding author E. Hartveit: University of Bergen, Department of Biomedicine, Jonas Lies vei 91, N-5009 Bergen, Norway. Email: espen.hartveit{at}biomed.uib.no
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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50% of all amacrine cells (Pourcho & Goebel, 1985; Menger et al. 1998; reviewed by Slaughter, 2004). Amacrine cells are local circuit interneurons that receive synaptic input from bipolar cells and other amacrine cells and send output to bipolar cells, ganglion cells and other amacrine cells. Glycine activates receptors with an integral chloride-selective ion channel. Molecular biological studies have revealed the existence of five different receptor subunits (1–4 and ; reviewed by Lynch, 2004). A functional glycine receptor is a pentameric receptor, either an homomeric receptor or an heteromeric receptor. Depending on the exact subunit composition, glycine receptors display marked functional variability, including differences in kinetic properties and single-channel conductance (reviewed by Legendre, 2001). The most striking example of the functional consequences of differential expression of glycine receptor subunits is the developmental speeding of glycinergic synaptic transmission in the spinal cord and brain stem (Takahashi et al. 1992; Singer et al. 1998). The change from slowly to rapidly decaying synaptic responses is accompanied by a change in glycine receptor subunit expression from slow 2 receptors (homomeric 2 or heteromeric 2 receptors) to fast 1 heteromeric receptor channels (Becker et al. 1988; Takahashi et al. 1992). The 2-subunit is therefore considered an embryonic and juvenile subunit, and at most a minor constituent of glycine receptors in the mature spinal cord and brain stem (Legendre, 2001; Lynch, 2004). The change in receptor expression and synaptic kinetics is considered to play a critical role in the maturation of motor functions (reviewed by Takahashi, 2005). The 3-subunit has a similar developmental expression pattern to that of the 1-subunit, but at all developmental stages the 3-subunit expression level is less intense (Malosio et al. 1991). The 4-subunit is expressed in embryonic spinal cord, sympathetic and dorsal root ganglia, but not in the adult brain and spinal cord (Harvey et al. 2000).
The status of the 2-subunit as an embryonic receptor in the spinal cord and brain stem contrasts with the patterns of expression observed in the adult mammalian retina. As for adult spinal cord and brain stem, there is strong evidence for expression of the 1-subunit (Grünert & Wässle, 1993). In addition, however, immunocytochemical experiments have detected the presence of 2-, 3- and 4-subunits in mature retinal tissue, with strong evidence for a differential expression of these subunits among retinal neurons (Grünert & Wässle, 1993; Haverkamp et al. 2003, 2004; Heinze et al. 2006). This raises the question whether there might be a corresponding variation of functional properties of retinal glycine receptors. Mammalian retinas contain 55 separate neuronal types and it is generally considered that each type, as defined by morphological criteria, carries out a specific physiological function (Masland, 2001). For amacrine cells, 30 different types have been distinguished. It has been suggested that amacrine cells only express glycine receptors with slow kinetic properties (Frech et al. 2001). However, a recent investigation of AII amacrine cells found evidence for very fast decay kinetics of glycine receptor currents in these cells, both for synaptic currents and for currents evoked in outside-out patches (Gill et al. 2006). Here, we have investigated the functional characteristics of glycine receptors in wide-field amacrine cells. These cells display common morphological characteristics with long, radiating processes that stratify narrowly at different levels of the inner plexiform layer (Perry & Walker, 1980; Famiglietti, 1992a,b; MacNeil & Masland, 1998; MacNeil et al. 1999; Völgyi et al. 2001; Badea & Nathans, 2004; Lin & Masland, 2006). As a group, wide-field amacrine cells are generally considered to play a role in larger-scale integration across the retina, with each type transmitting inhibition over long distances (reviewed by Baccus, 2007). In the wide-field amacrine cells we recorded from, spontaneous inhibitory postsynaptic currents (spIPSCs) displayed very slow decay kinetics. Importantly, this was parallelled by even slower deactivation kinetics of glycine-evoked responses measured in outside-out patches. This suggested that the slow time course of the spIPSCs is a consequence of the kinetic properties of the glycine receptors expressed by wide-field amacrine cells. A comparison of the present results for wide-field amacrine cells with those obtained previously for AII amacrine cells suggests that unique glycine receptor properties may differentially shape glycinergic input to different types of amacrine cells.
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Methods |
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for recordings in the whole-cell configuration and 5–8 M for recordings in the outside-out patch configuration. All patches were isolated from the soma of the recorded cells. For some outside-out recordings, pipettes were coated with dental wax and fire-polished immediately before use. Recordings were carried out at room temperature (20–23°C). Solutions and drugs
The extracellular perfusing solution was continuously bubbled with 95% O2–5% CO2 and had the following composition (mM): 125 NaCl, 25 NaHCO3, 2.5 KCl, 2.5 CaCl2, 1 MgCl2, 10 glucose, pH 7.4. The recording pipettes (whole-cell and patch) were filled with (mM): 130 KCl, 10 Hepes, 1 CaCl2, 8 NaCl, 5 EGTA, 4 MgATP, 2 N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide (QX-314; Tocris Bioscience, Bristol, UK). pH was adjusted to 7.3 with KOH. Lucifer yellow was added at a concentration of 1 mg ml–1 to the intracellular solution for visualization of cells at the end of the recordings (whole-cell and patch). Theoretical liquid junction potentials were calculated with the computer program JPCalcW (Molecular Devices, Sunnyvale, CA, USA) and membrane holding potentials were automatically corrected online for liquid junction potentials.
Drugs were added directly to the extracellular solution used to perfuse the slices. For recordings of spIPSCs, the extracellular solution contained (µM; Tocris Bioscience): 3 bicuculline methchloride, 10 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 0.3 tetrodotoxin (TTX). Solutions were either made up freshly for each experiment or prepared from concentrated aliquots stored at –20°C. CNQX was first dissolved at 100 mM in dimethylsulfoxide (Sigma, St Louis, MO, USA) and then diluted to the final concentration.
Fast drug application
Ultrafast drug application was performed according to the description of Jonas (1995) and as detailed in Veruki et al. (2003). Drugs were applied from a theta-tube application pipette (septum thickness 117 µm; final tip diameter 300 µm; Hilgenberg, Malsfeld, Germany). The pipette tip with the outside-out patch was positioned near the interface between the control solution and agonist-containing solution continuously flowing out of each barrel, about 100 µm away from the tip of the application pipette. Concentration jumps of agonist to the patch were performed by rapidly moving the application pipette and thus the interface between the two solutions. Drugs were dissolved in Hepes-buffered solution containing (mM): 145 NaCl, 2.5 KCl, 2.5 CaCl2, 1 MgCl2, 5 hemisodium-Hepes, 10 glucose, pH adjusted to 7.4 with HCl. For any concentration of glycine (May and Baker Ltd, Dagenham, UK), it replaced an equimolar concentration of NaCl. Agonist pulses were applied every 5–10 s. The solution exchange time was measured as previously described (Veruki et al. 2003). Under optimal conditions, the 20–80% rise time of the solution exchange ranged from 200 to 400 µs.
Electrophysiological recording and data acquisition
Voltage-clamp recordings were made with either an EPC9-dual or EPC10-triple amplifier (HEKA Elektronik, Lambrecht, Germany) controlled by either Pulse or PatchMaster software (HEKA Elektronik). Cells and patches were held at a potential of –60 mV. The signals were typically low-pass filtered with a corner frequency (–3 dB) of 5 kHz, and sampled at 50 kHz. Capacitative currents caused by the recording pipette capacitance (Cfast) and the cell membrane capacitance (Cslow) were measured with the automatic capacitance neutralization network feature of the amplifier that also estimated the series resistance (Rseries). The average capacitance in the whole-cell recordings was 3.4 ± 0.3 pF (mean ±
S.E.M.) (n
= 19). Throughout every continuous recording of spontaneous synaptic currents, Rseries was regularly monitored (every 60 s) by acquiring the responses to a series of 20 mV hyperpolarizing voltage pulses (16 ms duration). During such stimulation, the Cslow neutralization circuitry was transiently disabled. The capacitative transients were analysed off-line by averaging consecutive responses (typically 15–20) and fitting the decay with double or triple-exponential functions in order to estimate the peak capacitative current and calculate Rseries. The average Rseries in whole-cell recordings of cells with spIPSCs was 15.5 ± 1.6 M (n
= 8). The average holding current in whole-cell recordings was –5.6 ± 1.6 pA (n
= 19).
General data analysis
Data were analysed with PulseFit/PulseTools, FitMaster (HEKA Elektronik), IGOR Pro (WaveMetrics, Lake Oswego, OR, USA), AxoGraph (AxoGraph Scientific, Sydney, Australia) and TAC (Bruxton Corp., Seattle, WA, USA). Spontaneous postsynaptic currents (spPSCs) were detected with a threshold of 5–7 pA depending on the noise level (MiniAnalysis; Synaptosoft, Decatur, GA, USA) and verified by eye. For amplitude and interevent interval analyses, complex PSCs, i.e. PSCs consisting of temporally overlapping events, were analysed as described by Veruki et al. (2003). For kinetic and non-stationary noise analyses, we included only well-separated (interevent intervals 
200 ms), monophasic PSCs which appeared to rise in a monotonic fashion without visible deviation of the rising phase (cf. Traynelis et al. 1993) and which decayed exponentially. For kinetic analysis of averaged PSCs, the number of individual events for each cell ranged between 35 and 465. Before averaging, individual spPSCs were aligned along the point of steepest rise.
The decay time course of individual and averaged PSCs, as well as responses evoked by the ultrafast application of agonist, was estimated by curve fitting with exponential functions. For single-exponential functions we used the function:
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| (1) |
is the time constant, and Iss is the steady-state current amplitude (typically zero). For double-exponential functions we used the function:
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| (2) |
1 and 2 are the time constants of the first (fast) and second (slow) exponential components and Iss is the steady-state current amplitude (typically zero). Fitting was started 300–750 µs after the peak amplitude (typically 500 µs for PSCs). For double-exponential functions the amplitude contribution was calculated as 100%
x (Ax/(A1
+
A2)). The weighted decay time constant was calculated as (a11
+
a22), where a1 and a2 are the relative amplitudes of the two exponential components, and 1 and 2 are the corresponding time constants. For waveforms fitted with double-exponential functions (spontaneous PSCs and evoked responses in patches), we defined time 0 as the start of the response (determined by eye as the point in time at which the current rose from the baseline noise).
In cases where step transitions corresponding to single-channel gating could be directly observed in the later portions of glycine responses, all-point amplitude histograms were constructed from selected epochs and fitted with sums of Gaussian distributions to obtain the mean current of the open level (TAC; maximum likelihood algorithm). The single-channel current was taken as the difference between the open-channel peak and the baseline. The single-channel chord conductance (
) was calculated as:
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| (3) |
Data are presented as means ± S.E.M. (n = number of cells, events or patches) and percentages are presented as percentage of control. Statistical analysis of correlations (either between time and a parameter for stability analysis or between two parameters) was performed using Spearman's rank order correlation test. Differences were considered significant at the P < 0.05 level. For illustration purposes, most raw data records were low-pass filtered (digital non-lagging Gaussian filter; –3 dB at 1–2 kHz; 500 Hz for traces with discrete single-channel transitions). Unless otherwise noted, the current traces in the figures represent individual traces.
Non-stationary noise analysis
To obtain the conductance of glycine receptor channels in outside-out patches, we applied non-stationary noise analysis (Sigworth, 1980) to responses evoked by ultra-fast applications of glycine (3 mM; 2 or 5 ms pulses; 12–189 repetitions evoked every 10 s). The sampling frequency was 50 kHz and the records were low-pass filtered at 5 kHz. For patches with no obvious rundown, the ensemble variance was calculated as the variance about each sample point in the ensemble mean. When there was evidence of rundown, the ensemble variance was calculated from the differences of overlapping pairs of successive responses as 
2
=
2
/2, where is the difference of successive records, and
2 is the ensemble average (expectation value) of the squared differences of successive records (Heinemann & Conti, 1992). The ensemble mean response was binned into 50 equal segments along the ordinate, such that each bin, on average, corresponded to an equal number of channel closings during the decay phase (Traynelis et al. 1993). The ensemble mean response and variance were averaged within each bin. Finally, the ensemble variance was plotted against the mean current (omitting the rising phase of the response) and fitted with the function:
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| (4) |
2b is the variance of the background noise. The open probability (Popen) at any given time is determined by the equation Popen
=
I/iN. The single-channel conductance was calculated by eqn (3). To obtain the conductance of synaptic glycine receptor channels, we applied peak-scaled non-stationary noise analysis (Traynelis et al. 1993) to ensembles of spPSCs (low-pass filtered at 5 kHz and sampled at 50 kHz). For selection of spPSCs for noise analysis, we followed the procedure described by Momiyama et al. (2003). Briefly, individual spPSCs were first low-pass filtered at 2 kHz (digital Gaussian filter) and analysed by measuring peak amplitude, 20–80% rise time and decay time constant. For the latter measurement, we used a single-exponential function, despite the finding (see Results) that ensemble averages were best fitted with double-exponential functions. Measured parameters were then numbered according to the event number and tested by Spearman's rank order correlation test for time stability (using a variable size, sliding window algorithm implemented in IGOR Pro, code adapted from that in the NeuroMatic package; http://www.physiol.ucl.ac.uk/research/silver_a/). From the complete recording of an individual cell, the algorithm returned a maximum number of consecutive spPSCs whose parameters did not display any significant correlations (P > 0.05). After testing for correlations between rise time and amplitude and between rise time and decay time constant (Spearman's rank order correlation test; see Results), spPSCs were used for noise analysis without the imposed digital (2 kHz) Gaussian filter. The number of events for each cell that passed these criteria and were used for noise analysis represented 70–100% of the total number of events recorded from a given cell.
To correct for quantal variability (Traynelis et al. 1993), the synaptic currents were analysed with peak-scaled non-stationary noise analysis. Before calculating the ensemble mean PSC, the individual spPSCs were aligned along the point of steepest rise between onset and peak. The peak of the mean current response waveform was scaled to the response value at the corresponding point in time of each individual event before subtraction to generate the difference waveforms. The ensemble mean PSC was binned (see above) and variance versus mean curves were plotted for the decay phase of the PSC. The curve was fitted with eqn (4) to obtain an estimate for the single-channel current (i). Depending on the shape of the variance versus mean curve (parabolic versus skewed; see Hartveit & Veruki (2006)), either the whole or a subregion (75%; excluding the rightmost data points) of the curve was used for curve fitting. The single-channel chord conductance was calculated from eqn (3).
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Results |
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This study includes results from 30 wide-field amacrine cells (19 whole-cell recordings and 11 outside-out patch recordings). The criteria used to visually target wide-field amacrine cells for recording were identical to those used to target AII amacrine cells (Gill et al. 2006). The targeting criteria were as follows: (1) location of the cell body at and across the border between the inner nuclear layer and the inner plexiform layer; (2) medium size of the cell body; and (3) a relatively thick primary dendrite that tapers as it descends into the inner plexiform layer. In most cases, these criteria resulted in recordings from AII amacrine cells, but in a smaller number of cases, we recorded instead from amacrine cells that displayed the characteristic morphology of wide-field amacrine cells. During whole-cell recordings from wide-field amacrine cells, we never observed unclamped action currents similar to those characteristically seen in AII amacrine cells (5 mV depolarizing test pulses from a holding potential of –60 mV; Mørkve et al. (2002)). All cells were filled with Lucifer yellow and after both whole-cell and outside-out patch recordings, fluorescence microscopy allowed visualization of each cell's morphology (Fig. 1A). For all the wide-field cells included in this study, fluorescence microscopy revealed long, thin processes, narrowly monostratifying in a single stratum (S) of the inner plexiform layer. The cells recorded from in this study stratified in S2, S3 or S4 (see examples in Fig. 1A). Laterally, the processes could extend beyond the microscope's field of view or could appear truncated at some distance from the soma. We often observed small varocisities and spine-like protrusions along the length of the processes. Based on the general morphological characteristics, as observed in the vertical slice preparation, we hypothesize that these cells are the rat counterparts to some of the types of wide-field amacrine cells described in detail in rabbit and mouse retina (Famiglietti, 1992a,b; MacNeil & Masland, 1998; MacNeil et al. 1999; Völgyi et al. 2001; Badea & Nathans, 2004; Lin & Masland, 2006). On the basis of Golgi-staining, the morphology of a few types of wide-field amacrine cells has also been described in whole-mounts of rat retina (Perry & Walker, 1980); however, the level of stratification in the inner plexiform layer was not reported with sufficient precision that an identification can reliably be made for cells only observed in vertical sections, as in our study. On this background, we have not attempted to classify each cell recorded from as the rat equivalent of a particular type of wide-field amacrine cell. We were not able to determine if the wide-field amacrine cells recorded from were axon-bearing or not.
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Spontaneous PSCs in wide-field amacrine cells
To isolate the action of glycine receptors on wide-field amacrine cells, whole-cell recordings were carried out in the presence of CNQX, TTX and bicuculline in order to block non-NMDA receptors, voltage-gated Na+ channels and GABAA receptors, respectively. Bicuculline was used at a concentration of 3 µM that presumably does not block glycine receptors (Protti et al. 1997; Jonas et al. 1998; Wang & Slaughter, 2005; Gill et al. 2006). Of the 19 wide-field amacrine cells that we made whole-cell recordings from, eight cells displayed spPSCs in this condition. All of these cells had processes stratifying in S2 of the inner plexiform layer. The spPSCs occurred at a low, irregular frequency (1.4 ± 0.3 Hz, range 0.29–2.3 Hz, n
= 8). Seven of these cells had enough spPSCs to contruct histograms of interevent intervals, which were well fitted by single-exponential functions (average time constant
= 0.50 ± 0.08 s), suggesting individual events occurred independently (Fig. 1D). Amplitude histograms were skewed towards larger values. For the example shown in Fig. 1E, the mode was 12.5 pA and the mean was 21.5 ± 1.1 pA (range 5–142 pA). The coefficient of variation was 0.79 for this cell. For the eight wide-field amacrine cells (range 50–921 events), the mode occurred at 14.5 ± 2.3 pA (range 7–31 pA) and the mean was 23 ± 5 pA (range 12–55 pA). The coefficient of variation varied between 0.28 and 1.0 (average 0.63 ± 0.09).
In AII amacrine cells, 300 nM of the specific glycine receptor antagonist strychnine is sufficient to block both spIPSCs and responses evoked by application of glycine to outside-out patches (Gill et al. 2006). In wide-field amacrine cells, spPSCs were completely and reversibly blocked by 300 nM strychnine (Fig. 1F; n = 4). The small, inwardly directed noise transients remaining in the presence of strychnine (Fig. 1F) could not be reliably identified as spIPSCs because the amplitude was too low, but could suggest that 3 µM bicuculline is not sufficient to completely block GABAA receptors in wide-field amacrine cells (cf. Protti et al. 1997). Based on this evidence, we consider the spPSCs recorded in the conditions described here to be glycinergic IPSCs. The low incidence of recording of wide-field amacrine cells precluded a more detailed pharmacological analysis.
Time course of spontaneous glycinergic IPSCs in wide-field amacrine cells
To examine the kinetic properties of the glycinergic spIPSCs, we selected well-separated events with monophasic waveforms. For five wide-field amacrine cells, each with more than 100 spIPSCs, we measured kinetic parameters for individual events and for ensemble averages. For the representative example illustrated in Fig. 2, the peak amplitude of individual spIPSCs varied from 6 to 70 pA (Fig. 2A; average 32.6 ± 0.7 pA). The 20–80% rise time of the individual spIPSCs varied from 150 to 910 µs (Fig. 2B; average 326 ± 7 µs). The decay phase of individual spIPSCs was moderately well fitted by single-exponential functions (Fig. 2D) with decay varying from 10 to 139 ms (Fig. 2C; average 42 ± 1 ms). For analysis of the average spIPSC, the decay phase was better fitted with a double-exponential function (Fig. 2E; fast
= 19 ms, slow
= 85 ms; amplitude contribution 66% and 34%, respectively) than with a single-exponential function (not shown; decay
= 37 ms).
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decay (Fig. 2F), suggesting the absence of differential electrotonic filtering. Furthermore, for this and another cell there was no correlation between 20%–80% rise time and peak amplitude (Fig. 2G), but for three of the five cells, there was a significant negative correlation between these parameters, suggesting the presence of differential electrotonic filtering (Spearman's rank order correlation test; Spearman's R varied between –0.54 and –0.30; P < 0.0001). Although it is very likely that spIPSCs originating at distal locations in the dendritic tree of a wide-field amacrine cell would be subject to marked electrotonic filtering, the lack of correlation between 20%–80% rise time and decay suggests that the range of observed values for decay cannot solely be explained by differential electrotonic filtering of identical, fast events originating at different locations in the dendritic tree. It is also likely that a substantial fraction of the recorded spIPSCs originated from relatively proximal regions of the dendritic tree of the wide-field amacrine cells, either because of a corresponding spatial restriction in the location of glycinergic synaptic input, or, alternatively, because of truncation of cellular processes during the slicing procedure and a corresponding removal of distant synaptic input. A proximal origin for many spIPSCs is supported by the relatively fast rise times of the majority of spIPSCs.
Kinetic properties of ensemble averages were measured for all eight wide-field amacrine cells that displayed glycinergic spIPSCs, with the number of events ranging between 35 and 465. The population average of the 20–80% rise time was 340 ± 53 µs (range 250–630 µs). For comparison with other published reports, the average 10–90% rise time was 570 ± 110 µs (range 390–1200 µs). For each cell, the decay phase of the average spIPSC was best fitted by a double-exponential function (fast
= 15 ± 2 ms; 77 ± 2% amplitude contribution; slow
= 57 ± 7 ms; 23 ± 2% amplitude contribution). The amplitude-weighted decay was 25 ± 3 ms. When the decay phase was fitted with a single-exponential function, the average decay was 26 ± 4 ms (not shown).
Non-stationary noise analysis of glycinergic spIPSCs in wide-field amacrine cells
For the five wide-field amacrine cells with more than 100 events (see above), we tested for time stability by systematically searching the recording for a consecutive series of spIPSCs with no time-dependent change in either peak amplitude, 20–80% rise time or decay (Fig. 3A–C; cf. Momiyama et al. 2003). For each of the five cells, such regions were identified, with Spearman's R varying between –0.13 and 0.10 for peak amplitude, between –0.01 and 0.13 for 20–80% rise time and between –0.03 and 0.18 for decay (P
= 0.25 ± 0.10 for peak amplitude; P
= 0.61 ± 0.14 for 20–80% rise time; P
= 0.42 ± 0.16 for decay).
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60% of the curve), gave a unitary current (i) of 2.2 pA, corresponding to a unitary chord conductance () of 37.1 pS. For the five cells, peak-scaled non-stationary noise analysis gave a unitary chord conductance of 34 ± 4 pS (range 24–48 pS). Deactivation and desensitization kinetics of glycine receptors in outside-out patches from wide-field amacrine cells
The analysis of spIPSCs in wide-field amacrine cells demonstrated markedly slow decay kinetics compared to spIPSCs in AII amacrine cells (Gill et al. 2006). The slow decay could be due to the expression of synaptic glycine receptor channels with slow kinetic properties. Alternatively, the slow decay could be due to slow clearance of transmitter from the synaptic cleft. A third possibility is that the slow decay is related to the neuronal morphology with long, thin processes, giving rise to inadequate space-clamp and pronounced electrotonic filtering. In order to directly investigate the kinetic properties of glycine receptor channels in wide-field amacrine cells, we measured responses evoked by ultrafast application of glycine to somatic outside-out patches. Figure 4A illustrates the response evoked by a short (5 ms) pulse of glycine (3 mM) to a patch from a wide-field amacrine cell. The response rose rapidly to a peak with a 20–80% rise time of 360 µs. For nine patches, the 20–80% rise time of responses evoked by brief (2–5 ms) pulses was 610 ± 79 µs (range 360–1020 µs). At the end of the pulse, the response decayed very slowly. This deactivation, which reflects the closure of channels after removal of agonist, was well fitted by a double-exponential function. For the patch shown in Fig. 4A, fast was 74 ms and slow was 410 ms. The amplitude contributions were 66% and 34%, respectively. The average fast was 45 ± 5 ms (64 ± 7% amplitude contribution) and the average slow was 350 ± 56 ms (36 ± 7% amplitude contribution; n
= 9). For the patch illustrated in Fig. 4A, the amplitude weighted decay was 190 ms (average = 170 ± 39 ms). When the time course of deactivation was fitted with a single-exponential function, we obtained a decay of 190 ms (average 160 ± 47 ms). The deactivation kinetics of patches from wide-field amacrine cells were considerably slower than the deactivation kinetics previously observed for patches from AII amacrine cells (Gill et al. 2006; Fig. 4B).
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fast
= 24 ms and slow
= 670 ms. The amplitude contributions were 35% and 65%, respectively. For a total of eight patches, the average fast was 43 ± 10 ms (53 ± 8% amplitude contribution) and the average slow was 680 ± 170 ms (47 ± 8% amplitude contribution). The average amplitude-weighted decay was 380 ± 150 ms. The equilibrium response to 3 mM glycine was measured as the current at the end of the 1000 ms application, and was on average 32 ± 3% of the peak response. While glycinergic spIPSCs were only observed in wide-field amacrine cells stratifying within S2, glycine-evoked responses in outside-out patches were obtained in all types of wide-field amacrine cells recorded from, including those with processes stratifying in S2, S3 and S4. Although our material is too small for statistical testing of potential differences in response kinetics between cells stratifying at different levels of the inner plexiform layer, the data did not suggest the presence of such differences.
Non-stationary noise analysis of transient currents evoked by glycine in outside-out patches from wide-field amacrine cells
When spIPSCs were analysed by non-stationary noise analysis, peak-scaling precluded any measurement of Popen. In order to estimate the single-channel current and maximum Popen of glycine receptors in wide-field amacrine cells, we used non-stationary noise analysis of responses evoked by ultrafast application of brief pulses of 3 mM glycine to outside-out patches from wide-field amacrine cells. Figure 5A shows three individual, successive records evoked by glycine, and the ensemble mean is shown in Fig. 5C. Because of moderate rundown of the response, we estimated the ensemble variance by calculating the differences of overlapping pairs of successive responses (see Methods). Two such pairwise difference traces (for the responses in Fig. 5A) are shown in Fig. 5B, and the resulting ensemble variance is shown in Fig. 5D. The variance versus mean plot of the data points corresponding to the decaying phase is shown in Fig. 5E and has a clear parabolic shape, indicating that the maximum Popen reached a value larger than 0.5. When the data points were fitted with eqn (4), the apparent glycine-activated single-channel current was 2.0 pA, corresponding to an apparent single-channel chord conductance of 33.4 pS. The number of active channels was estimated as 28.8, corresponding to a maximum Popen at the peak response of 0.72 (Fig. 5E). For eight patches tested with glycine (3 mM), the mean single-channel chord conductance was 31 ± 3 pS (range 19–39 pS), and the mean number of available channels was 34 ± 4 (range 19–48). The average maximum Popen was 0.76 ± 0.03 (range 0.63–0.85).
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The estimates from non-stationary noise analysis potentially represent weighted averages of different conductance levels, irrespective of whether they correspond to different channels or different (sub)conductance levels of the same channels. Cull-Candy et al. (1988) demonstrated that the weights are determined by the relative number of channels, the single-channel conductance(s) for each channel type and their open probability. It is therefore important to investigate the relation between the apparent single-channel conductance values estimated by non-stationary noise analysis (spIPSCs or outside-out patches) and directly observed single-channel conductance levels. For several outside-out patches with low noise levels, discrete transitions between open and closed states of varying levels were apparent during later phases of individual current responses. Figure 6A shows the response of an outside-out patch from a wide-field amacrine cell to a brief pulse of glycine (3 mM) with several discrete transitions between open and closed states. We estimated the single-channel conductance by constructing an all-point amplitude histogram (Fig. 6A; inset) from a selected segment in a late phase of the response (Fig. 6A; legend). For the analysed segment, the single-channel current was 2.8 pA, corresponding to a single-channel chord conductance of 47 pS. Observations of similar single-channel openings (42–52 pS) were made for 11/11 patches. In addition, however, in several patches we also observed lower and higher current levels, corresponding to conductance levels of approximately 25–28 pS, 32–38 pS and 70–83 pS. Figure 6B shows a response with a segment containing repeated single-channel openings of 1.9 pA, corresponding to a single-channel chord conductance of 32 pS. Single-channel transitions with even lower amplitude were observed, but not analysed further. We never observed single-channel transitions with conductance levels around 100–110 pS, the reported main conductance level of 2 homomeric receptors (Bormann et al. 1993).
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2.8 pA (47 pS), as well as a lower amplitude of 2.1 pA (35 pS), as illustrated by the examples in Fig. 6C. In glycinergic spIPSCs of AII amacrine cells, we observed single-channel transitions with an amplitude of 5 pA (Gill et al. 2006), corresponding to the reported main conductance state of homomeric 1 receptors (83 pS; Bormann et al. 1993). We never observed transitions corresponding to this, or higher conductance levels, in spIPSCs in wide-field amacrine cells. |
Discussion |
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The decay time course of spIPSCs in wide-field amacrine cells was best fitted by double-exponential functions, with fast
15 ms and slow
57 ms. A double-exponential decay has also been found in other glycinergic synapses (Legendre, 1998; Singer et al. 1998; Singer & Berger, 1999; Gill et al. 2006). The amplitude contribution of the fast component was 77%. Because of the special cellular morphology of wide-field amacrine cells, with long, thin processes, it is relevant to consider whether the slow decay kinetics of spIPSCs could be caused by inadequate voltage-clamp control in the dendritic tree. In this case, the slow decay kinetics would simply correspond to pronounced electrotonic filtering. While synaptic currents originating from distant sites of the dendritic tree of wide-field amacrine cells undoubtedly would undergo a considerable degree of electrotonic filtering when the cells are voltage clamped at the soma, other observations suggest instead that the glycine receptor channels of wide-field amacrine cells have genuinely slow decay kinetics. First, although the spIPSCs in wide-field amacrine cells displayed a range of rise times, long decay times were also observed for the spIPSCs displaying the fastest rise times (<500 µs). Second, directly resolved single-channel gating activity could be observed in spIPSCs with long decay times, suggesting that these events originated relatively close to the soma, with little electrotonic filtering. Finally, responses evoked by ultrafast application of brief pulses of glycine to outside-out patches displayed very slow deactivation kinetics. The decay phase could be well fitted by double-exponential functions, with fast
45 ms and slow
350 ms.
A more detailed comparison of the decay kinetics of spIPSCs and the deactivation kinetics of patch responses evoked by brief pulses of glycine suggests that the synaptic receptors and the extrasynaptic receptors are similar, but not identical (Table 1). Both fast and, in particular, slow were longer for the patch responses than for the spIPSCs. This could be due to differences in the exact subunit composition of synaptic versus extrasynaptic receptors or differences in the modulatory state of specific subunits. The decay kinetics of glycinergic spIPSCs and the deactivation kinetics of glycine responses in patches were considerably slower for wide-field amacrine cells than for AII amacrine cells (Table 1; Gill et al. 2006). We are aware of only one other report that has measured kinetic properties of glycinergic spIPSCs in mammalian amacrine cells. Frech et al. (2001) recorded glycinergic spIPSCs in four unidentified mouse amacrine cells and reported a decay time constant of 25 ms. It is unclear, however, if the decay was best fitted by a single- or double-exponential function.
Non-stationary noise analysis of spIPSCs and patch responses in wide-field amacrine cells yielded an average apparent single-channel conductance of 34 pS and 31 pS, respectively. With an average spIPSC peak amplitude of 23 pA, this corresponds to 11 glycine receptor channels being open at the peak of a single spIPSC. With a saturating concentration of agonist, the glycine receptors in patches reached a maximum Popen of 0.76. If the synaptic receptors reach a similar maximum Popen, these estimates suggest that 14–15 receptors are available to bind transmitter after release of a single vesicle.
Glycine receptors expressed by wide-field amacrine cells
Individual estimates of glycine receptor single-channel conductance by non-stationary noise analysis ranged from 24 pS to 48 pS for spIPSCs and from 19 pS to 39 pS for patches. We directly identified single-channel transitions between closed and open states in both types of responses. For patch responses, we observed openings corresponding to a single-channel conductance of 47 pS (42–52 pS) in 11/11 patches. In addition, we observed conductance levels at 25–28 pS, 32–38 pS and 70–83 pS, but none at 100–110 pS. The multiple conductance levels could reflect a corresponding receptor heterogeneity, but could also be related to the documented presence of subconductance levels of glycine receptors in the outside-out patch configuration (Bormann et al. 1987; Takahashi & Momiyama, 1991; Beato & Sivilotti, 2007). For spIPSCs, the signal-to-noise ratio was lower, but we could directly identify openings with a single-channel conductance of 35 pS and 47 pS. Given that the directly resolved channel openings were observed during later phases of the responses, it is possible that additional conductance levels remained undetected if the corresponding channels displayed faster decay kinetics and thereby contributed primarily to the peak of the response.
The slow decay kinetics of spIPSCs and patch responses suggest that the 2-glycine receptor subunit contributes to both synaptic and extrasynaptic receptors in wide-field amacrine cells (Takahashi & Momiyama, 1991). In immunocytochemical studies of the distribution of glycine receptor subunits in mouse retina (Haverkamp et al. 2003, 2004), many 2- and 3-immunoreactive clusters were found in register with amacrine cell processes labelled for the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). Because wide-field amacrine cells presumably are GABAergic (Pourcho & Goebel, 1983; reviewed by Vaney, 1990), this suggests that these and other GABAergic amacrine cells receive glycinergic synaptic input mediated via receptors containing these subunits. At the same time, it is difficult to exclude a contribution of receptors with the kinetically faster 1-subunit to wide-field amacrine cells, particularly for synaptic receptors which displayed faster decay kinetics than extrasynaptic receptors (see above). Notably, in a study of glycine receptors in zebrafish embryos, Ali et al. (2000) demonstrated that simulations of evoked currents with varying relative proportions of glycine receptors with slow (‘2’) and fast (‘1’) decay kinetics resulted in responses with intermediate decay kinetics. On the average, the decay kinetics of glycinergic spIPSCs in wide-field amacrine cells are most similar to glycinergic spIPSCs of ‘intermediate’ kinetics in zebrafish embryos (Ali et al. 2000). Both types of spIPSCs displayed faster decay kinetics than ‘slow’ spIPSCs in zebrafish embryos. On the other hand, the decay kinetics of glycine-evoked patch responses in wide-field amacrine cells are most similar to the decay kinetics of ‘slow’ patches in zebrafish embryos, and, to our knowledge, these are the slowest responses reported for glycine receptors. Both spIPSCs and patch responses in wide-field amacrine cells display slower decay kinetics than spIPSCs and patch responses in neonatal brain stem motoneurons where the 2-subunit is thought to be involved (Singer et al. 1998; Singer & Berger, 1999). We are not aware of any reports that have measured deactivation kinetics of responses mediated by receptors involving the 3- and 4-subunits. Accordingly, the possibility that these subunits are expressed by the wide-field amacrine cells we have recorded from and contribute to their spIPSCs has to be kept open.
A single-channel conductance of 47 pS, as observed in both spIPSCs and in patch responses, suggests the presence of heteromeric receptors (44–54 pS; Bormann et al. 1993), as opposed to homomeric receptors. Previous evidence has suggested the presence of homomeric receptors in extrasynaptic, but not synaptic glycine receptors (Takahashi et al. 1992). The lack of single-channel openings at the level characteristic of homomeric 2 receptors (100–110 pS; Bormann et al. 1993) suggests the absence of such receptors in these cells. This is consistent with evidence that the specific properties of homomeric 2 receptors impairs a proper synaptic functioning (Mangin et al. 2003), in particular the low Popen attained at transmitter concentrations thought to be reached in the synaptic cleft (1–3 mM). In patches from wide-field amacrine cells we found an average peak Popen of 0.76 with 3 mM glycine. We are not aware of any reports that have measured Popen for 2 heteromeric receptors. On the basis of these observations, and assuming that 2 receptor subunits contribute to the synaptic glycine receptors of the wide-field amacrine cells studied here, we propose that the receptors are dominated by heteromeric 2, as opposed to homomeric 2, receptors. Because the apparent single-channel conductance estimates (for both spIPSCs and patches) are lower than the presumed main conductance state for heteromeric receptors (47 pS), it is likely that lower conductance states make a significant contribution to glycine-evoked activity.
Functional consequences of the kinetic properties of glycine receptor channels in wide-field amacrine cells
The slow decay kinetics of spIPSCs in wide-field amacrine cells will impact the integrative properties of these cells and determine how inhibitory inputs shape their response properties. With perfectly synchronous release, the slow kinetics of spIPSCs set a lower limit on the time course of evoked IPSCs. This means that summation of input will occur at relatively low input frequencies, and that wide-field amacrine cells might be effective integrators of glycinergic input. Without knowledge of the activity patterns of their presynaptic inputs, the potential physiological relevance is difficult to predict. Another implication is that the timing of the inhibitory input, relative to that of the excitatory input, may not be very critical for the functional impact of glycinergic inhibition in several types of wide-field amacrine cells. If inhibitory input to wide-field amacrine cells primarily acts as shunting inhibition, the long duration of glycinergic synaptic events will lead to a correspondingly long-lasting inhibition. It will therefore be important to investigate the kinetic properties of the receptors mediating glutamatergic input from bipolar cells to wide-field amacrine cells.
For wide-field amacrine cells in rat retina, we are not aware of previous morphological or physiological studies that have directly addressed the type(s) of cells we have recorded from. In general, however, electron microscopic investigations have revealed that the dendrities of wide-field amacrine cells typically are both presynaptic (with synaptic vesicles) as well as postsynaptic (with corresponding membrane specializations) (Dowling & Boycott, 1965). Because of the proximity of pre- and postsynaptic sites along the dendrites of many wide-field amacrine cells, it is likely that local glycinergic, inhibitory input will directly modulate transmitter release from neighbouring presynaptic sites. In the thin dendritic processes of these cells, the input resistance is likely to be high such that small excitatory synaptic currents can give rise to relatively large depolarizations, amplified by activation of voltage-gated currents (Koizumi et al. 2001). The long-lasting glycinergic inhibition observed here could be a very effective mechanism for counteracting activation of these voltage-gated currents. For rat amacrine cells in culture, there is evidence that propagating action potentials can be generated both locally in dendritic regions as well as in the soma (Koizumi et al. 2001), and that propagation from the soma to individual dendrites can be regulated by inhibitory input (Yamada et al. 2002). If similar mechanisms operate in wide-field amacrine cells in vivo, it is tempting to speculate that the glycinergic inhibitory input we have observed in the present study could be involved in dynamic regulation of the output characteristics of these cells. In wide-field amacrine cells of tiger salamander retina, there is physiological evidence that excitatory input from bipolar cells is concentrated to a narrow area of processes close to the soma, while inhibitory input is distributed over a larger area of the dendritic tree (Cook & Werblin, 1994). Similar mapping of the spatial sensitivity to excitatory and inhibitory transmitters of wide-field amacrine cells in the mammalian retina remains to be performed. In light of the slow decay kinetics of the glycinergic IPSCs observed in the wide-field amacrine cells and the location of their dendritic processes in a specific stratum of the inner plexiform layer, it is also possible that some types of wide-field amacrine cells, and their presynaptic glycinergic partners, could be involved in a disinhibitory circuit with operational characteristics similar to those proposed to generate transient visual responses of ganglion cells in the amphibian retina (Roska et al. 1998).
Of the glycinergic amacrine cells described in the rat retina (Menger et al. 1998), all are narrow-field cells. Some have dendritic trees restricted to the OFF-sublamina, some have dendritic trees restricted to the ON-sublamina, and others have dendritic trees spanning both the ON- and OFF-sublamina. In addition, the processes of all glycinergic amacrine cells span at least two strata of the inner plexiform layer. Thus, the narrow stratification of wide-field amacrine cells, like the ones studied here, is not matched by a correspondingly narrow stratification of potential candidates for glycinergic input. The morphology of the dendritic trees of glycinergic amacrine cells suggests that these cells have the potential to communicate between different strata and sublaminae (ON versus OFF) of the inner plexiform layer (reviewed by Wässle, 2004), and also means that we cannot predict the visual response polarity (OFF versus ON versus ON/OFF) of the glycinergic input to wide-field amacrine cells stratifying at a specific level of the inner plexiform layer.
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