Characterization of the Spontaneous Synaptic Activity of Amacrine Cells in the Mouse Retina

Moritz J. Frech, Jorge Pérez-León, Heinz Wässle, and Kurt H. Backus

Max-Planck-Institut für Hirnforschung, Neuroanatomische Abteilung, D-60528 Frankfurt am Main, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Frech, Moritz J., Jorge Pérez-León, Heinz Wässle, and Kurt H. Backus. Characterization of the Spontaneous Synaptic Activity of Amacrine Cells in the Mouse Retina. J. Neurophysiol. 86: 1632-1643, 2001. Amacrine cells are a heterogeneous class of interneurons that modulate the transfer of the light signals through the retina. In addition to ionotropic glutamate receptors, amacrine cells express two types of inhibitory receptors, GABAA receptors (GABAARs) and glycine receptors (GlyRs). To characterize the functional contribution of these different receptors, spontaneous postsynaptic currents (sPSCs) were recorded with the whole cell configuration of the patch-clamp technique in acutely isolated slices of the adult mouse retina. All amacrine cells investigated (n = 47) showed spontaneous synaptic activity. In six amacrine cells, spontaneous excitatory postsynaptic currents could be identified by their sensitivity to kynurenic acid. They were characterized by small amplitudes [mean: -13.7 ± 1.5 (SE) pA] and rapid decay kinetics (mean tau : 1.35 ± 0.16 ms). In contrast, the reversal potential of sPSCs characterized by slow decay kinetics (amplitude-weighted time constant, tau w, >4 ms) was dependent on the intracellular Cl- concentration (n = 7), indicating that they were spontaneous inhibitory postsynaptic currents (sIPSCs). In 14 of 34 amacrine cells sIPSCs were blocked by bicuculline (10 µM), indicating that they were mediated by GABAARs. Only four amacrine cells showed glycinergic sIPSCs that were inhibited by strychnine (1 µM). In one amacrine cell, sIPSCs mediated by GABAARs and GlyRs were found simultaneously. GABAergic sIPSCs could be subdivided into one group best fit by a monoexponential decay function and another biexponentially decaying group. The mean amplitude of GABAergic sIPSCs (-42.1 ± 5.8 pA) was not significantly different from that of glycinergic sIPSCs (-28.0 ± 8.5 pA). However, GlyRs (mean T10/90: 2.4 ± 0.08 ms) activated significantly slower than GABAARs (mean T10/90: 1.2 ± 0.03 ms). In addition, the decay kinetics of monoexponentially decaying GABAARs (mean tau w: 20.3 ± 0.50), biexponentially decaying GABAARs (mean tau w: 30.7 ± 0.95), and GlyRs (mean tau w = 25.3 ± 1.94) were significantly different. These differences in the activation and decay kinetics of sIPSCs indicate that amacrine cells of the mouse retina express at least three types of functionally different inhibitory receptors: GlyRs and possibly two subtypes of GABAARs.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

GABA and glycine are the most abundant inhibitory neurotransmitters in the mammalian CNS. By activating specific receptors, GABA and glycine can modulate the membrane resistance by controlling a Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> conductance of the cell membrane. GABAA receptors (GABAARs) and glycine receptors (GlyRs) are ligand-gated channels assembled from five specific subunits that form the receptor/ion channel complex. A broad variety of distinct subunits of GABAARs and GlyRs have been characterized that provide a structural basis for many different receptor subtypes (McKernan et al. 1991; Möhler et al. 1996a). Recently the distribution of these subunits in the mammalian nervous system was investigated with in situ hybridization and immunohistochemical studies (Fritschy and Möhler 1995; Laurie et al. 1992; Persohn et al. 1992; Wisden et al. 1992). With electrophysiological experiments, some pharmacological and biophysical properties of several recombinant receptor subtypes have been characterized (Mihic et al. 1995; Möhler et al. 1996b). However, our knowledge of the molecular composition and the functional significance of endogenous GABAAR and GlyR subtypes is still poor. To characterize the functional differences of these receptors and their physiological roles, we used retinal amacrine cells as a model system. Amacrine cells are a heterogeneous class of 20-40 types of interneurons that modulate the transfer of light signals in the mammalian retina (MacNeil and Masland 1998; Vaney 1990). About one half of the amacrine cell population is known to release GABA while the other half was found to be glycinergic. GABAergic and glycinergic amacrine cells provide multiple inhibitory inputs to retinal bipolar cells and ganglion cells, thereby mediating some of the most important functions in light signal processing such as lateral inhibition and direction selectivity (Cook and McReynolds 1998; Taylor et al. 2000; Wässle and Boycott 1991). In addition, amacrine cells themselves receive synaptic input from other amacrine cells and express GABAARs and GlyRs (Greferath et al. 1994a,b, 1995; Sassoè-Pognetto and Wässle 1997; Sassoè-Pognetto et al. 1994; Wässle et al. 1998). Thus the reciprocal inhibition mediated by GABAARs and GlyRs between amacrine cells might play an important role in the fine tuning of the inhibition directed to bipolar cells and ganglion cells.

To investigate the functional contributions of GABAARs and GlyRs in the inhibitory network formed by amacrine cells, we have applied the patch-clamp technique in acutely isolated slices of the adult mouse retina to record the spontaneous inhibitory postsynaptic currents (sIPSCs) of amacrine cells. Besides rapidly decaying spontaneous excitatory postsynaptic currents (sEPSCs), slowly activating glycinergic and fast activating GABAergic sIPSCs were found. At least two types of GABAergic sIPSCs could be identified that significantly differed with respect to their decay kinetics. A portion of these results has been presented in abstract form (Frech and Backus 2000).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of retinal slices, visualization and identification of amacrine cells

Retinal slices were prepared as previously described (Boos et al. 1993; Euler et al. 1996). Adult mice (older than 7 wk) were killed by cervical dislocation. The eyes were enucleated and opened by an encircling cut along the ora serrata. The vitreous body was removed, and the retina was carefully dissected free and cut into four pieces. Vertical slices (thickness ca. 200-300 µm) of the retina were cut with a scalpel or a microslicer (DSK, Kyoto, Japan), stored at room temperature in extracellular saline, and bubbled with 95% O2-5% CO2. Electrophysiological experiments were started 30 min after preparation of the slices. Retinal neurons were visualized by differential interference contrast microscopy using a ×40 water-immersion objective and a digital camera (PCO Computer Optics GmbH, Kelheim, Germany) mounted on an upright microscope (Axioskop FS; Zeiss, Oberkochen, Germany). Recordings were taken from visually identified amacrine cells in the inner nuclear layer (INL) of the retina. All cells were filled with Lucifer yellow during recording to confirm their identity as amacrine cells based on their cell body position in the INL and on morphological criteria such as the dendritic ramification pattern (Fig. 1A). However, we did not find any correlation between any morphological characteristics of the amacrine cells investigated and the properties of sEPSCs or sIPSCs analyzed. In addition, electrical membrane properties were checked (Fig. 1B). Voltage-dependent Na+ currents were found in only one amacrine cell, which was not included in our sample. Bipolar cells also did not express significant voltage-dependent inward currents but could be easily distinguished by their characteristic morphology. Amacrine cells located in the ganglion cell layer (displaced amacrine cells) were not included in this study.



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Fig. 1. Whole cell currents in identified amacrine cells of the mouse retina. A: micrograph showing the patch pipette and an amacrine cell filled with Lucifer yellow with a typical dendritic tree stratifying in the inner plexiform layer. B: typical current responses to depolarizing voltage steps of an amacrine cell recorded in the whole cell configuration comprising mainly voltage-dependent outwardly rectifying K+ currents; Vh = -60 mV; step increment +10 mV; from -100 to +40 mV; duration 100 ms. C: continuous recording of the spontaneous postsynaptic activity of an amacrine cell in the whole cell configuration. Two types of postsynaptic currents were observed: fast decaying small sEPSCs (some of them indicated by up-arrow ) and slowly decaying sIPSCs. Bottom: enlarged display of sEPSCs as indicated.

Solutions and chemicals

Slices were continuously superfused with a physiological extracellular saline that contained (in mM): 125 NaCl, 2.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 25 glucose, 2 CaCl2, and 1 MgCl2. To maintain the extracellular pH at 7.4, the saline was bubbled with 95% O2 and 5% CO2. The patch pipette solution contained (in mM): 140 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, and 11 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA); pH adjusted to 7.2 with KOH. Stock solutions of strychnine (1 mM in distilled water) and bicuculline (10 mM in distilled water) were prepared shortly before the experiments and added to the extracellular saline in defined concentrations. To block sEPSCs mediated by ionotropic glutamate receptors, 1 mM kynurenic acid was added to the extracellular saline in some experiments.

Patch-clamp recordings

Patch pipettes were pulled from borosilicate glass tubing (2.0 mm OD, 0.5-mm wall thickness; Hilgenberg, Malsfeld, Germany). When filled with internal solution, they had a resistance of 6-8 MOmega . Amacrine cells were approached under visual control by maintaining a moderate positive pressure in the patch pipette. Membrane currents were recorded in the whole cell configuration of the patch-clamp technique using an EPC-9 amplifier (Heka, Lambrecht, Germany). Patch pipette capacitance and cell capacitance were canceled and series resistance was compensated by about 80% using the internal compensation circuits of the amplifier. Recordings were done at a holding potential (Vh) of -60 mV. If the holding current exceeded ±20 pA, the recording was discarded. The sampling frequency was 10 kHz. Currents were filtered at 2 kHz using the internal low-pass filter of the amplifier. Data were digitized and stored on-line using the Pulse software (Heka). All recordings were made at room temperature (20-24°C).

Analysis of postsynaptic currents and statistics

Spontaneous postsynaptic currents (sPSCs) were detected (threshold 8-10 pA) by using the MiniAnalysis software (Synaptosoft, Leonia, NJ). Peak amplitudes, rise times, and decay time constants were estimated for single sPSCs and further analyzed on a Pentium-based personal computer using the MiniAnalysis, Igor (Wavemetrics, Lake Oswego, OR), and Origin software (MicroCal, Northampton, MA). Mean amplitudes and frequencies of sPSCs were computed from all sIPSCs and all sEPSCs observed in an amacrine cell. Only events that did not show any signs of multiple peaks (i.e., contamination of rise or decay phases by subsequent events) were selected for analysis of the kinetics and for exponential fitting. The rise times of the sPSCs were determined by calculating the time in which the current increased from 10 to 90% of the peak amplitude denoted as T10/90. To compare our T10/90 values with the activation kinetics of recombinant GABAARs from another study (Zhu et al. 1998), we converted their 20-80% rise time values (T20/80) into T10/90 values by assuming a monoexponential rise, which gives T10/90 = T20/80 * 1.585. Decay kinetics of single sIPSCs were determined by least-square fits of the decay phase after the peak current using a monoexponential and a biexponential decay function. The number of exponentials necessary for a good fit of the data were determined by visual inspection. To allow the comparison of events best fitted with a different number of exponentials, the weighted time constant, tau w = (A1tau 1 + A2tau 2)/(A1 + A2), was calculated, where A1 and tau 1 are the amplitude and the time constant of the fast component and A2 and tau 2 are the amplitude and the time constant of the slow component of the biexponential fit, respectively. To calculate the decay kinetics of sEPSCs, at least 50 sEPSCs were randomly selected, averaged, and fitted using a monoexponential decay function.

All data are given as means ± SE. The significance of the difference between the mean values of two samples was determined using the two-tailed Student's t-test for unpaired data. The correlation between two samples, i.e., between tau w and T10/90, was determined by linear regression analysis. If not stated otherwise, data were denoted as statistically significant when P < 0.01.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have used the whole cell configuration of the patch-clamp-technique to characterize the sPSCs in retinal amacrine cells. Amacrine cells were filled with Lucifer yellow during the recordings to identify them by the position of their cell bodies, their arborization pattern and other morphological criteria (Fig. 1A). Amacrine cells also expressed a characteristic set of outwardly rectifying K+ currents (Fig. 1B) as previously described (Boos et al. 1993; Menger and Wässle 2000). The sPSCs recorded from 34 amacrine cells could be subdivided due to pharmacological criteria (see following text) into sEPSCs mediated by ionotropic glutamate receptors and into sIPSCs mediated by GABAARs and GlyRs (Fig. 1C).

EPSCs

In 6 of 34 amacrine cells, we observed sPSCs that could be clearly separated into a group that was characterized by small amplitudes and rapid decay kinetics (Fig. 1C, up-arrow ) and another group with significantly higher amplitudes and slower decay kinetics (Fig. 1C). The rapid sPSCs persisted in the presence of 1 µM strychnine and 10 µM bicuculline but completely disappeared in the presence of 1 mM kynurenic acid (not shown), indicating that they were sEPSCs mediated by ionotropic glutamate receptors. The other group of sPSCs could be blocked by bicuculline or strychnine, indicating that they were sIPSCs mediated by GABAARs and GlyRs (described in detail in the following text). The appearance of the sEPSCs was very consistent. Their amplitude distributions showed a typical narrow distribution ranging from 5 to 25 pA (Fig. 2A, inset) with a mean peak amplitude of -13.7 ± 1.5 pA (n = 6). sEPSCs occurred at a mean frequency of 0.49 ± 0.26 Hz (n = 4) and decayed with a mean tau  of 1.35 ± 0.16 ms (n = 6). sEPSCs and sIPSCs could be easily distinguished by the size of their amplitudes (Fig. 2B) and, in particular, by their different decay kinetics. Superimposed and averaged traces of sEPSCs and the corresponding cumulative fraction plot were compared with sIPSCs recorded in the same amacrine cell (Fig. 2, C and D), showing that the decay kinetics of sEPSCs and sIPSCs were significantly different.



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Fig. 2. Comparison of spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs) in an amacrine cell. A: wide-ranged polymodal amplitude histogram of all sPSCs recorded from an amacrine cell (mean amplitude -54.7 ± 3.0; binwidth: 3 pA; n = 1045). Inset: narrow-ranged unimodal amplitude histogram of sEPSC amplitudes (mean amplitude -13.0 ± 0.2; binwidth: 1 pA; n = 235). B: the corresponding cumulative fraction plot of the amplitudes shown in A indicates the significant difference between sEPSC and sIPSC amplitudes. C, top: superimposed traces of sIPSCs and sEPSCs (normalized for better representation) as indicated. Bottom: averaged traces of sEPSCs and sIPSCs were normalized and superimposed to show their different decay kinetics. Thin lines represent fitted curves (sEPSCs: tau  = 1.04 ms; n = 235; sIPSCs: tau w = 15.0 ms; n = 206). D: cumulative fraction plot of the decay time constants of sEPSCs and sIPSCs indicating the significantly different distribution.

Unfortunately, the use of kynurenic acid to block sEPSCs also strongly decreased the frequency of sIPSCs. However, the distinctive properties of sEPSCs and sIPSCs allowed us to perform most experiments in the absence of kynurenic acid. In addition, events characterized by a tau w < 4 ms were discarded while analyzing sIPSCs.

IPSCs

To resolve currents mediated by Cl- permeable GABAARs and GlyRs at a Vh close to the resting potential of amacrine cells (Bloomfield 1992; Boos et al. 1993; Zhou and Fain 1995), a pipette solution was used that contained 144 mM Cl- (ECl- = 3 mV). The reversal potential of sIPSCs was determined by recording sIPSCs at different Vh values and plotting their mean peak amplitudes against Vh. With the 144 mM Cl--containing pipette solution, sIPSCs reversed their polarity at +3.7 mV (n = 6; Fig. 3C) as approximated by linear regression, a value close to the ECl- of 3 mV. When a low-Cl--containing pipette solution was used (14 mM Cl-; ECl- = -57 mV; Fig. 3B), the reversal potential of the sIPSCs was -47.1 mV (n = 7; Fig. 3D), thus significantly shifted toward the new ECl-, indicating that sIPSCs were dependent on the intracellular Cl- concentration as expected for currents mediated by GABAARs and GlyRs. The deviation of the estimated reversal potential from ECl- is likely due to a significant HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> permeability of GABAARs and GlyRs (Backus et al. 1998; Frech et al. 1999; Kaila 1994).



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Fig. 3. Reversal potential of sIPSCs in amacrine cells. sIPSCs of an amacrine cell continuously recorded in the whole cell configuration using a 144 mM Cl--containing pipette solution (A; ECl- = 3 mV) and another amacrine cell using a 14 mM Cl--containing pipette solution (B; ECl- = -57 mV) at different Vh as indicated. C and D: the mean peak amplitudes were calculated and plotted against Vh. The different symbols represent recordings from different amacrine cells. The reversal potentials were estimated by linear regression as indicated (---). Reversal potential with the 144 mM Cl--containing pipette solution was 3.7 mV (C). With the 14 mM Cl--containing pipette solution, the reversal potential was shifted toward the new ECl- to -47.1 mV (D), indicating that these sIPSCs were mediated by GABAARs and GlyRs.

GABAergic sIPSCs

To characterize the properties of the inhibitory receptor types mediating sIPSCs, we utilized the selective antagonists of GABAARs, bicuculline, and of GlyRs, strychnine. A typical experiment is presented in Fig. 4, where sIPSCs mediated by GABAARs could be pharmacologically isolated. Figure 4A shows the variation of the peak amplitudes of sIPSCs as a function of the recording time in an amacrine cell that did not exhibit sEPSCs. In the absence of any antagonist (control, Fig. 4B), sIPSCs were observed at a frequency of 3.1 Hz. The corresponding amplitude distribution of these sIPSCs showed several peaks and was skewed toward lower values (Fig. 4C) with a mean amplitude of -46.3 ± 0.6 pA (n = 2,177). The addition of 1 µM strychnine to the extracellular saline did not inhibit the sIPSCs but increased their frequency to 4.7 Hz (Fig. 4, A and B). The corresponding amplitude histogram (Fig. 4C) was also skewed toward lower values with a mean amplitude of -33.9 ± 0.3 pA (n = 3,406) that was significantly different from the mean amplitude in the absence of strychnine as confirmed by the cumulative amplitude fraction plot in Fig. 4D. The application of 1 µM strychnine in combination with 10 µM bicuculline resulted in a nearly complete inhibition of the sIPSC activity in this amacrine cell (Fig. 4, A and B). Thereafter, bicuculline was applied alone and no recovery of the sIPSCs was observed, indicating that these sIPSCs were mediated by GABAARs. The analysis of the decay kinetics using monoexponential fits (see METHODS section) revealed two similar tau  values in the absence and presence of strychnine (control: tau  = 14.2 ms; in strychnine: 16.3 ms; P > 0.05; Fig. 4, E and F), suggesting that they were mediated by the same type of receptors, namely GABAARs. It is also possible that strychnine blocked a subclass of GlyRs with kinetics similar to the GABAARs expressed in this cell. However, sIPSCs observed in the presence of strychnine were mediated by GABAARs. The increase in sIPSCs frequency observed in the presence of strychnine was most likely due to disinhibition of some GABAergic neurons that receive inhibitory inputs from glycinergic amacrine cells. The decrease in amplitude (Fig. 4A), which was observed in a few amacrine cells, may be the result of a rundown of the sIPSCs during the experiment or caused by a desensitization of GABAARs due to accumulated GABA in the synaptic cleft.



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Fig. 4. Characteristics of sIPSCs mediated by GABAARs in amacrine cells. A: sIPSCs were continuously recorded in the whole cell configuration in the absence and presence of strychnine (Stry; 1 µM) and bicuculline (Bic; 10 µM) as indicated. Plot of sIPSC peak amplitudes against the recording time. In the presence of strychnine, sIPSC frequency increased, whereas in the presence of bicuculline, sIPSCs were blocked, indicating that they were mediated by GABAARs. B: display of current traces obtained in the absence and presence of strychnine and bicuculline. C: amplitude histogram of sIPSCs in the absence (left) and in the presence of strychnine (right). D: cumulative fraction plot of sIPSC amplitudes corresponding to C. The decrease of the amplitudes in the presence of strychnine was rather due to a general rundown in the beginning of the experiment than an effect of strychnine. E: averaged and normalized traces of sIPSCs in the absence (control) and presence of strychnine showing similar decay kinetics. F: cumulative fraction plot of the decay time constants corresponding to D. The bicuculline sensitivity and the similar decay kinetics of sIPSCs in the absence and in the presence of strychnine indicates that they were mediated by the same receptor type, namely by GABAARs.

sIPSCs mediated by GABAARs, because they were blocked by bicuculline in the presence of strychnine, were isolated in seven amacrine cells. The mean peak amplitude of these GABAergic sIPSCs was -41.5 ± 8.6 pA, and their mean rise time (T10/90; see METHODS) was 1.4 ± 0.2 ms. In addition, in another seven amacrine cells, sIPSCs that were blocked by bicuculline in the absence of strychnine showed a mean peak amplitude of -39.8 ± 7.7 pA and a mean rise time of 1.3 ± 0.2 ms. The mean peak amplitudes and mean rise times of these sIPSCs were not significantly different from those recorded in the presence of strychnine.

To characterize the kinetics of these sIPSCs, the current decays were fitted with one or two exponentials. sIPSCs could be subdivided into two groups: one group that comprised sIPSCs best fitted with a monoexponential decay function (monoexponential group) and another group, comprising sIPSCs best fitted biexponentially (biexponential group) characterized by a fast (tau 1) and a slow (tau 2) decay time constant. Strychnine-resistant, bicuculline-sensitive sIPSCs best fitted monoexponentially were characterized by a tau  of 18.3 ± 3.3 ms and those best fitted biexponentially by a tau 1 of 6.7 ± 1.0 ms and tau 2 of 62.7 ± 12.1 ms. Bicuculline-sensitive sIPSCs recorded in the absence of strychnine best fitted monoexponentially were characterized by a tau  of 17.7 ± 3.2 ms and those best fitted biexponentially by a tau 1 of 6.9 ± 0.9 ms and tau 2 of 58.1 ± 9.7 ms. Like the mean peak amplitudes and the rise times (see preceding text), all corresponding time constants were not significantly different from each other indicating that the bicuculline-sensitive sIPSCs recorded in the presence, and those recorded in the absence of strychnine were not different from each other. Therefore these samples were pooled as GABAergic sIPSCs.

To allow a comparison of the monoexponential and the biexponential groups, the amplitude-weighted time constants (tau w; see METHODS) were calculated. In 4 of 14 amacrine cells, the monoexponential group of sIPSCs was not different from the biexponential group (mean tau w = 21.3 ± 4.7 ms; pooled data from the monoexponential and biexponential group). However, in 10 of 14 amacrine cells, the tau w values of the monoexponential sIPSC group (tau w = 19.4 ± 2.3) were significantly different from those of the biexponential group (tau w = 35.0 ± 3.6 ms). The following findings are shown for one representative amacrine cell in Fig. 5. In this amacrine cell, the monoexponential group was characterized by a unimodal narrow ranged tau w distribution (Fig. 5, A and C), whereas the tau w values of the biexponential sIPSC group showed a polymodal distribution (Fig. 5, B and C). In addition, we found that the tau 1 values and the tau 2 values of the biexponential group were bimodally distributed. The distribution histograms were fit with two Gaussians showing peaks at 2.8 and 5.7 ms for tau 1 (Fig. 5D) and at 20.2 and 79.1 ms for tau 2 (Fig. 5E), respectively. With respect to the Gaussian fits shown in Fig. 5D, the tau 1 values were subdivided further into the tau 1 values <4.5 ms (Fig. 5D, ) and the tau 1 values >= 4.5 ms (Fig. 5D, ). When the tau 2 values were plotted against the tau 1 values (Fig. 5F), we found that the tau 1 values <4.5 ms were paired with fast tau 2 values (see Fig. 5F, open circle ) and the tau 1 values >= 4.5 were paired with slow tau 2 values (Fig. 5F, +), suggesting that the biexponential group is composed of fast and slowly decaying subgroups.



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Fig. 5. GABAergic sIPSCs of an amacrine cell with different decay kinetics. All data (A-F) are from the same amacrine cell. A: unimodal frequency distribution of tau w values of GABAergic sIPSCs best fit monoexponentially. B: the frequency distribution of tau w values of GABAergic sIPSCs best fit biexponentially shows more than 1 peak. C: cumulative fraction plot of the tau w values displayed in A and B confirming their different distributions. The tau 1 values (D) and the tau 2 values (E) of GABAergic sIPSCs of the biexponential group (see text) are bimodally distributed. The lines in D and E indicate the fitting of the data with 2 Gaussians (peak values of tau 1 at 2.8 and 5.7 ms, and of tau 2 at 20.2 and 79.1 ms, respectively). F: according to D, the tau 1 values were subdivided into tau 1 < 4.5 ms () and tau 1 >=  4.5 ms (). The tau 2 values were plotted against the tau 1 values. The tau 1 values <4.5 ms were paired with fast tau 2 values (open circle ) and the tau 1 values >= 4.5 were paired with slow tau 2 values (+), indicating that the biexponential group is composed of fast and slowly decaying subgroups. These findings suggest that an amacrine cell could express at least 2 functionally different subtypes of GABAARs.

To investigate whether GABAAR-mediated sIPSCs with different decay kinetics were due to dendritic filtering, the correlations of the peak amplitudes and the decay time constants with the rise times (T10/90; Fig. 6) were analyzed. However, neither the peak amplitudes nor the tau w values were correlated with their corresponding T10/90 values (Fig. 6, A and B). In addition, no correlation of the tau 1 values with the corresponding T10/90 values was found, indicating that there was also no significant influence of dendritic filtering on either the fast decaying sIPSCs (Fig. 6C, ) nor the slowly decaying sIPSCs (Fig. 6C, open circle ). In summary, the lack of correlation between peak current amplitudes and decay times with rise times in any amacrine cell investigated suggested that dendritic filtering was not the cause of the occurrence of distinct groups of GABAARs with different decay kinetics.



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Fig. 6. Correlation between the mean peak amplitudes and the decay kinetics of GABAergic sIPSCs with the corresponding rise times (T10/90). A: plot of the T10/90 values of sIPSC mediated by GABAARs of an amacrine cell against the corresponding tau w values (top) and mean peak amplitudes (bottom). Lines represent fits of the data by linear regression (top: r = 0.093; P = 0.347; n = 105; bottom: r = -0.010; P = 0.918; n = 109). Same evaluation from another amacrine cell (top: r = -0.025, P = 0.730, n = 195; bottom: r = 0.014, P = 0.822, n = 249). The amacrine cells in A and B are representative, indicating that there was no correlation of the T10/90 values with the tau w values and the mean peak amplitudes. C: to test for a correlation of the decay kinetics of the fast decaying (; tau 1 < 4.5 ms; cf. Fig. 5D and text) and the slowly decaying biexponential sIPSCs (open circle ; tau 1 >=  4.5 ms; cf. Fig. 5D and text), the sIPSCs of the cell displayed in C and in Fig. 5, C and D, were subdivided as described in the text. No correlation was found between tau w values and T10/90 values (for tau 1 < 4.5 ms: r = -0.194, P = 0.341; n = 26, · · · ; for tau 1 >=  4.5 ms: r = 0.018, P = 0.922; n = 31, - - -; lines represent fits by linear regression), indicating that the different decay kinetics of GABAergic sIPSCs was not due to dendritic filtering.

Glycinergic IPSCs

In one amacrine cell, two pharmacologically distinct types of sIPSCs were observed simultaneously (Fig. 7A). In the absence of antagonists, one type of sIPSC was characterized by fast decay kinetics (Fig. 7B, a) and another type by a slow current decay (Fig. 7B, b). In the presence of 10 µM bicuculline, the fast decaying sIPSCs disappeared completely while the amplitude and the frequency of the slow decaying sIPSCs increased significantly (Fig. 7, A and B). When bicuculline was applied in combination with 1 µM strychnine, the sIPSCs were completely blocked. These results indicate that the fast decaying, bicuculline-sensitive sIPSCs were mediated by GABAARs while the slowly decaying, strychnine-sensitive sIPSCs were mediated by GlyRs. The comparison of averaged and normalized sIPSCs indicated that the sIPSCs mediated by GABAARs (Fig. 7C, a) were characterized by a faster rise time and a faster decay kinetics than those mediated by GlyRs (Fig. 7C, b). The corresponding cumulative fraction plots of the T10/90 and tau  values are shown in Fig. 7D. Since no correlation between decay times and T10/90 values of both GABAergic and glycinergic sIPSCs was found in this amacrine cell (Fig. 7E), it is concluded that the differences in kinetics of GABAAR- and GlyR-mediated sIPSCs are not due to an impairment of the recordings by dendritic filtering. Interestingly, the application of bicuculline induced an increase in frequency and amplitude of the sIPSCs. When the sIPSCs in the presence of bicuculline were compared with those in its absence, a significant difference in mean amplitude and frequency was found that was likely mediated by disinhibition (Fig. 7A).



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Fig. 7. GABAAR- and GlyR-mediated sIPSCs in an amacrine cell. A: sIPSCs were continuously recorded in the whole cell configuration in the absence and presence of bicuculline (Bic; 10 µM) and strychnine (Stry; 1 µM) as indicated. Plot of sIPSC peak amplitudes against the recording time. In the presence of bicuculline the frequency and amplitude of a subpopulation of sIPSCs increased. In the presence of bicuculline and strychnine the remaining sIPSCs were blocked. B: in the absence of antagonists (control) fast decaying (a) and slowly decaying (b) sIPSCs were present. Fast decaying sIPSCs were blocked in the presence of bicuculline, indicating that they were mediated by GABAARs. In the presence of bicuculline and strychnine also, the slowly decaying sIPSCs were blocked, indicating that they were mediated by GlyRs. C, top and middle: superimposed GABAergic (a) and glycinergic (b) sIPSCs. Bottom: averaged and normalized traces of GABAergic (a) and glycinergic (b) sIPSCs indicate that GABAARs in this amacrine cell activate and decay significantly faster than GlyRs. D: cumulative fraction plots of T10/90 values (top) and tau w values (bottom) of GABAergic (a) and glycinergic (b) sIPSCs. E: plot of the tau w values of GABAergic () and glycinergic () sIPSCs against the corresponding T10/90 values. · · · : regression line of the data from GABAergic sIPSCs: r = -0.112; P = 0.366; n = 67; - - -: regression line of the data from glycinergic sIPSCs: r = -0.059; P = 0.647; n = 63. The lack of correlation indicates that the different decay kinetics of GABAergic and glycinergic sIPSCs were not due to dendritic filtering.

GlyR-mediated sIPSCs could be pharmacologically isolated in four amacrine cells (Fig. 8, A and B). In the absence of any antagonist (control), sIPSCs were observed at a frequency of 0.59 Hz in this cell. Their peak amplitude distribution was skewed to lower values characterized by a mean amplitude of -10.0 ± 0.3 pA (n = 140; Fig. 8C). The application of 10 µM bicuculline induced an increase of the frequency to 0.88 Hz and a significant increase of the mean amplitude (mean: -11.2 ± 0.2 pA; n = 415; P < 0.001; Fig. 8, C and D). When 10 µM bicuculline was applied in combination with 0.5 µM strychnine, sIPSC frequency was 0.59 Hz, while the mean amplitude remained significantly increased at -11.2 ± 0.3 pA (n = 281; P < 0.001; Fig. 8, A-D) compared with the sIPSCs of the control. However, 1 µM strychnine produced a complete inhibition of sIPSCs (Fig. 8A), indicating that these sIPSCs were mediated by GlyRs. The analysis of the current decay kinetics revealed similar tau w values (Fig. 8E) in control (tau w = 35.8 ms), in 10 µM bicuculline (tau w = 40.5 ms), and in 10 µM bicuculline plus 0.5 µM strychnine (tau w = 40.6 ms) that were not significantly different from each other as indicated by the corresponding cumulative fraction plots (Fig. 8F). In summary, glycinergic IPSCs were found in only 4 of 34 amacrine cells. They were characterized by a mean peak amplitude of -28.0 ± 8.5 pA that was not significantly different from that of GABAARs-mediated IPSCs. However, the mean rise times of the sIPSCs mediated by GlyRs (mean T10/90: 2.9 ± 0.6 ms; n = 4) were significantly slower than those mediated by GABAARs (mean T10/90: 1.2 ± 0.03 ms; n = 13; see preceding text). Cumulative fraction plots of the pooled T10/90 values confirmed this significant difference in the activation kinetics (GlyRs: mean T10/90 = 2.4 ± 0.08 ms; n = 336 from 4 amacrine cells; GABAARs: mean T10/90 = 1.2 ± 0.03 ms; n = 1933 from 13 cells; P < 0.001; Fig. 9A).



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Fig. 8. Characteristics of sIPSCs mediated by GlyRs in amacrine cells. A: sIPSCs were continuously recorded in the whole cell configuration in the absence and presence of bicuculline (Bic; 10 µM) and strychnine (Stry; 0.5 µM and 1 µM) as indicated. Plot of sIPSC peak amplitudes against the recording time. In the presence of bicuculline, sIPSC frequency and peak amplitude increased. In the presence of bicuculline and 0.5 µM strychnine, sIPSCs were only weakly inhibited, while in the presence of bicuculline and 1 µM strychnine, sIPSCs were completely blocked, indicating that they were mediated by GlyRs. B: display of current trace obtained in the absence and presence of bicuculline and strychnine. C: amplitude histogram of sIPSCs in the absence (con) and in the presence of bicuculline (Bic) and 0.5 µM strychnine plus bicuculline (0.5 µM Stry + Bic). D: the cumulative fraction plot corresponding to C indicates the increase of the peak amplitudes in the presence of bicuculline. E: averaged and normalized traces showing the similar decay kinetics of sIPSCs recorded in the absence and presence of bicuculline and strychnine as indicated. F: cumulative fraction plots of the decay time constants corresponding to E. The bend of the gray curve, which corresponds to the data obtained in the presence of bicuculline plus 0.5 µM strychnine, indicates that a part of the glycinergic sIPSCs are blocked. D-F: a, control; b, bicuculline; c, 0.5 µM strychnine plus bicuculline.



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Fig. 9. sIPSCs mediated by GABAARs and GlyRs are functionally different in amacrine cells. A: cumulative fraction plot of T10/90 values pooled from all cells indicate the significant difference in the activation kinetics between GABAARs and GlyRs expressed in amacrine cells. GlyRs (thin line; mean T10/90 = 2.4 + 0.08 ms; n = 336 from 4 amacrine cells) activated significantly slower than GABAARs (thick line; mean T10/90 = 1.2 ± 0.03 ms; n = 1933 from 13 cells; P < 0.001). B: cumulative fraction plot of tau w values pooled from all cells indicate the significant difference in the decay kinetics between monoexponentially decaying GABAARs (thick line; mean tau w = 20.3 ± 0.50; n = 456 from 13 cells), biexponentially decaying GABAARs (dashed line; mean tau w = 30.7 ± 0.95; n = 1,064 from 13 cells) and GlyRs (thin line; mean tau w = 25.3 ± 1.94; n = 183 from 4 cells).

The tau w values from all cells with sIPSCs mediated by GABAARs and those mediated by GlyRs were also analyzed by comparing the corresponding cumulative fraction plots. The mean tau w values of the glycinergic sIPSCs (24.3 ± 6.7 ms; n = 4) were not significantly different from the GABAergic sIPSCs (28.2 ± 3.0 ms; n = 13). However, the tau w values of GABAergic sIPSCs of the monoexponential group (tau w = 20.3 ± 0.5 ms; n = 456; pooled data from 13 cells) and the biexponential group (30.7 ± 0.95 ms; n = 1064; pooled data from 13 cells) were significantly different from each other and from the tau w values of the glycinergic sIPSCs (tau w = 25.3 ± 1.94 ms; n = 183; pooled data from 4 cells; Fig. 9B), indicating that these groups were functionally different with respect to their decay kinetics.

PSCs remaining in the presence of bicuculline and strychnine

In some amacrine cells, sPSCs that persisted in the presence of 1 µM strychnine and 10 µM bicuculline could be observed. These PSCs were always characterized by significantly smaller peak amplitudes (less than -20 pA) compared with the peak amplitudes recorded in the absence of strychnine and bicuculline (control). The analysis of their kinetics never showed any significant difference to the kinetics of the control sIPSCs of the same cell. Therefore these sPSCs might have persisted because of an incomplete exchange of bicuculline and strychnine. However, because these sPSCs were not studied in detail, it cannot be excluded that they were mediated by receptors other than GABAARs or GlyRs.


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To characterize the functional contribution of the different synaptic inputs to amacrine cells, their postsynaptic activity was recorded in the absence and presence of specific receptor antagonists. Rapidly decaying sPSCs that could be blocked with kynurenic acid were mediated by ionotropic glutamate receptors. sPSCs with significantly higher peak amplitudes and slow decay kinetics were identified as sIPSCs. Based on their pharmacological and kinetic properties, sIPSCs could also be further subdivided. While sIPSCs sensitive to strychnine were recognized as glycinergic, those inhibited by bicuculline were mediated by GABAARs. A few sPSCs that remained in the presence of strychnine and bicuculline were reduced in amplitude and showed similar kinetics to control sIPSCs. Since higher concentration of bicuculline and strychnine have not been used, because they were reported to affect both receptors (Cohen et al. 1989; Protti et al. 1997), these sPSCs could have been mediated by receptors other than GABAARs or GlyRs. A contribution of sIPSCs mediated by GABAC receptors is unlikely because the GABAC receptor-specific rho  subunits are not expressed by amacrine cells (Bormann and Feigenspan 1995; Enz et al. 1996).

sEPSCs in amacrine cells

In amacrine cells, sEPSCs and sIPSCs were characterized by significantly different time constants (cf. Fig. 2D) and could thus be separated for analysis easily. The sEPSCs were characterized by their small amplitudes and rapid decay kinetics (tau  = 1.35 ms). The sEPSC amplitudes showed a narrow unimodal distribution, suggesting that only one or a few vesicles were spontaneously released at excitatory synapses. Indeed we never found more than one peak or a significantly skewed amplitude distribution of sEPSCs in amacrine cells. Since only PSCs were analyzed that did not show any contamination of subsequent events, it is assumed that a single PSC resulted from the activation of a single synapse. Thus a variation in the number of vesicles should produce a different number of peaks in the amplitude distribution of the EPSCs. However, the lack of multiple peaks in the amplitude distributions of sEPSCs in amacrine cells suggests that the number of vesicles released was relatively constant and/or that the transmitter concentration released was sufficient to saturate the excitatory postsynaptic receptors.

At present, ionotropic glutamate receptors are subdivided into three subclasses: the N-methyl-D-aspartate receptors (NMDARs), the alpha -amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs), and the kainate receptors. Amacrine cells express many subunits that are specific for these different subclasses. The subunits GluR6, GluR7, KA2, and delta 1/2, which are specific for kainate receptors, have been found in amacrine cells of the adult rat retina. In a subset of adult rat amacrine cells, the NR1, NR2A, and NR2B subunits of NMDARs were found (Fletcher et al. 2000), and in many amacrine cells, including AII amacrine cells, the AMPAR-specific subunits GluR2 and GluR4 were present (Qin and Pourcho 1999). The decay kinetics of sEPSCs in amacrine cells were similar to that of EPSCs mediated by AMPARs in rat retinal ganglion cells (Protti et al. 1997; Tian et al. 1998) and in rat hippocampal and cortical pyramidal cells (Jonas et al. 1993; Stern et al. 1992). Because the decay kinetics of the sEPSCs we recorded in amacrine cells were also similar to that of recombinant AMPARs (Jonas and Sakmann 1992; Mosbacher et al. 1994) but significantly faster than that of NMDARs (Barnes-Davies and Forsythe 1995; Taschenberger et al. 1995), we conclude that the sEPSCs we observed in amacrine cells were probably mediated by AMPARs. However, NMDA-activated currents have been previously observed in AII amacrine cells (Hartveit and Veruki 1997). Therefore it is likely that amacrine cells express NMDA receptor-mediated EPSCs that were inhibited at the Vh of -60 mV in the Mg2+-containing saline used in this study.

sIPSCs

Since kynurenic acid, a broad spectrum antagonist of ionotropic glutamate receptors, did not affect PSCs with a tau w > 4 ms, these PSCs were not mediated by ionotropic glutamate receptors. All sIPSCs recorded at different holding potentials were characterized by a reversal potential that depended on the ECl-. In addition, the majority of sIPSCs were blocked either by bicuculline or strychnine. Therefore we conclude that PSCs with a tau w > 4 ms in the amacrine cells of this study were mainly sIPSCs mediated by GABAARs or GlyRs. In contrast to the sEPSCs, the amplitude distributions of sIPSCs showed several peaks and were strongly skewed to lower values (cf. Figs. 2B, 4C, and 8C), indicating a strong variation in the number of vesicles released. The question arises whether the amount of transmitter activating an IPSC is sufficient to saturate the postsynaptic receptors. Our results suggest that this is not the case in all amacrine cells. Inhibitory inputs onto amacrine cells originate from other glycinergic or GABAergic amacrine cells. In five amacrine cells, the application of strychnine or bicuculline induced a significant increase in frequency and amplitude of sIPSCs (e.g., Figs. 7A and 8A). This was likely due to a disinhibition of the presynaptic cell. Indeed, multiple synapses between amacrine cells have been found in the inner plexiform layer (Chun and Wässle 1989; Koontz and Hendrickson 1990). Since a single sIPSC results from the spontaneous activation of a single synapse, the increase of the mean sIPSC amplitudes evoked by disinhibition is likely due to an increase in the number of activated receptors caused by an increase in the transmitter concentration. On-bipolar cells depolarize in response to light stimuli. Since they are nonspiking interneurons, they produce a tonic and graded glutamate release onto amacrine cells that is proportional to the light intensity (Matsui et al. 1998). A nonsaturating vesicle content combined with a high number of postsynaptic receptors would broaden the operating range of the synapses. This would enable amacrine cells to respond to tiny differences of light signals providing a basis for the fine tuning of inhibition mediated by these cells.

GABAergic IPSCs

In situ hybridization and immunohistochemical experiments showed a strong heterogeneity in the expression of GABAAR subunits in amacrine cells (Greferath et al. 1995). Since the functional properties of ligand gated channels vary with their subunit combinations, it was not surprising that amacrine cells expressed GABAergic sIPSCs with strongly varying properties. For example, the T10/90 values ranged from a few hundred microseconds to a few milliseconds with a mean value of 1.2 ms. This is significantly faster than that of 2.63 ms found in cultured chick amacrine cells (Gleason et al. 1993). Rapid GABA application experiments with recombinant receptors composed of alpha 1beta 3gamma 2 subunits revealed T10/90 values of 0.4-1.27 ms (Zhu et al. 1998; calculated from T20/80 = 0.25-0.8 ms; see METHODS). For receptors composed of alpha 1beta 1gamma 2, the mean T10/90 value was 2.1 ms and for alpha 2beta 1gamma 2 receptors 1.0 ms (Lavoie et al. 1997), indicating that different subunit assemblies result in distinct activation kinetics. Thus the wide range of the T10/90 distribution of GABAergic sIPSCs in amacrine cells may reflect the contribution of more than one GABAAR subtype. The decay kinetics of recombinant GABAARs was also found to be dependent on the subunit composition. The decay kinetics of GABAergic sIPSCs of amacrine cells from this study showed a strong variability. In most amacrine cells, sIPSCs could be subdivided into a group best fit monoexponentially and another best fit biexponentially (Fig. 5C). The tau w values of these groups were significantly different, suggesting the occurrence of different GABAAR subtypes in amacrine cells. In addition, the bimodal distributions of the tau 1 and the tau 2 values within the biexponential group suggest that this last group comprises two subgroups, a fast and a slowly decaying group (Fig. 5, D and E). If these distributions would be at random, then the tau 1 values should be randomly paired with tau 2 values. However, fast tau 1 values were highly correlated with fast tau 2 values and slow tau 1 values with slow tau 2 values, indicating that there are two populations of GABAergic sIPSCs with distinct decay kinetics. Interestingly, the tau w value of the monoexponential group is similar to the tau w value of the fast biexponential group (Fig. 5, A and B), suggesting that these subpopulations might be similar. Thus at least two types of differentially decaying GABAergic sIPSCs are present in amacrine cells, suggesting the expression of more than one GABAAR subtype within the same cell. The time constants we found in amacrine cells did not exactly match with those obtained from recombinant receptors. Thus the question of which subunits contribute to GABAARs in amacrine cells still remains unsolved. Immunocytochemical studies have shown that amacrine cells express different GABAARs subtypes. While cholinergic amacrine cells likely express GABAARs assembled from alpha 2, beta 1, beta 2/3, delta , and possibly gamma 2 subunits, dopaminergic amacrine cells were found to express alpha 1, alpha 2, alpha 3, and gamma 2 subunits (Greferath et al. 1995; Gustincich et al. 1999). GABAARs composed of alpha 1, beta 2/3, and gamma 2 are expressed in glycinergic amacrine cells (Greferath et al. 1995). The tau w values of the monoexponential group of GABAARs in amacrine cells of this study was 20.3 ms, which is close to the tau w values of 19.3 ms reported for spontaneous quantal events mediated by GABAARs of cultured chick amacrine cells (Gleason et al. 1993), of native GABAARs expressed in juvenile ICC neurons (tau w = 22.4 ms) (Backus et al. 2000), and of recombinant alpha 1beta 1gamma 2 receptors (tau w = 20.5 ms) (calculated from Lavoie et al. 1997), but faster than that of alpha 2beta 1gamma 2 receptors (tau w = 198.7 ms) (calculated from Lavoie et al. 1997), suggesting the contribution of the alpha 1 subunit in a subpopulation of GABAARs most likely expressed in glycinergic amacrine cells.

The activation and decay kinetics of sIPSCs are not exclusively caused by the ligand on- and off-binding rates, which are intrinsic properties of specific receptor subtypes, but also by the transmitter exchange rate at the synapse. Since the GABA exchange rate is dependent on the morphological particularities of a release site, i.e., the width of the synaptic cleft, the GABA reuptake rate, the degree of clustering of postsynaptic receptors and the contribution of glial cells, it cannot be excluded that the different kinetics were caused by one of these factors. Given the fact that we did not find a correlation between amplitudes and decay or rise times, the different kinetics reflect the intrinsic properties of distinct GABAAR subtypes rather than dendritic filtering.

Glycinergic sIPSCs

In some cells, we recorded sIPSCs that were sensitive to strychnine but persisted in the presence of bicuculline, indicating that they were mediated by GlyRs. Whether amacrine cells also express different subtypes of GlyRs remains unclear. In situ hybridization experiments showed strong signals for the alpha 2 subunits, whereas only low amounts of mRNA coding for the alpha 1 subunit was found in the amacrine cell layer. Transcripts of beta  subunits were found in the INL but their distribution could not yet be resolved on the cellular level (Greferath et al. 1994a). These findings may suggest the expression of different GlyRs subtypes in amacrine cells. Although glycinergic sIPSCs showed some heterogeneity with respect to their kinetics, the low number of amacrine cells that showed glycinergic sIPSCs did not allow a more complete analysis. This was surprising because when glycine was applied by bath application, all amacrine cells tested responded with an inward current (data not shown) as previously reported by others (Boos et al. 1993; Menger and Wässle 2000). Horizontal, bipolar, and ganglion cells do not release glycine onto amacrine cells, suggesting that the most likely presynaptic cells are glycinergic amacrine cells. Most glycinergic amacrine cells are nonspiking neurons because the sIPSCs recorded in ganglion cells, which were mediated by glycinergic amacrine cells, were not tetrodotoxin sensitive (Protti et al. 1997). Thus the spontaneous release of glycine, which depends on the membrane potential and membrane conductance, might have been low under our experimental light-adapted conditions. In addition, glycinergic amacrine cells have smaller dendritic fields than GABAergic amacrine cells (Pourcho and Goebel 1985; Vaney 1990; Wässle and Boycott 1991), suggesting that they provide a lower coverage than GABAergic amacrine cells and that there is a lower number of glycinergic synapses. Indeed, immunocytochemical labeling with antibodies specific for GABAAR and GlyR subunits have shown that there are four times more GABAergic than glycinergic synapses in the plexiform layers of the mammalian retina (Fischer et al. 2000).

Are GABAergic and glycinergic IPSCs functionally different?

The mean amplitudes of GlyRs were not significantly different from those of GABAergic sIPSCs, but their activation and decay kinetics were different from those of the presumed GABAARs subtypes (cf. Fig. 9). In one amacrine cell, the glycinergic sIPSCs were significantly slower decaying than the GABAergic sIPSCs (Fig. 7), suggesting that at least a subpopulation of GABAARs is functionally different from GlyRs. In addition, the mean T10/90 values of glycinergic sIPSCs were significantly slower than that of GABAergic sIPSCs. These differences may be due to different association rate constants for GABAARs and GlyRs. However, glycinergic synapses may also differ with respect to several other properties, such as slower transmitter exchange rate, which could be due to different morphological properties, different glial ensheathment, or different transmitter reuptake rate. Finally, glycinergic synapses might be characterized by a different form of receptor clustering resulting in a more desynchronized activation of the GlyRs, thus leading to a slower rise time. Differences in rise times have also been observed in recombinant GABAARs subtypes of different subunit composition (Lavoie et al. 1997) and between GABAARs and GlyRs in nucleated patches obtained from neurons of the inferior colliculus (Backus et al. 2000). Although we have found clear functional differences between GABAARs and GlyRs in amacrine cells, at present, the physiological significance of these differences remains unknown.


    ACKNOWLEDGMENTS

We thank Drs. D. A. Protti and B. O'Brien for critically reading the manuscript. We are grateful to F. Boij for excellent technical assistance.

This study was supported by grants to K. H. Backus [Deutsche Forschungsgemeinschaft (DFG)-Schwerpunkt 1026; BA 1311/6-2] and H. Wässle (DFG-Sonderforschungsbereich 269/B4).


    FOOTNOTES

Present address and address for reprint requests: K. H. Backus, Institute of Physiology II, Cellular Neurophysiology, University of Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany (E-mail: backus{at}em.uni-frankfurt.de).

Received 18 December 2000; accepted in final form 13 June 2001.


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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society