Identification and classification of bipolar cells

By using Nomarski optics, individual neurons within the different layers of the retinal slice can be recognized (Fig.1A). RB cell bodies are found more often in the outermost part of the INL, cell bodies (CBs) more toward the center. Hence, to some extent it was possible to select RBs or CBs according to their cell body position. We filled the cells with LY during the recordings and thus were able to verify their identity according to the classification scheme proposed by Euler and Wässle (1995a) and summarized in Fig. 1D. The CB cells are arranged according to the stratification level of their axons—from outer to inner—and are numbered accordingly. Figure 1Ashows the slice and the recording pipette contacting an RB cell body. The same bipolar cell using fluorescence illumination is shown in Fig.1B,C. Its axon terminates close to the ganglion cell layer and has varicosities, which are the features of RB cells (Fig.1D, RB).

Altogether we recorded 78 bipolar cells in the present study; of these, 59 could be classified according to the scheme in Fig. 1D. The remaining cells were filled only partially. The resting potentials of RB (−45 ± 13 mV; n = 21) and CB cells (−49 ± 10 mV;n = 38) were in the broad range given in the literature for dissociated RB cells (Karschin and Wässle, 1990; Suzuki et al., 1990; Yeh et al., 1990; Yamashita and Wässle, 1991b). No significant differences were observed between RB cells or different CB cells. Twenty-three RB and 34 CB cells could be analyzed in detail, and their relative numbers are indicated in Table 1. The type 9 CB cell, which is a putative blue CB cell (Euler and Wässle, 1995a), was not encountered.

All cells showed GLU-induced whole-cell currents. The cells could be divided into two major groups (Fig.2A,B): bipolar cells in which GLU elicited inward currents (Fig. 2A) and bipolar cells in which GLU elicited outward currents (Fig. 2B). In many instances, however, these currents were biphasic, suggesting direct and indirect actions of GLU on the recorded cells. It was not possible here to perform a detailed pharmacological analysis of the GluRs expressed by the different types of bipolar cells. Recordings of these extremely small cells in a slice are difficult, the time course of the drug application system is relatively slow, and the exact concentration of the drugs at the cell membrane is unknown. Our major aim is to show which cells are putative ON- and which are OFF-bipolar cells.

Responses of bipolar cells to different GLU agonists

Four possible direct actions of GLU and related agonists on bipolar cells are known from nonmammalian retinae (for review, seeShiells, 1995). (1) GLU can open nonspecific cation channels of the KA/AMPA type, which is the action found in OFF-bipolar cells (Attwell et al., 1987; Hensley et al., 1993; Kim and Miller, 1993). (2) GLU can close nonspecific cation channels by binding to a metabotropic (AP-4-type) receptor, which was described for ON-bipolar cells (Nawy and Jahr, 1990, 1991; Shiells and Falk, 1990, 1992 a,b). (3) GLU can open specific channels (such as K⁺ channels), as has been described for some fish and tiger salamander ON-bipolar cells (Saito et al., 1979, 1981; Nawy and Copenhagen, 1987, 1990; Hirano and MacLeish, 1991). (4) GLU can activate a chloride channel by a receptor with GLU-transporter-like pharmacology (Grant and Dowling, 1995).

In addition to the direct action of GLU, indirect actions are produced when GLU acts on neurons presynaptic to the bipolar cells. Such indirect actions might involve activation of amacrine cells, horizontal cells, and photoreceptors. ECO was used to block Ca²⁺-dependent transmitter release from photoreceptors and amacrine cells. Transmitter release from horizontal cells is more problematic because it has been shown in nonmammalian retinae to be Ca²⁺-independent (Schwartz, 1982) and thus might not be blocked by Co²⁺ ions. Therefore, BIC and STRY were included in the bathing solution to block GABAergic input from horizontal and amacrine cells and also glycinergic input from amacrine cells; however, the recent discovery of BIC and picrotoxin-insensitive GABA_C-like responses in mammalian RB cells (Feigenspan et al., 1993) creates an additional problem. Therefore, symmetrical (I1) and low Cl⁻ concentrations (I2, I3) were applied to determine whether GABA_C receptors were involved in the responses measured.

Perforated-patch whole-cell recordings from an RB cell are shown in Fig. 3. The cell, filled with LY after the recordings (Fig. 3A), had the characteristic shape of an RB cell (Fig.1D). The electrode contained a low concentration of Cl⁻ (I3). The cell was voltage-clamped to V_C = −45 mV, and a steady inward current of 11 pA was measured (Fig.3B, arrow). Application of AP-4 evoked a net outward current and a reduction in membrane noise (Fig. 3B) in 11 of 23 RB cells tested (Table 1). The maximum amplitudes of AP-4-induced outward currents were between 5 and 20 pA. This outward current did not change in these 11 cells when Co²⁺, BIC, or STRY were applied with the bathing solution (not shown). The current–voltage relation (I–V curve) was steeper in the absence of AP-4 than during the application of AP-4; hence AP-4 caused a conductance decrease (190 ± 10 pS; n = 4). The reversal potential was −6 ± 16 mV (n = 4) and was shifted to more negative values when a low concentration of external Na⁺ was puffed onto the cell. GABA responses were measured in 18 of 19 RB cells tested (Fig.3C). Similar currents also have been found in dissociated RB cells of rat and cat retinae (Yamashita and Wässle, 1991a; de la Villa et al., 1995), and they quite likely represent direct actions of the drug on the cell.

The cell also responded weakly to the application of KA, with an outward current that persisted in the presence of external Co²⁺ (Fig. 3D). As shown in Fig. 3D, however, it was blocked by co-application of CNQX and KA, suggesting the involvement of KA/AMPA receptors. The response to KA could also be blocked by the co-application of KA and a mixture of BIC and STRY (Fig.3D). Together these results suggest that the KA/AMPA receptors are not on the RB cell but on neurons presynaptic to RB cells, possibly horizontal cells, where release of GABA is not blocked by Co²⁺. Such presynaptic effects of KA were observed in 7 of 21 RB cells tested and ranged in amplitude between 5 and 60 pA. The reversal potential agreed with the Cl⁻ reversal potential.

Whole-cell recordings from a CB cell are shown in Fig.4. The cell had an axon branching in the inner portion of the IPL, close to the ganglion cell layer (Fig. 4A), and was identified as a type 8 CB cell (Fig. 1D). Similar to the responses of the RBs in 13 of the 29 CB cells tested, AP-4 evoked net outward currents between 5 and 20 pA, always accompanied by a reduction in membrane noise (Fig. 4B). Twelve of the 13 CB cells had axon terminals in the inner part of the IPL (Table 1). The currents were resistant to the application of BIC and STRY or to superfusion with Co²⁺ (data not shown) and quite likely represented a direct action of AP-4. The reversal potential of the AP-4-evoked current averaged −13 ± 10 mV (n = 4). GABA induced an inward current at the holding potential (V_C = −53 mV) because of the symmetrical Cl⁻ concentration (I1) (Fig.4C). KA also induced a strong inward current; however, because Co²⁺ nearly completely blocked this current, it was very likely mediated via activation of a presynaptic cell (Fig.4D). CNQX blocked the KA responses.

Whole-cell recordings from a CB cell that behaved differently are shown in Fig. 5. The axon terminal of this cell was confined to the outer IPL, and the cell was classified as a type 2 CB cell (Figs. 5A, 1D). AP-4 did not elicit any current in this cell, but GLU evoked an inward current (Fig. 5B). KA evoked an even stronger inward current than GLU did, and this inward current was not significantly changed by using Co²⁺containing bathing solution; however, it was abolished by CNQX (Fig.5C). We interpret these findings as a direct action of GLU and KA on this CB cell. Such KA responses were observed in 14 of 29 CB cells tested (Table 1). Thirteen of the CB cells showing direct KA responses had axon terminals in the outer part of the IPL. The maximum amplitude of KA-induced inward currents was between 40 and 120 pA. Reversal potentials were measured in 13 CB cells; they shifted to more negative values when a low external Na⁺ concentration was applied, and the average reversal potential was −4 ± 7 mV. In four of the outer stratifying bipolar cells, AP-4-induced currents could be observed; however, they were always indirect currents, which could be blocked by BIC and/or STRY.

In summary, there seems to be a clear dichotomy of bipolar cells with respect to their GluRs. Bipolar cells with axons terminating in the inner half of the IPL express AP-4 type receptors; bipolar cells with axon terminals in the outer half of the IPL express KA/AMPA type receptors (Table 1).

GABA-induced currents of retinal bipolar cells

RB cells dissociated from mammalian retinae show prominent GABA-gated Cl⁻ conductances (mouse: Suzuki et al., 1990; rat: Karschin and Wässle, 1990, Yeh et al., 1990; Yamashita and Wässle, 1991a,b; rabbit: Gillette and Dacheux, 1995). The Cl⁻ reversal potential is ∼−70 mV (Yamashita and Wässle, 1991a). The recent cloning of a retina-specific subunit of GABA-receptors, the ρ-subunit, (Polenzani et al., 1991), and the finding of GABA responses that are resistant to BIC (Feigenspan et al., 1993; Qian and Dowling, 1993), suggest that different types of GABA-gated Cl⁻ channels might exist in bipolar cells.

GABA-activated Cl⁻ currents recorded from amacrine cells in the organotypic slice culture and in the acute slice preparation could be blocked reliably by the co-application of 100 μmBIC (Boos et al., 1993; Feigenspan et al., 1993). Similarly, glycine-induced currents were blocked by the application of 1 μm STRY. The situation was different, however, for bipolar cells: a fraction of the GABA-activated current was resistant to BIC (Feigenspan et al., 1993).

Perforated-patch recordings from an RB cell with an electrode filled with a low Cl⁻ concentration (I3) are shown in Fig.6A. At very negative holding potentials, an inward current was elicited by GABA, and at holding potentials more positive than −70 mV, the current was outward. Co-application of BIC and STRY strongly reduced the GABA-activated current, but was not able to block it completely (Fig. 6B). The current–voltage relation is shown in Fig. 6C. Both the total (open circle) and the STRY/BIC-resistant (solid circle) GABA-induced currents have a reversal potential at −70 mV, which corresponds to the Cl⁻ reversal potential. Six of seven RB cells tested in this way showed such STRY/BIC-insensitive GABA-induced currents. The peak amplitudes of GABA-activated currents of RB cells clamped at the resting potential were between 10 and 80 pA. Application of STRY/BIC reduced the currents by 20–65%. We also measured GABA-activated currents of 11 CB cells. The current amplitudes were in the same range as those of RB cells. In four CB cells, GABA-activated currents were blocked effectively by STRY/BIC; in seven CB cells, they could not be blocked but were reduced by 50–80%. We also applied BAC, an agonist at GABA_B receptors, to 18 bipolar cells. Of these, only four CB cells gave weak, noisy, and delayed responses.

ON-bipolar cells of the rat retina

RB and CB cells with axons terminating in the inner half of the IPL (CB types 6, 7, 8) express an AP-4 type GluR. In the absence of GLU, the channels are open and the cells are in a depolarized state. On binding of GLU, the channels close and the cells are hyperpolarized. Photoreceptors release GLU in darkness, and release is suppressed by light (Cervetto and MacNichol, 1972; Murakami et al., 1975; Marc and Lam, 1981; Ayoub et al., 1989; Copenhagen and Jahr, 1989). Consequently, the present findings suggest that RB cells and type 6, 7, and 8 CB cells are depolarized by light and hyperpolarized in the dark and that they are ON-bipolar cells.

The metabotropic GluR mGluR6 cloned from rat retina (Nakajima et al., 1993) was activated potently by AP-4 in an artificial expression system. In situ hybridization analyses indicated that mGluR6 mRNA expression is restricted to the INL of the rat retina (Nakajima et al., 1993; Hartveit et al., 1995). Recently, specific antibodies against mGluR6 produced punctate labeling on the dendritic tips of rat RB cells (Nomura et al., 1994). Immunostaining of CB cell dendritic tips was not studied by Nomura et al. (1994); however, when mGluR6 was deleted genetically in mice (knock out), the b wave of the electroretinogram was absent and no ON-responses could be recorded from optic tract terminals, suggesting that the ON-pathway in such animals is nonfunctioning (Masu et al., 1995). The identification of the AP-4 receptor as a metabotropic GluR was supported further by its pharmacological similarity with metabotropic receptors (Tian and Slaughter, 1994; Thoreson et al., 1995; Thoreson and Ulphani, 1995). Therefore the evidence is strong that RB cells and ON-CB cells express the mGluR6 receptor.

In situ hybridization studies of dissociated RB cells indicate that these cells might also express ionotropic GluRs (Hughes et al., 1992; Müller et al., 1992). In the present study, we also observed weak responses to KA in some RB cells, which might represent direct effects on the RB cells. In previous studies of dissociated RB cells, after washing out the second messenger system of the AP-4 responses (Nawy and Jahr, 1990), we sometimes observed channel openings induced by GLU (Karschin and Wässle, 1990). Therefore it is possible that RB cells in addition to mGluR6 express other GluRs. Whether these receptors are involved in physiological responses is still open to question.

Occasionally in the present study we recorded putative ON-bipolar cells, in which the dendrites were cut off during the slicing procedure. This became apparent after the cells were filled with LY. Such truncated cells never showed AP-4 responses, suggesting that the receptors are on the dendrites, as shown by Nomura et al. (1994). In only approximately half of the recorded RB cells did we observe responses to the application of AP-4. There is the possibility that the nonresponding half might express different GluRs; however, there was no evidence for such a dichotomy, and we assume that mechanical damage during the slicing procedure might have broken the finest dendrites of these cells. This was also true for dissociated RB cells (Yamashita and Wässle, 1991b) whenever their dendritic terminals were lost during dissociation.

OFF-bipolar cells of the rat retina

CB cells with axons terminating in the outer half of the IPL (CB type 1, 2, 3, 4, 5) express KA/AMPA type GluRs. Binding of GLU or KA to this receptor causes the opening of nonspecific cation channels. Because photoreceptors release GLU in the dark (Murakami et al., 1972), such cells are depolarized in darkness: they are OFF-bipolar cells (Murakami et al., 1975).

Molecular cloning has revealed several families of ionotropic GluRs (for review, see Hollmann and Heinemann, 1994). Traditionally these receptors have been classified into three broad subtypes: KA receptors, AMPA receptors, and NMDA receptors. This classification now has been extended, however, and 16 different ionotropic GluRs have been found. Anatomical studies using in situ hybridization techniques and immunocytochemistry (Hughes et al., 1992; Müller et al., 1992; Hamassaki-Britto et al., 1993; Brandstätter et al., 1994;Hartveit et al., 1994; Peng et al., 1995; Watanabe et al., 1994) have shown a differential distribution of the GluR isoforms in the rat INL and IPL. They show that there might be many different subunits, isoforms, and combinations of ionotropic GluR in bipolar cells of the rat retina. In the absence of specific antagonists, these isoforms cannot be distinguished pharmacologically. It is known that different GluRs have different desensitization times (Dudel et al., 1990), and it is possible that the different CB cells may express kinetically different GluRs. Unfortunately application of drugs using puffer pipettes is too slow to obtain kinetically meaningful data. Analysis of light responses may provide additional clues.

Further actions of GLU on rat retinal bipolar cells

Because bipolar cells release GLU as their transmitter (for review, see Massey, 1990), they may have a high-affinity uptake system for GLU. This is supported by the staining of type 5 and 6 CB cells with antibodies against the GLU transporter GLT-1 (Rauen and Kanner, 1994; Euler and Wässle, 1995a). Therefore, it is possible that the GLU responses shown might have been caused by electrogenic GLU uptake at bipolar cell axon terminals. This is probably not so, for the following two reasons. The agonists used in most of the recordings presented here were AP-4 or KA. There is no evidence for the electrogenic transport of AP-4 and KA through GLU transporters in the retina (Eliasof and Werblin, 1993; Danbolt, 1994). We also applied L-tPDC (Rauen et al., 1992), which is transported with high affinity by GLU transporters. Only weak effects were observed in six of seven bipolar cells tested, and these always differed from the responses elicited by AP-4, GLU, or KA. Grant and Dowling (1995) recently described Cl⁻ channels in ON-bipolar cells of the white perch retina that were activated by a GluR with transporter-like pharmacology; however, this channel was not activated by KA or AP-4 and therefore cannot account for the results reported here.

Another possible source of GLU-induced currents are metabotropic GluRs other than mGluR6, the AP-4 receptor. A proposed specific agonist, which binds to mGluR5, is tADA (Favaron et al., 1993; Thoreson and Ulphani, 1995). Five of 10 bipolar cells, all putative ON-bipolars, showed weak effects, which were not investigated in greater detail. This supports the contention that mGluR6 is the major GluR in ON-bipolars.

GABA receptors of rat retina bipolar cells

In the present study, BIC-sensitive GABA-induced currents were found in almost all bipolar cells, suggesting that all bipolar cells express GABA_A receptors. Recently we have investigated rat retinae with subunit-specific antibodies against GABA_Areceptors (Greferath et al., 1995) and have found multiple GABA_A receptors in bipolar cells of the rat retina. Hence, several different GABA_A receptor subtypes might contribute to the BIC-sensitive currents recorded in the present study.

GABA_C receptors have been described to gate BIC-insensitive Cl⁻ currents in various parts of the vertebrate brain (for review, see Johnston, 1994; Bormann, 1995). In the retina, GABA_C receptors are present on rat RB cells (Feigenspan et al., 1993; Feigenspan and Bormann, 1994; Pan and Lipton, 1995), on tiger salamander bipolar cells (Dong et al., 1994; Lukasiewicz et al., 1994), and on horizontal cells of the white perch (Qian and Dowling, 1993, 1994). In the present study, we show evidence for GABA_C-receptor expression in CB cells. GABA_Creceptors are likely to be composed of the recently discovered ρ1 and ρ2 subunits (Cutting et al., 1991, 1992). In situhybridization of retinal sections revealed the presence of ρ1 and ρ2 transcripts in the INL and in dissociated RB cells of the rat retina (Enz et al., 1995; Pan et al., 1995). Such GABA_Creceptors are more sensitive than conventional GABA_Areceptors (Feigenspan and Bormann, 1994); however, more information concerning the precise localization of GABA_A and GABA_C receptors on bipolar cells is needed before their different roles in visual processing can be determined.