Relative contributions of rod and cone bipolar cell inputs to AII amacrine cell light responses in the mouse retina
- Ji-Jie Pang1,
- Muhammad M. Abd-El-Barr1,2,
- Fan Gao1,
- Debra E. Bramblett3,
- David L. Paul4 and
- Samuel M. Wu1,2
- Departments of 1Ophthalmology and 2Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA3Biology Department, University of St Thomas, Houston, TX 77006, USA4Department of Neurobiology, Harvard Medical School, Boston, MA 02135, USA
- Corresponding author S. M. Wu: Cullen Eye Institute, Baylor College of Medicine, One Baylor Plaza, NC-205, Houston, TX 77030, USA. Email: swu{at}bcm.tmc.edu
Abstract
AII amacrine cells (AIIACs) are crucial relay stations for rod-mediated signals in the mammalian retina and they receive synaptic inputs from depolarizing and hyperpolarizing bipolar cells (DBCs and HBCs) as well as from other amacrine cells. Using whole-cell voltage-clamp technique in conjunction with pharmacological tools, we found that the light-evoked current response of AIIACs in the mouse retina is almost completely mediated by two DBC synaptic inputs: a 6,7-dinitro-quinoxaline-2,3-dione (DNQX)-resistant component mediated by cone DBCs (DBCCs) through an electrical synapse, and a DNQX-sensitive component mediated by rod DBCs (DBCRs). This scheme is supported by AIIAC current responses recorded from two knockout mice. The dynamic range of the AIIAC light response in the Bhlhb4−/− mouse (which lacks DBCRs) resembles that of the DNQX-resistant component, and that of the connexin36 (Cx36)−/− mouse resembles the DNQX-sensitive component. By comparing the light responses of the DBCCs with the DNQX-resistant AIIAC component, and light responses of the DBCRs with the DNQX-sensitive AIIAC component, we obtained the input–output relations of the DBCC→AIIAC electrical synapse and the DBCR→AIIAC chemical synapse. Similar to other glutamatergic chemical synapses in the retina, the DBCR→AIIAC synapse is non-linear. Its highest voltage gain (approximately 5) is found near the dark membrane potential, and it saturates for presynaptic signals larger than 5.5 mV. The DBCC→AIIAC electrical synapse is approximately linear (voltage gain of 0.92), consistent with the linear junctional conductance found in retinal electrical synapses. Moreover, relative DBCR and DBCC contributions to the AIIAC response at various light intensity levels are determined.
One of the most striking features of the visual system is its ability to encode light images of an enormous range of intensities (Hecht et al. 1942; Dowling, 1987). Photoreceptors in the retina partition this task into two ranges: rods encode dim images and cones encode bright images. Rod and cone signals are transmitted in parallel pathways to the inner retina via rod and cone bipolar cells. In mammals, several types of depolarizing and hyperpolarizing cone bipolar cells (DBCCs and HBCCs) have been identified, but only depolarizing rod bipolar cells (DBCRs) exist (Kolb & Famiglietti, 1974; Wassle et al. 1991; Kolb, 1994). DBCCs and HBCCs make direct synapses on ganglion cells (GCs) whereas DBCRs send signals to GCs indirectly through the AII amacrine cells (AIIACs): AIIACs receive chemical synaptic inputs from DBCRs, then relay the DBCR signal to DBCCs via gap junctions, and subsequently transmit the signal to the on-centre GCs through the DBCC–onGC synapses (Famiglietti. & Kolb, 1976; Nelson, 1982; Strettoi et al. 1990; Strettoi et al. 1992). Moreover, AIIACs make glycinergic chemical synapses on HBCC axon terminals (Bolz et al. 1984) and off-centre GCs (Kolb & Famiglietti, 1974), resulting in rod-mediated hyperpolarizing signals in offGCs. Therefore the AIIAC is a crucial relay station for rod-mediated signals in the mammalian retina.
Electrophysiological studies have shown that AIIACs give rise to depolarizing responses to light stimuli with much wider dynamic range (5–6 log units) than rods and DBCRs (Xin & Bloomfield, 1999; Pang et al. 2004), suggesting that these cells receive not only rod-mediated signals but also cone inputs. While rod input to AIIACs is carried mainly by the DBCR–AIIAC chemical synapse, cone signals can also be transmitted to the AIIACs through the DBCC–AIIAC electrical synapse (Strettoi et al. 1992; Tsukamoto et al. 2001; Veruki & Hartveit, 2002b), although how well this electrical synapse functions in the DBCC→AIIAC direction continues to be studied (Xin & Bloomfield, 1999; Trexler et al. 2001). Therefore, in addition to its role as a relay station for rod signals to ganglion cells, AIIAC may integrate rod- and cone-mediated signals passing through a chemical and an electrical synapse for providing wide-range output signals to GCs (Pang et al. 2003). It is important to determine synaptic properties and the relative contributions of these two inputs to AIIAC light responses.
In this report, we study DBCR→AIIAC and the DBCC→AIIAC synaptic inputs to dark-adapted mouse AIIACs in two ways. We first used pharmacological tools to separate the DBCC from the DBCR inputs, and then studied AIIAC light responses in two strains of knockout mice, the connexin36−/− (Cx36−/−), which lacks the DBCC→AIIAC electrical synapses (Deans et al. 2002) and the Bhlhb4−/−, which lacks DBCRs (Bramblett et al. 2004). The two approaches give remarkably similar results on the DBCR and the DBCC synaptic inputs to AIIACs. By comparing the light responses of the DBCRs and the DBCCs (from our previous publication) with the AIIAC responses, we obtained the input–output relations and voltage gains of the DBCR→AIIAC and the DBCC→AIIAC synapses.
Methods
Preparations and light stimulation
The wild-type (WT) mouse used in this study was C57Black6J from Jackson laboratory (Bar Harbour, ME, USA). Generation of the Bhlhb4−/− and Cx36−/− mice was described in previous publications (Deans et al. 2002; Bramblett et al. 2004). All animals were handled in accordance with Baylor College of Medicine's policies on the treatment of laboratory animals. Mice were dark-adapted for 1–2 h prior to the experiment. To maintain the retina in the fully dark-adapted state, all further procedures were performed under infrared illumination with dual-unit Nitemare (BE Meyers, Redmond, WA, USA) infrared scopes. Animals were killed by a lethal injection of Ketamine + xylazine + acepromazine (0.1 ml, 100 mg ml−1) and the eyes were immediately enucleated and placed in oxygenated Ames' medium (Sigma, St Louis, MO, USA) at room temperature. Dissection and preparation of living retinal slices followed essentially the procedures described in previous publications (Werblin, 1978; Wu, 1987). Oxygenated Ames solution (adjusted at pH 7.3) was introduced continuously to the recording chamber by a gravity superfusion system, and the medium was maintained at 34°C by a temperature control unit (TC 324B; Warner Instruments, CT, USA). All pharmacological agents were dissolved in Ames' medium with a superfusion time of 45–120 s.
A photostimulator was used to deliver light spots (of diameter 600–1200 μm) to the retina via the epi-illuminator of the microscope.
The intensity of unattenuated (log I
= 0) 500 nm light was 1.4 × 106 photons μm−2 s−1. The number of photoisomerizations per rod per second (Rh*rod−1 s−1) was calculated from a rod cross section of 0.5 μm−2 (Howes et al. 2002), and a rod integration time of 0.4 s (Baylor, 1987). The peak amplitude of light-evoked current responses was plotted against light stimulus intensity, and data points were
fitted to the Hill equation (Naka & Rushton, 1966):
(1)where R is the current response amplitude, Rmax is the maximum response amplitude, σ is the light intensity that elicits a half-maximal response, N is the Hill coefficient, tanh is the hyperbolic tangent function, and log is the logarithmic function of base 10. In this
study, we used the R
– log I plot for our analysis (the right-hand term of the above equation), and for such plots the light intensity span (dynamic range
(DR): range of intensity that elicits responses between 5 and 95% of Rmax) of a cell equals to 2.56/N (Thibos & Werblin, 1978). We define response threshold as the intensity of light that elicits 5% of Rmax.
Voltage-clamp recordings
Voltage-clamp recordings were made with an Axopatch 200A amplifier connected to a DigiData 1200 interface and pClamp 6.1 software (Axon Instruments, Union City, CA, USA). Whole-cell voltage-clamp recordings were made with patch electrodes made with Narishige or Sutter patch electrode pullers that were of 5–7 MΩ tip resistance when filled with internal solution containing 118 mm Cs methanesulphonate, 12 mm CsCl, 5 mm EGTA, 0.5 mm CaCl2, 4 mm ATP, 0.3 mm GTP, 10 mm Tris, 0.8 mm Lucifer yellow, adjusted to pH 7.2 with CsOH. The chloride equilibrium potential (ECl) with this internal solution was about −60 mV. Estimates of the liquid junction potential at the tip of the patch electrode prior to seal formation varied from −9.2 to −9.6 mV (Pang et al. 2002). For simplicity, we corrected all holding potentials by 10 mV.
Visualization of cell morphology
Cell morphology was visualized in retinal slices through the use of Lucifer yellow fluorescence with a confocal microscope (Zeiss 510). Images were acquired with a ×40 water immersion objective (NA = 1.20), using the 458 nm excitation line of an argon laser, and a long pass 505 nm emission filter. Consecutive optical sections were superimposed to form a single image using the Zeiss LSM-PC software, and these compressed image stacks were further processed in Adobe Photoshop 6.0 to improve the signal-to-noise ratio. Since signal intensity values were typically enhanced during processing to improve visibility of smaller processes, the cell bodies and larger processes of some cells appear saturated due to their larger volume of fluorophore. The level at which dendritic processes stratified in the IPL was characterized in retinal vertical sections by the distance from the processes to the distal margin (0%) of the IPL. For immunostaining (connexin 36 and protein kinase C (PKC) staining of the rod bipolar cells), retinas were fixed in fresh 4% paraformaldehyde with 0.05% gluderaldehyde for 15 min, transferred to 4% paraformaldehyde for another 2 h, and then incubated with primary (mouse monoclonal antibody against Cx35/36 from Chemicon, and rabbit anti-PKCα from Oxford Biochemical Research) and secondary antibodies (donkey antimouse IgG from Molecular Probes).
Electroretinograms.
Before testing, mice were allowed to adapt to the dark overnight. Under dim red light, mice were anaesthetized with an intraperitoneal injection of a solution of ketamine (95 mg ml−1) and xylazine (5 mg ml−1). The pupils were dilated with a single drop of 1% tropicamide and 2.5% phenylephrine. A drop of 0.5% proparacaine hydrochloride was applied for corneal anaesthesia. Mice were placed on a heating pad, maintained at 39°C, inside a Ganzfeld dome coated with highly reflective white paint (Munsell Paint, New Windsor, NY, USA). A small amount of 2.5% methylcellulose gel was applied to the eye, and a platinum electrode was placed in contact with the centre of the cornea. Similar platinum reference and ground electrodes were placed in the forehead and tail, respectively. After placement in the dome, mice were allowed to remain in complete darkness for 5 min. Signals were amplified with a Grass P122 amplifier (bandpass 0.1–1000 Hz; Grass Instruments, West Warwick, RI, USA). Data were acquired with a National Instruments Laboratory personal computer Data Acquisition board (sampling rate 10 000 Hz; National Instruments, Austin, TX, USA). Traces were averaged and analysed with custom software written in Matlab (Mathworks, Natick, MA, USA).
Flashes were calibrated in a manner similar to that described by Lyubarsky & Pugh (1996). Our protocol has been described in detail previously (Pennesi et al. 2003b). Flashes for scotopic measurements were generated by a Grass PS-33 photostimulator. Light was spectrally filtered with a 500 nm interference filter (Edmund Industrial Optics, Barrington, NJ, USA). A series of metal plates with holes of varying diameters and glass neutral density filters were used to attenuate the flash. As the intensity of the flash increased, the number of trials was decreased and the time between each flash was increased. To remove oscillatory potentials before fitting, the scotopic b-wave was digitally filtered using the filtfilt function in Matlab (low-pass filter; Fc = 60 Hz). At higher intensities, where the a-wave became significant, the b-wave amplitude was defined as the amplitude of the trough of the unfiltered a wave amplitude to the peak of the filtered b-wave amplitude. The relationships between b-wave amplitude and flash intensity were fit by eqn (1) and plotted as continuous lines.
Results
Light responses of AIIACs are mediated by two ON bipolar cell inputs: a DNQX-sensitive (rod DBC) and a DNQX-resistant (cone DBC) component
Light-evoked current responses of AIIACs were recorded under voltage-clamp conditions in living retinal slices of the wildtype mouse. AIIACs were identified by their characteristic morphology (thick globular dendrites in the distal half and pyramidally branching dendrites in the proximal half of the IPL, and with dendritic width less than 30 μm; Tsukamoto et al. 2001). Figure 1A shows an AIIAC in a retinal slice filled with Lucifer yellow, and Fig. 1B displays the light-evoked current responses at various holding potentials. In darkness, before the light stimulus, the cell exhibited inward spontaneous excitatory postsynaptic currents (sEPSCs) at negative holding potentials and outward inhibitory postsynaptic currents (sIPSCs) above −60 mV (ECl). A 2.5 s, 500 nm, −3 (700 Rh*rod−1 s−1) light step elicited inward current responses (ΔI, with a transient onset and a offset rebound) at all holding potentials, typical in AIIACs as reported in a previous study (the lack of light response reversal is probably caused by extensive coupling among AIIACs and between AIIACs and DBCCs (Strettoi et al. 1992; Veruki & Hartveit, 2002a,b) so that the inward currents from unclamped neighbouring cells dominate the cell's response at all holding potentials (Pang et al. 2004)). When 20 μml-2-amino-4-phosphonobutyric acid (l-AP4) was added to the bath (Fig. 1C), sEPSCs and sIPSCs were largely suppressed and the light response was almost completely abolished (except for a small transient current at the light step offset, possibly mediated by inputs from hyperpolarizing bipolar cells (Tsukamoto et al. 2001)). These l-AP4 actions were reversible as shown by wash 1 in Fig. 2D. Since l-AP4 selectively blocks light responses of DBCs (Slaughter & Miller, 1981), our interpretation of this result is that the light response (ΔI) as well as the majority of the sPSCs in mouse AIIACs are predominately mediated by DBC-mediated inputs.
Anatomical evidence has suggested that AIIACs in mammalian retina receive chemical synaptic inputs from DBCRs (presumably glutamatergic) (Morkve et al. 2002; Trexler et al. 2005), and are connected with DBCCs via electrical synapses (Veruki & Hartveit, 2002b). In order to determine whether and how much these two DBC inputs contribute to AIIAC light responses, we examined the effects of 6,7-dinitro-quinoxaline-2,3-dione (DNQX), an AMPA/kainate glutamate receptor antagonist (Gao & Wu, 1999), on AIIAC light responses (Fig. 1E). DNQX (100 μm) completely blocked all sEPSCs and the vast majority of sIPSCs. It also reduced and slowed down ΔI onset and completely suppressed ΔI offset rebound. We also tried higher concentrations of DNQX (200–500 μm) and ΔI and the sPSCs were not further reduced (but with longer wash time for recovery), suggesting that 100 μm DNQX elicited the maximum effect. These DNQX actions were reversible as shown by wash 2 in Fig. 1F. These data demonstrate that AIIAC light responses consist of two components: one is DNQX-resistant (Fig. 1E) and the other is DNQX-sensitive (control − DNQX see below). (Note, in several AIIACs, we added 100 μm carbenoxolone in the presence of DNQX and the DNQX-resistant responses were largely abolished. But we were unable to reverse the carbenoxolone action after prolonged wash, and thus we are uncertain whether carbenoxolone abolished the DNQX-resistant responses by blocking the gap junctions between DBCCs and AIIACs (Trexler et al. 2005), or the cells were damaged by this compound. For this reason, we do not show the carbenoxolone experiments in Fig. 1). The current–voltage relations of the light-evoked responses under control conditions, in 20 μml-AP4, and in 100 μm DNQX are plotted in Fig. 1G. Results similar to Fig. 1A–G were obtained from all AIIACs examined (n = 10), and are consistent with the notion that two parallel sources of DBC inputs, one glutamatergic (presumably DBCR) and the other not (presumably DBCC), are responsible for mediating AIIAC light responses in the mouse retina.
We next studied the response dynamic range of the DNQX-sensitive and DNQX-resistant components of the AIIAC light-evoked currents. Figure 2 shows the current responses of an AIIAC to a 2.5 s light steps of increasing intensities (−7.5 to −3.5, or 0.022–221 Rh*rod−1 s−1) at holding potential −60 mV in normal medium (control, Fig. 2A) and in 100 μm DNQX (Fig. 2B). Figure 2C displays the difference responses (control − DNQX). We perform these experiments on eight AIIACs and the average (±s.d.) response–intensity (ΔI − log I) of the current responses in control, DNQX and control − DNQX are illustrated in Fig. 2D. The curves were fitted by eqn (1) in Methods. The response thresholds and dynamic ranges (defined in Methods) of the control response (dash–dot curve) are −8.7 (0.0014 Rh*rod−1 s−1) and 3.97 log units, respectively. The response thresholds and dynamic ranges of the DNQX-sensitive component (control − DNQX, continuous curve) are −8.5 (0.0022 Rh*rod−1 s−1) and 2.56 log units, and the corresponding values of the DNQX-resistant component (DNQX, dashed curve) are −8.7 (0.0014 Rh*rod−1 s−1) and 4.27 log units. Additionally, the DNQX-resistant component has slower time-to-peak than the control at all light intensities, which is why the difference between the peak control responses and the peak responses in DNQX in Fig. 2D does not equal to the peak control − DNQX responses.
In Fig. 2E we plot the ratio of the postsynaptic response of the DBCR inputs (control AIIAC response − AIIAC response in DNQX, continuous curve in Fig. 2D) and that of the DBCC inputs (AIIAC response in DNQX, dashed curve in Fig. 2D). This gives the relative contribution of the DBCR and DBCC inputs to AIIAC responses at various light intensities. For dim lights (I < −9), DBCC contribution appears to be larger than DBCR, but as light intensity increases, DBCR contribution takes over, giving a peak ratio of 2.7 near −7, and then it decreases again with a ratio of nearly one at −4 and −3.
DBCR and DBCC responses in Cx36−/− and Bhlhb4−/− mice
Results from the last section suggest that two parallel DBC inputs, one DNQX-sensitive and the other DNQX-resistant, mediate AIIAC light responses. Since we cannot rule out contributions of some DBCC-mediated glutamatergic synaptic circuits, such as the DBCC–AC–AIIAC pathway, to the AIIAC light response, we are not certain that the DNQX-sensitive input is mediated only by the DBCR–AIIAC synapse. Moreover, since there is no specific and reversible gap junction blocker available (e.g. carbenoxolone, see note in last section), it is uncertain that the DNQX-resistant input is mediated by the DBCC–AIIAC electrical synapse. In order to independently verify that DNQX-sensitive component is only mediated by DBCRs and the DNQX-resistant component is mediated by DBCCs, we used two strains of knockout mouse: Bhlhb4−/− and Cx36−/− mice (Deans et al. 2002; Bramblett et al. 2004). Figure 3 shows vertical retinal sections of WT (top), Cx36−/− (middle) and Bhlhb4−/− (bottom) mice immunostained with antibodies against Cx36 (left) and PKCa (right). It has been shown that PKC labels DBCRs in the mouse retina (Pennesi et al. 2003a), and Cx36 mediates photoreceptor coupling in the outer retina and AII–AII and AII–DBCC coupling in the inner retina (Deans et al. 2002). We found that DBCRs in the Cx36−/− mice are of the same morphology and density (Fig. 3Bb) as in the WT (Fig. 3Ba) whereas DBCRs in the Bhlhb4−/− mice completely disappear (Fig. 3Bc). On the other hand, the Cx36 antibody gives the same staining pattern in the Bhlhb4−/− mice (Fig. 3Ac) as in the WT, and it does not stain the Cx36−/− mouse retina (Fig. 3Ab). These results are consistent with the notion that the Cx36−/− mice lack Cx36, but with DBCRs of normal morphology and density, and that the Bhlhb4−/− mice lack DBCRs, but have normal distribution of Cx36.
In order to use these two knockout mice to separate the DBCR and DBCC inputs to AII amacrine cells, it is necessary to examine whether DBCRs in the Cx36 mice and DBCCs in the Bhlhb4 mice maintain normal light responses. We employed the electroretinogram method because it allows measurement of the eye's overall DBCR and DBCC light responses (ERG b-wave) and avoids the cell sampling problem. Figure 4A shows scotopic ERG b-wave responses to 500 nm light of increasing intensities recorded from WT, Cx36−/−and Bhlhb4−/−mice. Figure 4B shows the average response–intensity plots of the scotopic b-waves of the three types of mice over the intensity range 0.1–1000 R*rod−1 s−1. The Cx36−/− mice exhibit b-wave with very similar average amplitude as the WT, suggesting that the DBCRs and the DBCCs operating in this intensity range (DBCC1) (Pang et al. 2004) in these knockout mice maintain normal light responses. One the other hand, the Bhlhb4−/− mice give rise to much smaller scotopic b-wave, consistent with that DBCRs in these mice are absent (Bramblett et al. 2004). The average residual scotopic b-wave in Bhlhb4−/− mice is about 85 μV (significantly (P < 0.001) above zero) within the intensity range between 0.1 and 100 R*rod−1 s−1, and it increases at higher intensities within the cone operating range. This suggests that in addition to the DBCRs (which are absent in Bhlhb4−/− mice), some DBCCs (DBCC1) operate in this intensity range and thus contribute to the scotopic b-wave.
We then modelled the DBCR contribution to the scotopic b-wave by using the response–intensity curves of the PII light responses obtained previously (Saszik et al. 2002), and shown as the blue curve in Fig. 4B. To take into account absolute differences in ERG recordings between laboratories, we allowed the maximum amplitude of the PII component to be scaled by a constant factor, but maintained the sensitivity of this component. Subtracting the WT b-wave from the scaled DBCR contribution of the scotopic b-wave (blue curve), and calculating the scaling factor in a least-square method, we found that the difference fits the average Bhlhb4 b-wave amplitudes in the entire intensity range. This result suggests that DBCCs (scotopic b-wave) in the Bhlhb4 mice exhibit similar light response dynamic range and amplitude as the WT DBCCs (WT scotopic b-wave − WT DBCR b-wave).
AIIAC light response in Cx36−/− mice resembles the DNQX-sensitive component, and the AII light response in the Bhlhb4−/− mice resembles the DNQX-resistant component
We have shown that the DBCR and DBCC responses in Cx36−/− and DBCC responses in Bhlhb4 mice are normal under scotopic conditions. This allows us to use the two knockout mice to separate the DBCR and DBCC inputs to AIIACs. In Cx36−/− mice DBCC inputs to AIIACs (through Cx36-mediated gap junction) are curtailed where DBCRs are normal, and in the Bhlhb4−/− mice DBCRs are absent where DBCCs are normal. Figure 5 shows an AIIAC in a Cx36−/− retinal slice (Fig. 5Aa) and an AIIAC in a Bhlhb4−/− retinal slice (Fig. 5Ba) filled with Lucifer yellow, the light-evoked current responses at various holding potentials (Fig. 5Ab and Bb), and the current-voltage relations (Fig. 5Ac and Bc). The morphology of the AIIACs in these two knockout mice is very similar to the AIIAC in the WT, but the light responses are generally smaller. One noticeable difference is that the light-evoked current in Cx36−/− mice reverses near 0–10 mV (Fig. 5Ab and c), whereas that in the WT and Bhlhb4−/− mice never reverses (Figs 1B, D, F and G, and 5Bb). This is consistent with the notion that the lack of light response reversal in AIIACs is caused by extensive coupling among AIIACs and between AIIACs and DBCCs (Veruki & Hartveit, 2002a,b), and thus when coupling is deleted in the Cx36−/− mice, the reversal potential becomes observable.
In order to obtain independent support for the idea that the DNQX-sensitive component of the WT AIIAC light response is mediated by the DBCR input, and the DNQX-resistant component is mediated by the DBCC input, we compared the response dynamic ranges of AIIACs in Cx36−/− and Bhlhb4−/− mice with the data shown in Fig. 2. By using the same protocol as in Fig. 2, we recorded the current responses of eight AIIACs in the Bhlhb4−/− mice (an example is given in Fig. 6A) and nine AIIACs in Cx36−/− (an example is given in Fig. 6B) mice. The average (±s.d.) responses of the Cx36−/− AIIACs (shown as filled circle data points and thick continuous curve normalized against the maximum average response amplitude of the DNQX-sensitive component of the WT AIIAC in Fig. 2D) agree remarkably well with the dynamic range of DNQX-sensitive component of the WT AIIACs (with an average response threshold and dynamic range of −8.7 (0.0014 R*rod−1 s−1) and 2.87, thin continuous curve). The average (±s.d.) responses of the Bhlhb4−/− AIIACs (shown as filled diamond data points and thick dashed curve normalized against the maximum average response amplitude of the DNQX-resistant component of he WT AIIAC in Fig. 2D) match very well with the dynamic range of the DNQX-resistant component of the WT AIIACs (with an average response threshold and dynamic range of −8.5 (0.0022 R*rod−1 s−1) and 4.19, thin dashed curve). Moreover, the rise time of the AIIAC responses in Bhlhb4−/− mice (Fig. 6A) is slower than that in the Cx36−/− mice (Fig. 6B), similar to the slower rise time of the WT AIIAC response in DNQX (Fig. 2B). These results support the idea that the DNQX-sensitive component of the WT AIIACs is mediated by the DBCR input, and that the DNQX-resistant component of the WT AIIACs is mediated by the DBCC input.
Input–output relations of the DBCR→AIIAC and the DBCC→AIIAC synapses and the relative contributions of the DBCR and DBCC inputs to AIIAC light responses at various light intensities
In the last section, results obtained from the Cx36−/− and Bhlhb4−/− mice support the notion that the DNQX-sensitive component of the AIIAC light response in the WT mouse is mediated by the DBCR inputs, and the DNQX-resistant component is mediated by the DBCC input. In a previous publication, we obtained the average response–intensity relations of the DBCRs and DBCC1s (the DBCCs that are coupled with AIIACs (Pang et al. 2004)). By plotting the average DBCR response versus the average DNQX-sensitive AIIAC response (red curve in Fig. 2D), we obtain the input–output relation of the DBCR–AIIAC synapse (Fig. 7A, continuous curve). Similarly, plotting the average DBCC responses against the average DNQX-resistant AIIAC responses yields the input–output relation of the DBCC→AIIAC electrical synapse (Fig. 7A, dashed curve). These input–output relations are current responses of the postsynaptic/presynaptic cells, not the traditional voltage/voltage plots (Attwell et al. 1987), which better describe the synaptic behaviour of the DBC inputs to AIIACs under physiological conditions. Since the input resistance of the DBCs and AIIACs in the mouse retina are approximately linear near the resting potential (Pang et al. 2004), the light-evoked voltage responses can be estimated by multiplying the current responses by the input resistance (R) of the DBCRs, DBCCs and AIIACs. From our own unpublished results from the mouse and results by other investigators from the rodent retinas (Veruki & Hartveit, 2002a,b), the average RDBCR is 1.1 GΩ, RDBCC is 1.0 GΩ and RAIIAC is 0.4 GΩ. By using these values, the voltage input–output relations of the DBCR→AIIAC and DBCC→AIIAC were obtained (Fig. 7B). From Fig. 7A and B, it is evident that the DBCR–AIIAC synapse is non-linear with the highest gain near the DBCR dark membrane potential (with a current gain of 14 and voltage gain of 5) and the gain decreases progressively when the response becomes larger. The synapse saturates when the DBCR current response reaches about 5 pA (about 5.5 mV DBCR voltage response). These synaptic characteristics are very similar to those of other glutamatergic synapses in the retina (Attwell et al. 1987; Yang & Wu, 1993). The input–output relation of the DBCC→AIIAC electrical synapse, on the other hand, is approximately linear over the DBCC response span with a current gain of 2.3 and a voltage gain of 0.92 for small DBCC responses, and it reduces to a current gain of 1.6 and voltage gain of 0.64 for large DBCC responses. The near linear input–output relation of this electrical synapse is consistent with a DBCC–AIIAC gap junction that is ohmic (Veruki & Hartveit, 2002b).
Discussion
AIIAC light responses contain two components
Our results imply that the light-evoked current response of AIIACs in the mouse retina is mediated primarily by two parallel DBC inputs. The DBCR input is mediated by a DNQX-sensitive glutamatergic synapse and a DBCC input is mediated by a DNQX-resistant electrical synapse. This scheme is supported by the AIIAC responses recorded from the Cx36−/− and Bhlhb4−/− mice: the response threshold, kinetics and dynamic range of DNQX-sensitive DBCR input resemble the AIIAC response in the Cx36−/− mice in which the electrical synapse between DBCCs and AIIACs is absent, and response threshold, kinetics and dynamic range of DNQX-resistant DBCC input resemble the AIIAC response in the Bhlhb4−/−mice in which the DBCRs are absent. This is also consistent with a report which found that AIIAC response in the rabbit retina was reduced in the presence of NBQX (similar to DNQX) and the residual response was abolished in carbenoxolone (Trexler et al. 2005). However, our data differ from another study of the rabbit AIIACs, in which DNQX was found to increase the depolarizing light response (Bloomfield & Xin, 2000). In a recent study, mouse AIIAC responses to brief light flashes (10 ms) exhibited only a DBCR-mediated component (Dunn et al. 2006). This is inconsistent with the anatomical and physiological data reported previously (Famiglietti. & Kolb, 1975; Xin & Bloomfield, 1999; Tsukamoto et al. 2001) and the results presented in this study.
In Fig. 1, we show that most part of the AIIAC light response as well as the spontaneous postsynaptic currents (sPSCs) are suppressed in the presence of 20 μml-AP4, suggesting that the vast majority of the synaptic inputs to AIIACs are mediated by DBCs (as l-AP4 selectively blocks DBC light responses (Slaughter & Miller, 1981)). The residual response in l-AP4 is a small transient current at light offset (Fig. 1C), which is probably mediated by the HBC–AIIAC synapses in IPL sublamina as described in anatomical studies (Strettoi et al. 1992; Tsukamoto et al. 2001). Additionally, sPSCs of much reduced frequency are also observed in the presence of l-AP4, and the majority of them appeared to reverse near −60 mV, suggesting that they are sIPSCs, possibly from GABAergic/glycinergic amacrine cells (Chun & Wassle, 1989; Wassle et al. 1998). Anatomical studies reveal that the AIIAC receives inhibitory synaptic inputs from A17 amacrine cells (Kolb & Nelson, 1981; Nelson & Kolb, 1985). In the experiments analysing DBCR and DBCC contributions to AIIACs (Figs 2 and 6), we held the AIIAC membrane potential at −60 mV (near ECl, and near the dark potential of the adjacent unclamped, and electrically coupled AIIACs; Pang et al. 2004), and thus the contribution of inhibitory synaptic inputs from A17 and other amacine cells to AIIAC light responses was negligible.
Properties of the DBCR–AIIAC glutamatergic synapse
The input–output relations in Fig. 7 suggest that the DBCR–AIIAC synapse is non-linear, with the highest current gain of 14 and voltage gain of 5 for the small DBCR signals, and the synapse saturates when the DBCR response reaches about 5.5 mV. This resembles the input–output relations of several glutamatergic synapses in the retina (Attwell et al. 1987; Belgum & Copenhagen, 1988; Yang & Wu, 1993), and the narrow synaptic operating range probably reflects the narrow activation voltage window of the presynaptic Ca2+ current (Bader et al. 1982; Singer & Diamond, 2003).
The DBCR input to AIIACs has an average response threshold of −8.5 (0.0022 Rh*rod−1 s−1, Fig. 2), 1.7 log units (50 times) lower than that of the presynaptic cells (the average threshold of the DBCRs is −6.8 (0.11 Rh*rod−1 s−1); Pang et al. 2004). This means that a −8.5 light step (which on average generates a threshold response in one out of 50 DBCRs) is enough to generate a threshold response in AIIACs. Since on average each AIIAC receives synaptic inputs from only 3.5 DBCRs (Tsukamoto et al. 2001), the remaining DBCR inputs are mediated by DBCR inputs to adjacent AIIACs via AIIAC–AIIAC coupling (Veruki & Hartveit, 2002a). This is qualitatively consistent with our results from Cx36−/− mice in which the AIIAC–AIIAC coupling is absent, which show an average threshold lower than the WT DNQX-sensitive AIIAC response. Detailed models on DBC–AIIAC synaptic convergence and the AIIAC coupling network are needed for quantitative characterizations of DBCR and AIIAC response thresholds and signal detection.
Properties of the DBCC–AIIAC electrical synapse
The input–output relations of the DBCC–AIIAC synapse are near linear with a voltage gain of 0.92 for small DBCC signals and the gain decreased to 0.64 for large DBCC signals. This is consistent with the near ohmic junctional conductance of the electrical synapse between AIIACs and DBCCs in the rat retina (Veruki & Hartveit, 2002b). It has also been shown that the current flowing through the electrical synapse can be calculated by IJ= IDBC/[RDBC/(RAII × CC) + 1] = 0.11IDBC (Trexler et al. 2005), where IJ is the current from a DBCC to an AIIAC through the gap junction, IDBC is the light-induced current in the DBCC, RDBC and RAII are input resistance of the DBCC and the AIIAC (1.0 and 0.4 GΩ, respectively; Veruki & Hartveit, 2002a,b), and CC is the coupling coefficient (ratio of the voltage changes of the two coupled cells) of the DBCC→AIIAC electrical synapse (0.31; Veruki & Hartveit, 2002b). Since the current gain of the DBCC→AIIAC electrical synapse is about 2.3 (Fig. 7), there should be about 21 (2.3/0.11) DBCCs sending light-evoked current to an AIIAC.
The DBCC input to AIIACs has an average response threshold of −8.3 (0.0035 R*rod−1 s−1, Fig. 6C), 1.4 log units (25 times) lower than that of the presynaptic cells (the average threshold of the DBCCs is −6.9 (0.088 R*rod−1 s−1); Pang et al. 2004). Therefore, on average, a −8.3 light step (which generates a threshold response in one out of 25 DBCCs) is enough to generate a threshold response in the AIIAC. Since DBCCs–AIIACs and AIIAC–AIIACs are electrically coupled (Veruki & Hartveit, 2002a,b), this means that the coupled network is efficient enough to spread a threshold response to the AIIACs when one out of 25 DBCCs has a threshold response.
Light intensity-dependent DBCR and DBCC inputs to the AIIAC response
Figure 2E shows that the DBCR/DBCC input ratio in AIIACs has a peak value of 2.7 near −7 (0.07 R*rod−1 s−1), indicating that DBCR contribution to the AIIAC light response at this stimulus level is about three times stronger than the DBCC input. This is not surprising because the DBCR→AIIAC synapse at this intensity level has the highest voltage gain (about 5) whereas the DBCC→AIIAC synaptic voltage gain is only 0.92. At higher intensity levels, the DBCR→AIIAC voltage gain drops exponentially (Fig. 2E) whereas the DBCC→AIIAC synaptic gain stays almost constant. Therefore the DBCR/DBCC input ratio declines at these higher intensity levels. For light dimmer than −7, DBC signal convergence through AIIAC–AIIAC coupling plays a more important role in AIIAC light response, and the DBCR/DBCC contribution becomes more equal and thus the input ratio drops. This light-intensity dependence of DBCR/DBCC input ratio implies that AIIAC signal characteristics, such as response kinetics, discrete postsynaptic minis and signal/noise ratios, vary with levels of illumination.
The Cx36−/− and Bhlhb4−/− mice are useful models for analysing retinal circuitry
Our ERG results show that the scotopic b-wave of the Cx36−/− is the same as the WT mice for the entire DBCR dynamic range (−7 to −4 (0.7–70 R*rod−1 s−1); Pang et al. 2004), consistent with the ERG b-wave results from a previous Cx36−/− study (Robson et al. 2004). These results suggest that the DBCR response in this Cx36 knockout mouse is normal. By analysing the scotopic ERG b-wave of the Bhlhb4−/− mice, we found that the DBCC-mediated response is similar to its corresponding response in the WT mouse, although the scotopic b-wave amplitude is much lower, due to the absence of the DBCRs. We analysed the dark-adapted scotopic b-wave at intensity lower than −4 (70 R*rod−1 s−1), because this is the operation range of the AIIACs of the mouse retina (Pang et al. 2004). Therefore, the DBCC-mediated b-wave response is likely to be mediated by the high-sensitivity DBCCs (DBCC1; Pang et al. 2004). The similarities in dynamic ranges between the Cx36−/− AIIAC response and the DNQX-sensitive WT AIIAC response, and between the Bhlhlb4−/− AIIAC response and the DNQX-resistant WT AIIAC response suggest that the glutamatergic DBCR→AIIAC synapse in the Cx36−/− mice and the DBCC→AIIAC electrical synapse in the Bhlhb4−/− mice function normally.
It has been shown that neuronal organization and synaptic connections in many transgenic or knockout mice undergo rewiring (Marc et al. 2003). We are not certain how much rewiring occurs in the Cx36−/− and Bhlhb4−/− mice. It appears that at least at the age we used (2–3 months), the DBCs and the AIIACs are not much affected, although we cannot rule out some subtle changes that our experiments could not detect, or changes in synaptic pathways other than DBCs and AIIACs. Therefore, the Cx36−/− and Bhlhlb4−/− mice are useful animal models for studying the gap-junction and DBCR-specific synaptic pathways in the mammalian retina.
Results provided in this article suggest that light-evoked signals in AIIACs are mediated primarily by two bipolar cell input synapses: the DBCR→AIIAC glutamatergic chemical synapse and the DBCC→AIIAC connexin36-mediated electrical synapse. The DBCR→AIIAC synapse is non-linear with highest voltage gain (about 5) near the dark membrane potential that decreases as DBCR responses increase. The DBCC→AIIAC electrical synapse is approximately linear with a voltage gain of 0.92. These findings are largely consistent with previous anatomical and physiological data in the mammalian retina, and add to quantitative assessments on the relative contributions of the DBCR and DBCC inputs to AIIAC light responses. Additionally, by using pathway-specific knockout mice in conjunction with pharmacological blockers, our study provides direct physiological evidence illustrating how connexin36 and glutamate receptors mediate rod and cone signal convergence in third-order retinal neurons.
Acknowledgements
We thank Drs Robert Smith and Roy Jacoby for critically reading this manuscript. This work was supported by grants from NIH (EY 04446), NIH Vision Core (EY 02520), the Retina Research Foundation (Houston), the International Retinal Research Foundation, Inc., and Research to Prevent Blindness, Inc.
Footnotes
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(Received 8 September 2006; accepted after revision 22 January 2007; first published online 25 January 2007)
- 2007 The Authors. Journal compilation © 2007 The Physiological Society
References
Figure 1. Effects of AP4 and DNQX on AII amacrine cells A, an AII amacrine cell (AIIAC) in a retinal slice filled with Lucifer yellow. The smaller cell above the AIIAC (arrow) is a dye-coupled depolarizing cone bipolar cell (DBCC) soma. INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. B, light-evoked current responses the AIIAC to a −3 (700 R*rod−1 s−1) 2.5 s light step at various holding potentials. C, light-evoked current responses the AIIAC to the same light step (as in B) at various holding potentials in the presence of 20 μml-2-amino-4-phosphonobutyric acid (l-AP4). D, wash 1 shows that l-AP4 actions were reversible. E, light-evoked current responses the AIIAC to the same light step (as in B) at various holding potentials in the presence of 100 μm DNQX. F, wash 2 shows DNQX actions were reversible. G, current–voltage relations of the light-evoked responses under control conditions (○), in 20 μml-AP4 (•), and in 100 μm DNQX (▴).
Figure 2. DNQX-sensitive and DNQX-resistant components of AIIAC responses Current responses of an AIIAC to 2.5 s light steps of increasing intensities (−7.5 to −3.5, or 0.022–221 R*rod−1 s−1) at holding potential −60 mV in control medium (A), and in 100 μm DNQX (B). C, the difference responses (control − DNQX) were obtained by subtracting responses in B from responses in A at each intensity. D, average (±s.d.n = 8) response-intensity (ΔI − log I) of the current responses in control medium, in DNQX and control − DNQX. Curves were fitted by eqn (1) in Methods. The response thresholds and dynamic ranges of the control response (dash–dot curve) are −8.7 (0.0014 R*rod−1 s−1) and 3.97, respectively, for the DNQX-sensitive component (control − DNQX, continuous curve) they are −8.5 (0.0022 R*rod−1 s−1) and 2.56, and for the DNQX-resistant component (DNQX, dashed curve) they are −8.7 (0.0014 R*rod−1 s−1) and 4.27. E, ratio of the postsynaptic response of the depolarizing rod bipolar cell (DBCR) inputs (control AIIAC response − AIIAC response in DNQX, continuous curve in D) and that of the DBCC inputs (AIIAC response in DNQX, dashed curve in D) at various light intensities.
Figure 3. Immunostainings of mouse retinas with Cx36 and PKCa antibodies Vertical retinal sections of WT (a), Cx36−/− (b) and Bhlhb4−/− (c) mice immunostained with antibodies against Cx36 (A) and protein kinase C (PKC) (B). OPL, outer plexiform layer; IPL, inner plexiform layer; and GCL, ganglion cell layer.
Figure 4. Electroretinograms of wt.Cx36−/− and Bhlhb4−/− mice A, scotopic ERG b-wave responses to 500 nm light of increasing intensities (−6.3 to 2.6 log unit attenuation, or 0.35–1758 R*rod−1 s−1) recorded from WT (A), Cx36−/− (B) and Bhlhb4−/− (C) mice. B, average (±s.d.,n = 8) response–intensity plots of the scotopic b-waves of the three types of mice over the intensity span from −7 to −2 (500 nm, 0.07–7000 R*rod−1 s−1). WT, black; Cx36−/−, red; Bhlhb4−/−, green; WT PII, blue (Saszik et al. 2002); and difference b-wave (WT − WT PII), purple.
Figure 5. Light-evoked currents of AIIACs in Cx36−/− and Bhlhb4−/− mice Top, an AIIAC in a Cx36−/− retinal slice (Aa) and an AIIAC in a Bhlhb4−/− retinal slice (Ba) filled with Lucifer yellow. INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Middle, light-evoked current responses at various holding potentials of the AIIAC in Cx36−/− mouse (Ab) and in Bhlhb4−/− mouse (Bb). Bottom, current–voltage relations of the AIIAC in Cx36−/− mouse (Ac) and in Bhlhb4−/− mouse (Bc).
Figure 6. AIIAC response-intensity relations in Cx36−/− and Bhlhb4−/− mice Current responses of an AIIAC in the Bhlhb4−/− mouse retinal slice (A) and in the Cx36−/− mouse retinal slice (B) to 2.5 s light steps of increasing intensities (−7.5 to −3.5, or 0.022–221 R*rod−1 s−1) at holding potential −60 mV. The average (±s.d.) responses of nine Cx36−/− AIIACs (•, thick continuous curve (fitted by eqn (1)) normalized against the maximum average response amplitude of the DNQX-sensitive component of the WT AIIACs) with an average response threshold and dynamic range of −8.7 (0.0014 R*rod−1 s−1) and 2.87. The ΔI − log I of the DNQX-sensitive component of the WT AIIACs (from Fig. 2D) is shown as the thin dashed curve. The average (±s.d.) responses of eight Bhlhb4−/− AIIACs (♦, thick dashed curve (fitted by eqn (1)) normalized against the maximum average response amplitude of the DNQX-resistant component of the WT AIIACs) with an average response threshold and dynamic range of −8.5 (0.0022 R*rod−1 s−1) and 4.19. The ΔI − log I of the DNQX-resistant component of the WT AIIACs (from Fig. 2D) is shown as the thin dashed curve.
Figure 7. Input-out relations A, current input–output relation of the DBCR→AIIAC synapse (continuous curve) obtained by plotting the average DBCR response versus the average DNQX-sensitive AIIAC response (continuous curve in Fig. 2D), and current input–output relation of the DBCC→AIIAC synapse (dashed curve) obtained by plotting the average DBCC responses against the average DNQX-resistant AIIAC responses (dashed curve in Fig. 2D). B, voltage input–output relation of the DBCR→AIIAC synapse (dash–dot curve) and the DBCC→AIIAC synapse (dash–double-dot curve). The voltage responses were estimated by multiplying the current responses by the average input resistance (R) of the DBCRs, DBCCs and AIIACs (RDBCR, 1.1 GΩ; RDBCC, 1.0 GΩ; and RAIIAC, 0.4 GΩ).
