Koizumi, Mol Vis 2004; 10:328-340.

Molecular Vision 2004; 10:328-340 <http://www.molvis.org/molvis/v10/a42/>
Received 2 March 2004 | Accepted 13 May 2004 | Published 13 May 2004 Download
Reprint

Inward rectifying currents stabilize the membrane potential in dendrites of mouse amacrine cells: Patch-clamp recordings and single-cell RT-PCR

Amane Koizumi, Tatjana C. Jakobs, Richard H. Masland

Howard Hughes Medical Institute, Massachusetts General Hospital, Wellman 429, Boston, MA

Correspondence to: Amane Koizumi, Howard Hughes Medical Institute, Massachusetts General Hospital, Wellman 429, Boston, MA, 02114; Phone: (617) 726-3888; FAX: (617) 726-5336; email: akoizumi-ns@umin.ac.jp

Abstract

Purpose: To explore the possible existence of inward rectifying currents in the distal dendrites of amacrine cells.

Methods: Patch-clamp recordings were made from amacrine cells in a new horizontal slice preparation of mouse retina. Single-cell RT-PCR studies were performed after the patch-clamp recordings.

Results: In contrast to results from vertical slices or dissociated cells, all amacrine cells tested demonstrated inward rectifying currents, I_IR. Within the limits of our sample, this current did not depend on the morphological and physiological type of the amacrine cell. Amacrine cells from which the dendrites had been removed did not possess detectable amounts of I_IR. Pharmacological experiments with ZD7288 (100 μM) and single-cell RT-PCR from recorded cells revealed that I_IR includes an h-current (I_(H)) carried by hyperpolarization-activated cyclic nucleotide gated channels (HCN), HCN1 and/or HCN2 subtypes. In the presence of extracellular Cs⁺ (5 mM), which greatly suppressed I_IR, the resting membrane conductance was reduced. I_IR suppressed the generation of oscillatory potentials. Intracellular cAMP (8-cpt-cAMP, 1 mM) activated I_IR.

Conclusions: I_IR appears to occur within dendrites of many amacrine cells, where it tends to stabilize the resting membrane potential. HCN1 and/or HCN2 channels contribute to I_IR in amacrine cells. Dendritic I_IR would be expected to contribute to functional independence of the distal dendrites of amacrine cells that express it.

Introduction

Many previous studies of the electrophysiology of amacrine cells have used vertical slices or dissociated cells. These include starburst amacrine cells [1-3], dopaminergic amacrine cells [4], AII amacrine cells [5,6], and GABAergic amacrine cells [7,8]. However, the dendrites of amacrine cells are damaged in the process of vertical slice preparation or dissociation, a concern because dendrites of amacrine cells are reported to have many kinds of active conductances [8,9]. The input resistances of amacrine cells, which determine the passive spread of voltage changes into the dendrites [8], are often reported to be high (over 2 GΩ in dissociated dopaminergic amacrine cells [4]). If there is a large conductance at dendrites, however, the spread of voltage changes along dendrites would be restricted [8]. In support of this possiblity, Euler et al. [10] suggested, from observations using imaging of Ca²⁺ movements in the dendrites, that dendrites of starburst amacrine cells might be electrically isolated from each other. This could be due to specialized conductances localized in the dendrites. One candidate for the dendritic conductance is inward-rectifying current (I_IR). In the retina, I_IR is classically observed in photoreceptors [11], horizontal cells [12], and bipolar cells [13]. I_IR was thought to contribute to the amplification of light responses [11] in these non-spiking neurons. Previous reports on the electrophysiology of amacrine cells found only a small I_IR [4,14]. We wondered if this was, at least in part, because dendrites of amacrine cells are removed or damaged in these preparations. I_IR consists of multiple types of currents that show inward rectification, such as inward-rectifying K⁺ current and H currents (I_(H)). The inward rectifying K⁺ current is activated at more negative potentials than the K⁺ equilibrium potential and is selectively carried by K⁺ [15]. In contrast, I_(H) also shows inward rectification but is carried by K⁺ and also other cations. Recently, from physiological and molecular biological studies, I_(H) has been identified in photoreceptors and bipolar cells [16-18]. Muller et al. [17] suggested the existence of hyperpolarization-activated cyclic nucleotide gated channels (HCN channels) in amacrine cells of rat retina by immunohistochemical studies. In hippocampal and neocortical pyramidal neurons, dendritic I_(H) controls spontaneous spiking activity and modifies synaptic integration. A family of four mammalian genes, known as HCN1, 2, 3, and 4, contribute to I_(H) [19]. HCN channels are expressed at especially high levels in distal dendrites [20,21], where I_(H) shortens the decay time course of excitatory post-synaptic potentials (EPSP) and affects synaptic integration [22-25]. In those studies, the dendrites were an important source of I_(H). An aim of the present study was to explore the electrophysiology of amacrine cells when their dendrites are well preserved, concentrating on I_IR, and more specifically the possible existence of I_(H) in the dendrites of amacrine cells. We invented a horizontal slice preparation of the mouse retina and made whole-cell patch-clamp recordings from individual amacrine cells. We found that all recorded amacrine cells had I_IR, which increased the resting membrane conductance and stabilized the resting membrane potential. I_(H) partially contributes to I_IR, because ZD7288, a specific blocker for I_(H), suppressed I_IR. The types of HCN channels were evaluated with single-cell RT-PCR and were correlated with the observed recordings. Because of recent evidence of electrically isolated dendrites in some amacrine cells [10], the possible contribution of I_IR was studied by computer simulation.

Methods

Horizontal slice preparation of the mouse retina

All experiments were performed on horizontal slices of the mouse retina. Mice (C57BL/6) were deeply anesthetized and sacrificed before their eyes were removed, in accordance with National Institutes of Health guidelines for animal use. The retina was peeled away from the pigment epithelium and placed vitreous side up on a piece of filter paper (cellulose nitrate, 0.2 μm pore size, 13 mm diameter; Advantec Toyo, Japan). The retina was firmly attached to the filter paper and flattened by applying suction. The retina on filter paper was placed on an agar block (0.03 mg/ml; Sigma, St. Louis, MO) and covered with low-temperature-melting agarose (Agarose type VII-A; melting points 33 °C, 0.025 mg/ml; Sigma). After the agarose was completely solidified, the retina with the agarose was sliced horizontally by a vibratome (VT1000S; Leica, Germany) at the level of the inner nuclear layer (Figure 1A). With this method, sections may be cut at the level of different layers of the retina (Figure 1B-E). In slices sectioned at the outer nuclear layer (Figure 1B), the somas of photoreceptors were visible in a regular mosaic with consistent size. At the ganglion cell layer, we observed relatively large somas and large blood vessels (Figure 1D). For the work described here, a slice contained the inner part of the retina, from the nerve fiber layer to the inner third of the inner nuclear layer. At the inner nuclear layer, somas with a variety of sizes were located seemingly randomly (Figure 1C). These can include both bipolar cells and amacrine cells, but amacrine cells could be distinguished by both their relatively larger somas and their location at the border between the inner plexiform layer and inner nuclear layer. After patch-clamp recordings, Lucifer yellow was injected into the cell to identify the dendritic morphology of recorded amacrine cells.

Patch-clamp recordings

We performed whole-cell patch-clamp recordings on amacrine cells in the horizontal slice preparation. Patch pipettes were made by pulling Pyrex tubing on a micropipette puller (P-97; Sutter Instrument, Novato, CA). The tip diameter of the patch pipette was 1-2 μm, giving a resistance of approximately 10-15 MΩ when filled with the pipette solution (in mM): 125 K-gluconate, 5 KCl, 10 HEPES, 1 CaCl₂, 1 MgCl₂, and 11 EGTA (pH adjusted to 7.2 with KOH). Lucifer yellow (0.1%; Sigma) was included to stain and visualize the recorded cells. Slices were continuously perfused at a rate of 2 ml/min with an extracellular solution containing (in mM): 125 NaCl, 2.5 KCl, 2 CaCl₂, 1 MgCl₂, 26 NaHCO₃, 1.25 KH₂PO₄, and 12 glucose. The extracellular solution was continuously oxygenated with 5% CO₂/95% O₂ and kept between 32 and 35 °C. Slices were viewed using a microscope (Axioscope, Zeiss, Germany) equipped with a 60x water-immersion objective lens (LUMPLF L60x; NA 0.9, WD 2 mm; Olympus, Japan) and infra-red differential interference contrast optics. The recording pipette was connected to the input stage of a patch-clamp amplifier (PC-501A; Warner Instruments, Hamden, CT). Signals were sampled at 10 kHz with a DigiData 1322A interface and pCLAMP8 software (Axon Instruments, Foster City, CA). The liquid junction potential was corrected by (V_membrane = V_pipette -11 mV). Recorded data were analyzed with Igor Pro 4.0 software (WaveMetrics, Lake Oswego, OR) and additional user-written routines (AK).

Space clamp considerations

In our patch-clamp recordings, the space clamp was poor under voltage-clamp configurations. Evidence that the recorded cell was not a single compartment was that capacitive charging and discharging currents produced by voltage pulses were not fit with a single exponential. The slow capacitive components, following transient charging and discharging currents, indicate that dendrites of our recorded cells were not completely voltage-clamped. This was to be expected, as it has previously been reported that dendrites of amacrine cells are weakly voltage-clamped by a somatic patch-clamp pipettes [8]. In the present experiments this precluded quantitative examination of the electrophysiological characteristics of the channels.

Single-cell RT-PCR

Directly after patch-clamp recording, the contents of the cell were aspirated into the patch electrode. The electrode tip was then broken off into a thin-wall PCR reaction tube containing 10 μl ice cold reaction buffer and stored on dry ice until use. Reverse transcription and the first PCR were carried out for all four HCN subtypes and β-actin simultaneously using the Access PCR system from Promega (Madison, WI) according to the manufacturer's instructions. The reaction conditions were: 50 min at 48 °C for reverse transcription, followed by 28 cycles of 1 min at 94 °C, 1 min at 59 °C, 2 min at 68 °C, and a final extension of 7 min at 68 °C. Reaction products were diluted 20 fold and 1 μl was used for a secondary PCR of 32 rounds for each HCN channel subtype individually, using pairs of nested primers. Primers were used at a concentration of 2 μM, and all primers were designed to span at least one intron/exon boundary. Primers were used at a concentration of 2 μM, and all primers were designed to span at least one intron/exon boundary. For primer sequences see Table 1. Actin was used as a positive control, with the same primers for both rounds of PCR [26]. The predicted size (in bp) of the amplicons were as follows: HCN1 404; HCN2 658; HCN3 619; HCN4 604; actin 255. Reaction products were resolved on 2% agarose gels and photographed. Occasionally, bands were chosen at random, purified with QiaexII (Qiagen, Hilden, Germany) and subjected to diagnostic restriction enzyme digestions. The restriction enzymes and sites were PstI/157 for HCN1, PstI/363 for HCN2, BglII/229 for HCN3 and BglII/211 for HCN4. The protocol for single-cell RT-PCR from dissociated retina is described in detail elsewhere [27]. In brief, after isolation the retinas were kept in oxygenated Ames' medium. Small pieces of retina were cut off and dissociated in Hank's balanced salt solution (HBSS) containing 0.5 mg/ml papain for 10 min at 37 °C. The papain reaction was quenched with MEM/10% horse serum containing 200 U/ml DNAseI (Sigma). The tissue was gently triturated and approximately 50 μl of the suspension was put into one ring of a Gold Seal slide (Gold Seal Products, Portsmouth, NH). Individual cells were identified by morphology using a Zeiss IM35 inverted microscope, and aspirated into a microcapillary tube under microscopic control, then transferred to the other ring for washing in Ringer's solution containing 0.5% BSA. The cell was aspirated again and transferred into a thin-wall PCR tube containing 10 μl PCR buffer and 5 μl Gene Releaser (BioVentures, Portland, ME), also under microscopic control. A negative control for every cell was obtained by aspirating about 0.5 μl of the washing medium and processing it strictly in parallel to the cell. A fresh preparation of dissociated cells was used for every cell transfer. For some experiments, the dissociated cells were incubated with a FITC-labeled anti CD15 antibody (BD PharMingen, San Diego, CA) and CD15⁺ amacrine cells were identified by their fluorescence.

Computer simulation

We used the NEURON simulator (version 4.3.1) [28] to model dendritic responses and voltage spreads in amacrine cells.

Morphology

We reconstructed a mouse starburst amacrine cell from a photomicrograph after DiOlistic filling as per Rockhill et al. [29]. The reconstructed starburst amacrine cell was divided into 721 compartments.

Passive membrane parameters

Passive membrane parameters were obtained from dual-whole cell patch-clamp experiment on cultured amacrine cells described previously [30]. Membrane capacitance, cytoplasmic resistance and passive membrane conductance were determined as 1 μF/cm², 150 Ω-cm, and 3x10⁵ S/cm², respectively. These passive parameters were evenly distributed in a whole cell. In this study, to examine effects of passive membrane conductance to synaptic responses, passive membrane conductance was varied from 5x10⁶ to 5x10⁴ S/cm².

Synaptic stimulation

Synaptic stimulation was calculated as alpha functions, described by the following equations:

I_stim = gs(t) * (V - E_stim)

gs(t) = gsbar * (t/τ) * exp(1 - t/τ)

The reversal potential (E_stim) was set to 0 mV, maximum conductance (gsbar) was 1x10⁴ S/cm², and tau was 5 msec. To stimulate a dendritic branch, five points within the dendritic branch were selected randomly as synaptic input sites and they were stimulated simultaneously. The total maximum conductance was set to 1x10⁴ pS/cm².

Results

I_IR of amacrine cells in a horizontal slice preparation

We recorded from and successfully injected 33 cells. Their dendritic field diameters ranged from 50 to 300 μm, a range from the narrowest found for amacrine cells to the smaller of wide-field amacrine cells. Albeit with several varieties of responses to current injection (Figure 2A,B and Figure 3), all recorded cells showed a large membrane conductance around the resting membrane potential and I_IR at more negative potentials. Figure 2A,B shows an amacrine cell that generated a single slow action potential (n=10), recorded under voltage-clamp and current-clamp conditions (resting membrane potential, -69 mV). Under current-clamp, a single slow action potential was evoked by +100 pA current injection (Figure 2B, arrow). The threshold of such action potentials was above -30 mV. Under voltage-clamp (Figure 2C), they showed a hyperpolarization-induced I_IR (asterisk), as well as a transient inward current (arrow with TI), a transient outward current (arrow with TO), and a delayed outward current. In some amacrine cells (Figure 2D,E), dendrites were removed in the process of preparation, as shown by Lucifer yellow injection. These amacrine cells without dendrites showed only small resting membrane conductance, and especially I_IR was not observed (Figure 4B). We compared amacrine cells with dendrites and those without dendrites in regard to the input resistance and the conductance of I_IR. The input resistance was calculated under current-clamp configuration by current injection of -20 or -40 pA. The conductance of I_IR was determined in voltage-clamp configuration by voltage pulses to negative to the holding potential (-71 mV). The input resistances of amacrine cells without dendrites (2545±992 MΩ, mean±standard deviation, n=7) were significantly higher than amacrine cells with dendrites (526±351 MΩ, n=33). The conductances of I_IR in amacrine cells without dendrites (328±173 pS, n=7) were lower than in those with dendrites (3356±1893 pS, n=33). These results suggest that main portion of I_IR is evoked at the dendrites of amacrine cells rather than the somas, whether or not the somas have I_IR. Other amacrine cells with different electrophysiological properties possessed I_IR. Figure 3A,B shows an amacrine cell with oscillatory potentials (n=14). In this cell, the low-amplitude oscillatory potentials were observed in a steady depolarized state (+80 pA current injection) under current clamp (Figure 3A, arrow). Transient inward currents were observed when a depolarizing voltage pulse (from -40 mV to -20 mV) was applied under voltage-clamp (Figure 3B). Under voltage clamp, a hyperpolarization-activated inward rectifying current was observed (Figure 3B, asterisk). Non-spiking amacrine cells (n=9) generated slow potentials that might be mediated by slow inward current (Figure 3C,D). For these cells, I_IR was also prominent (Figure 3D, asterisk). Amplitudes and kinetics of I_IR were variable among recorded cells, but no correlation was apparent, within the limits of our sample, with the morphological and electrophysiological type of the cell. I_IR was suppressed markedly by external cesium ion (Cs⁺, 5 mM), which is known as non-selective blocker of any inward rectifying K⁺ channels (Figure 4A and Figure 4B). Thus, I_IR is mainly carried by K⁺. In summary, amacrine cells with intact dendrites possess I_IR, which appears to be located on dendrites of amacrine cells. The I_IR observed in these purely electrophysiological experiments might be a sum of classical inward rectifying K⁺ channels, I_(H) channels, and leak conductances.

I_IR decreases the input resistance and stabilizes the resting membrane potential

To evaluate the effect of I_IR on input resistances and membrane potential stability, Cs⁺ (5 mM) was added to the extracellular solution. Under voltage-clamp, extracellular Cs⁺ greatly suppressed I_IR (Figure 4A and Figure 4B). The steady state I-V relationship (arrows, Figure 4A and Figure 4B) showed that Cs⁺ mainly suppressed the hyperpolarization-activated inward rectifying component. Under current-clamp, Cs⁺ application increased input resistance calculated by current injection of -40 pA from 465 MΩ to 3927 MΩ (Figure 4D and Figure 4E) and amplified voltage changes. Under current-clamp in the control condition, +80 pA current injection was required to evoke oscillatory potentials (Figure 4D and Figure 4E, arrows), but during Cs⁺ application, as little as +20 pA current injection could evoke these oscillatory potentials. This is probably because external Cs⁺ application changed the efficiency of space clamp and modified the activation of other currents on the dendrites. We obtained similar results in all 9 cells tested (Figure 4F). I_IR thus decreases the input resistance and increases the current required to evoke oscillatory potentials.

I_(H) contributes to I_IR

To examine whether or not I_(H) contributes to I_IR, we carried out pharmacological experiments to isolate I_(H). ZD7288 (100 μM), a selective blocker for I_(H), was added to the extracellular solution. Under voltage-clamp, ZD7288 partially suppressed I_IR (Figure 5A and Figure 5B); the sustained component of I_IR was the most suppressed. Under current-clamp, ZD7288 application increased the input resistance calculated by current injection of -40 pA from 533 MΩ to 739 MΩ (Figure 5D and Figure 5E) and amplified the voltage changes. We obtained similar results in all 6 cells tested (Figure 5F). In all cases, ZD7288 partially suppressed I_IR. We compared the suppression ratios of I_IR by external Cs⁺ and by ZD7288 in Figure 5G. External Cs⁺ suppressed I_IR to 38±21% (n=9), and ZD7288 suppressed it to 52±20% (n=6). This result shows that I_(H) contributes to about half of I_IR, which is mainly carried by K⁺. The residual Cs⁺ insensitive portion of I_IR might be carried by other cations and non-selective leak conductances due to slice procedure. To examine the effect of intracellular cAMP to I_IR, we bath-applied a membrane-permeable analogue of cAMP (8-cpt-cAMP, Sigma, 1 mM, n=4). During cAMP application, I_IR amplitude was increased slightly and the neuron's input resistance was decreased. The input resistance decrease made it difficult to generate oscillatory potentials. In control conditions, current injection of +80 pA reliably evoked oscillatory potentials (Figure 6C, arrow); a minimum of +140 pA was required to evoke oscillatory potentials during the cAMP application (data not shown). The substantially higher threshold of oscillatory potentials was seen in the face of relatively small changes in input resistance and in the amplitude of total currents evoked by voltage steps (Figure 6E). In other systems, I_(H) can be enhanced by intracellular cAMP or cGMP [19,23,31]. Therefore, these pharmacological results in amacrine cells support the idea that I_(H) contributes to I_IR to some extent. However, we cannot rule out the effect of cAMP application on other K⁺ currents and leak currents. Because of space clamp problems described in Methods, it is difficult to voltage-clamp the cell to characterize electrophysiological properties of I_(H). For example, time course of I_(H) has well known as slowly sigmoid activating properties, but in some of our experiments we did not observe clearly these typical properties even when ZD7288 suppressed the current. Therefore, we identified I_(H) in amacrine cells by molecular techniques instead of further electrophysiological experiments.

To molecularly characterize the responsible HCN channels, we carried out single-cell RT-PCR on the cytoplasmic contents of the cell, which were aspirated at the end of some recording sessions (n=9). HCN1 and/or HCN2 subunit mRNAs were found in the amacrine cells (Figure 7A-F). Some tested cells had both HCN1 and HCN2 mRNAs (n=5; Figure 7A-C); while the others had HCN2 mRNA only (n=2; Figure 7D-F). No correlation was observed between types of HCN subunit mRNAs and the electrophysiological types of amacrine cells described above. HCN3 and HCN4 channel mRNAs were found in none of the amacrine cells from which we recorded. In two cells out of 9 cells tested, we did not find any HCN mRNA-positive bands, although we observed I_(H) in those cells electrophysiologically. This is probably because we failed to aspirate the cytoplasm into a narrow glass pipette after recording. To avoid these technical limits of single-cell RT-PCR after patch-clamp recordings and compare the subsets of HCN channel mRNAs with those of other cell types, we conducted a control experiment with freshly dissociated retinal neurons. In a sample of 12 dissociated cells, we found expression of HCN1 mRNA in 3/5 bipolar cells and 3/4 amacrine cells, HCN2 mRNA was expressed in 2 bipolar cells and 2/4 amacrine cells (Figure 7G). The CD15⁺ amacrine cells, which are among the wide-field amacrine cells, possessed both HCN1 and HCN2 mRNAs (Figure 7G, line 6 and 7). HCN3 or HCN4 mRNAs were not found in any bipolar or amacrine cells. All ganglion cells in our sample expressed HCN1, HCN2, and at least one of either HCN3 or HCN4 mRNAs. Taken together, our RT-PCR data from patch-clamp and dissociated cells indicate that HCN1, HCN2 or both are expressed in most amacrine cells, although we cannot rule out the possibility that there may be a small subpopulation among these highly diverse cells that does not express any HCN channels.

Functional consequences: I_IR sharpens the dendritic response temporally and spatially

What is the function of I_IR in amacrine cells? We conducted a computer simulation to explore whether I_IR would have a substantial effect on the electrical isolation of the stimulated dendrite in a morphologically realistic amacrine cells. We used the morphology of the mouse starburst amacrine cell. This cell has a size and general dendritic character representative of medium/wide-field amacrine cells. It has the advantage of an extremely well studied geometry, which is depicted in Figure 8A. Computer simulations were run on the virtual cell with a variety of passive membrane conductances (see Methods). To examine the effects of I_IR on synaptic responses, the passive membrane conductance was varied from 5x10⁶ to 5x10 S/cm². Two examples of voltage-clamp experiments with large passive membrane conductance (5x10⁵ S/cm², G1) and small conductance (5x10⁶ S/cm², G2) are shown in Figure 8B. The resting membrane potential of this cell was set to the reversal potential of passive membrane conductance (-66 mV). We simulated synaptic stimulation of the reconstructed starburst amacrine cell at dendritic branch "a" (Figure 8A). Voltage changes were calculated at the soma (filled circle "2"), the distal tips of dendritic branch "a" (filled circle "1") and the opposite dendritic branch (filled circle "3"). When dendritic branch "a" was stimulated, voltage changes that spread to the soma and the opposite dendritic branch were temporally sharpened and quantitatively suppressed in the cell with large passive membrane conductance compared with that with low conductance (Figure 8C). The amplitudes of local potentials in the dendritic branch "a" were not affected by conductance changes (Figure 8C, filled circle "1"). Figure 8D shows relationships of conductance changes with half durations and peak amplitudes of voltage changes at the opposite dendritic branch (filled circle "3"). Increasing the passive membrane conductance shortened the time course (half duration) of voltage changes and reduced their amplitudes. I_IR would thus enhances electrical isolation of dendritic responses in amacrine cells with middle-size dendritic fields, such as the starburst amacrine cells. If the cells have thick dendrites or small dendritic fields, the effect of I_IR should be much smaller.

Discussion

We carried out patch-clamp experiments on mouse amacrine cells in a horizontal slice preparation that preserves their dendritic structures. Across a wide variety of amacrine cell shapes and physiologies, almost all possessed I_IR. I_IR contributes to membrane potential stability and modifies dendritic responses. I_(H) contributes to I_IR. Single-cell RT-PCR studies suggest that the I_(H) in amacrine cells was carried through HCN1 and/or HCN2 channels.

I_IR and I_(H) in amacrine cells

In contrast to previous reports on mouse amacrine cells in retina slices [14], or dissociated amacrine cells [4], we found all of our recorded cells in horizontal slice preparations to have I_IR. Its major component was I_(H). The input resistance of isolated dopaminergic amacrine cells has been reported to be over 2 GΩ, very similar to our results in which I_IR was markedly suppressed when external cesium was applied or when dendrites were removed in a process of preparation. We therefore suspect that I_IR was partially lost in the previous reports of dissociated dopaminergic amacrine cells, when their distal dendrites were truncated. Taken together, these results suggest that I_IR may be preferably localized in distal dendrites in amacrine cells. Similar differences exist between the currents detected in the cerebellar slice preparation and these in isolated Purkinje cells. In acutely dissociated Purkinje neurons, I_(H) contributes little inward current at membrane potentials near the action potential threshold, and pharmacological blockade of I_(H) does not alter the frequency of tonic action potential firing [32]. However, in the cerebellar slice preparation, I_(H) is much more activated, and plays an important role in the control of tonic action potential firing in Purkinje neurons [33].

Is the membrane conductance of amacrine cells dynamically regulated?

In our present study, I_(H) contributes to I_IR in amacrine cells and increases membrane conductance. An interesting and important question involves the regulation of I_(H) in the inner plexiform layer in vivo. In other systems [23,31]. many neurotransmitters and modulators are known to modulate I_(H) via intracellular cAMP and cGMP activities. Increases in intracellular cAMP or cGMP, such as during β-adrenoceptor activation or application of nitric oxide, shift the I_(H) activation curve in a depolarized direction. An increase in resting I_(H) activation with increased cAMP concentrations has been reported in dendrites of CA1 pyramidal neurons [23]. DiFrancesco and Tortora [34] reported that I_(H) channel gating is directly regulated by cAMP binding to a site on the cytoplasmic surface of the channel, and cGMP also binds to this site to activate I_(H) channels. In the retina, intracellular cAMP is reported to modulate several phenomena. For example, in the developing retina, intracellular cAMP is key to sustaining developmental retinal waves [35]. In amacrine cells, GABA responses are enhanced by dopamine via an intracellular cAMP increase [36]. In the present study, we found that cAMP enhances I_IR and modulates the resting membrane properties of amacrine cells. Thus, intracellular cyclic-nucleotides such as cAMP (and cGMP [37]) may regulate the membrane conductance dynamically in retinal amacrine cells.

Function of I_IR in amacrine cell dendrites

The "dendrite" of an amacrine cell is both an input and output structure and these has been much discussion of possibly independent function by individual amacrine cell dendrites [10,38-40]. In hippocampal and cortical pyramidal neurons, I_(H) channels participate in generating a spatial gradient of EPSP and IPSP, and in shortening the length constant of the distal dendrite [22,41-43]. The presence of I_(H) in the distal dendrite is thought to modify the EPSP and IPSP time courses by enhancing the local resting membrane conductance. I_(H) provides a leakage path for current flow that decreases the local membrane time constant and speeds the decay of the distal EPSP. However, these are classically polarized neurons, and they are more than ten times larger than amacrine cells. Because differences of scale can have different electrotonic consequences, we carried out a computer simulation, using an anatomically realistic model of an amacrine cell, to verify that the prediction from brain neurons would hold for the very different geometry of amacrine cells. Our simulation on a reconstructed starburst amacrine cell confirmed that high membrane conductances should enhance the electrical isolation of dendritic voltage changes generated by synaptic input (as it does for the other neurons). This could contribute to the observed independence of evoked Ca²⁺ fluxes in different dendritic branches of starburst amacrine cells [10] and to dendritic independence in other amacrine cells.

Acknowledgements

The authors appreciate technical advice from Drs. Ryosuke Enoki and Taro Azuma (Department of Physiology, Keio University School of Medicine, Tokyo, Japan) during development of the horizontal slice preparation of the mouse retina. We would also thank Drs. Steve Stasheff, Guenther Zeck, and Ms. Kate Harmon for careful reading and discussion. This study is partly supported by the Japan Society for Promotion of Science (AK). RHM is a Senior Investigator of Research to Prevent Blindness.

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