Genetic Dissection of Rod and Cone Pathways

45 A monumental task of the mammalian retina is to encode an enormous range (> 10 fold) 46 of light intensities experienced by the animal in the natural environment. Retinal neurons 47 carry out this task by dividing labor into many parallel rod and cone synaptic pathways. 48 Here we study the operational plan of various rod and cone mediated pathways by 49 analyzing electroretinograms (ERGs), primarily b-wave responses, in dark-adapted 50 wildtype, connexin36 knockout, depolarizing rod-bipolar cell (DBCR) knockout, and rod 51 transducin knockout mice (WT, Cx36(-/-), Bhlhb4(-/-) and Trα(-/-)). To provide 52 additional insight into the cellular origins of various components of the ERG, we 53 compare dark-adapted ERG responses with response dynamic ranges of individual retinal 54 cells recorded with patch electrodes from dark-adapted mouse retinas published from 55 other studies. 56 Our results suggest that the connexin36-mediated rod-cone coupling is weak 57 when light stimulation is weak and becomes stronger as light stimulation increases in 58 strength, and that rod signals may be transmitted to some DBCCs via direct chemical 59 synapses. Moreover, our analysis indicates that DBCR responses contribute about 80% of 60 the overall DBC response to scotopic light, and that rod and cone signals contribute 61 almost equally to the overall DBC responses when stimuli are strong enough to saturate 62 the rod bipolar cell response. Furthermore, our study demonstrates that analysis of ERG 63 b-wave of dark-adapted, pathway-specific mutants can be used as an in vivo tool for 64 dissecting rod and cone synaptic pathways and for studying the functions of pathway65 specific gene products in the retina. 66 67 Introduction 68 The mammalian retina must encode an enormous range (> 10 fold) of light intensity as 69 encountered in the natural environment, from few photons in starlit sky to over 10 70 photons reflected from sunlit snow (Rodieck, 1998). This is a mon.umental task, as there 71 are a limited number of photoreceptor subtypes and synaptic pathways in the retina, and 72 each retinal neuron has limited dynamic range for registering visual signals of various 73 strengths e.g. for a study in mouse see (Pang et al., 2004). The first-order visual neurons 74 are rod and cone photoreceptors. Rods, which are more sensitive than cones, encode dim 75 light but saturate at lower light levels, whereas cones have low light sensitivity and 76 increase in amplitude as stimulus strength is increased to much higher levels. Rod and 77 cone signals, which under certain conditions can spread to each other through gap 78 junctions, are transmitted to second-order retinal cells, the bipolar cells, via glutamatergic 79 chemical synapses as reviewed in (Dowling, 1987). In mammalian retinas, rod signals 80 are conveyed to the inner retina through at least three bipolar cell pathways. The first 81 (primary) rod pathway transmits hyperpolarizing rod response through a sign-inversing 82 synapse to the ON or depolarizing rod bipolar cells (DBCRs) (Kolb and Famiglietti, 1974) 83 and DBCRs send signal through an excitatory glutamate synapse to AII amacrine cells 84 (AIIACs). AIIACs signal to cone depolarizing bipolar cells (DBCCs) through a 85 heterodimeric electrical synapse, consisting of Cx36 on the AIIAC side and Cx45 on the 86 DBCC side (Maxeiner et al., 2005;Dedek et al., 2006). AIIACs also signal to cone OFF, 87 or hyperpolarizing bipolar cells (HBCCs) through a sign-inverting glycinergic synapse 88 (Crooks and Kolb, 1992). DBCCs and HBCCs send signals to ON and OFF ganglion 89 cells (ONGCs and OFFGCs), respectively (Kolb and Nelson, 1983). 90 In the second (secondary) rod pathway, rod signals spread to cones, through an 91 electrical synapse, at least partially mediated by Cx36 (Deans et al., 2002; Zhang and 92 Wu, 2005), and then cones convey these rod signals to DBCCs and HBCCs via 93 glutamatergic synapses. The third (tertiary) rod pathway initially described for 94 contacts between rods and HBCCs (Soucy et al., 1998; Tsukamoto et al., 2001) has more 95 recently been shown to also include synaptic contacts between rod photoreceptors and a 96 certain subset of DBCCs (Tsukamoto et al., 2007). 97 In addition to the three rod pathways, a fourth pathway for ON and OFF 98 responses, perhaps overlapping with the secondary rod pathway, is the cone bipolar cell 99 pathway, which conveys hyperpolarizing cone responses to DBCCs and HBCCs, and 100 subsequently to ON and ON-OFF and OFF GCs (Kolb and Famiglietti, 1974). 101 Although the basic rod and cone bipolar cell synaptic pathways in mammalian 102 retina (described above) have been identified, many pieces of the puzzle are still missing. 103 For example, it is not clear how much each of the bipolar cell pathways contribute to the 104 overall job of the bipolar cells, to relay signals to retinal ganglion cells. Moreover, 105 because the pathways consist of many key elements, such as gap junction channels, 106 specific cells (e.g. DBCRs and DBCCs), and specific inputs (e.g. rod or cone inputs), it is 107 important to determine how each of these elements affects individual pathway functions 108 and the overall operation of the retina. 109 In this study, we examine full-field electroretinograms (ERGs), mainly the b110 wave responses, of dark-adapted mice to brief 500 nm flashes that ranged from flashes 111 weak enough to just elicit an ERG response, to stimuli that were 9 log units stronger. 112 Since the ERG b-wave represents the overall bipolar cell light responses of the retina 113 (Stockton and Slaughter, 1989; Tian and Slaughter, 1995; Robson et al., 2004), these 114 responses provided an in vivo tool for analyzing bipolar cell functional pathways over 115 the entire operating range of the visual system for flashes presented from darkness. The 116 more common situation in nature, when there is the transition from rod dominated to 117 cone dominated vision, is for the mean level of illumination to change as well, i.e. from 118 starlight to daylight. Using light flashes from darkness, rather than imposing them on 119 backgrounds of increasing mean illumination, isolates the responses of the pathways from 120 the effects of adaptational mechanisms that would otherwise be activated (Dunn et al., 121 2006; Dunn and Rieke, 2008). By comparing the dark-adapted b-wave responses with 122 the response dynamic ranges of individual retinal neurons recorded with patch electrodes 123 in the dark-adapted mouse retina published from our laboratory and others, we identified 124 plausible cellular origins of b-wave responses for different light zones. Moreover, we 125 took advantage of three pathway-specific knockout mice to study how the different 126 bipolar pathways affect the b-wave response to various ranges of light stimulation and the 127 corresponding bipolar cell pathways involved. To eliminate the primary rod pathway, we 128 used the basic helix-loop-helix transcription factor Bhlhb4 knockout mice, Bhlhb4(-/-), 129 which lose their DBCRs at P8-P12 (Bramblett et al., 2004). To eliminate the secondary 130 rod pathway (via rod-cone coupling), we used the connnexin36 knockout mouse, Cx36(131 /-). These mice lack, a gap junction that is expressed in the outer and inner plexiform 132 layers and is thought to mediate rod-cone coupling (Deans et al., 2002, (Volgyi et al., 133 2004). To isolate the cone pathway, we used the rod transducin alpha-subunit knockout 134 mouse, Trα(-/-), in which rod phototransduction is effectively eliminated, without 135 appreciable retinal degeneration at 13 weeks of age (Calvert et al., 2000). To find 136 evidence of the tertiary rod pathway (rod→DBCcs), we compared responses of the 137 Bhlhb4(-/-) mouse (in which the tertiary pathway should be intact, as DBCC populations 138 have been shown to be intact (Kim et al., 2008) and the Trα(-/-)mouse, which should not 139 have any functional rod pathways. 140 In this way, we were able to provide new insights into the functional organization 141 of rod and cone bipolar cell pathways in the dark-adapted mouse retina, and show how 142 these signaling pathways operate together in forming parallel information channels to 143 encode the wide range light that is encountered in nature. 144 145 146 147 Materials and Methods 148 Ethical Approval. Mice were cared for and handled following approved protocols from 149 the Animal Care and Use Committees of Baylor College of Medicine and in 150 compliance with NIH guidelines for the care and use of experimental animals. 151 152 Animals. Wildtype (WT) mice (C57BL/6) from Jackson Laboratories (Bar Harbor, ME) 153 aged 12-18 weeks were used for experiments. Bhlhb4(-/-) mice (99% C57BL/6 genetic 154 background)(Bramblett et al., 2004), Cx36(-/-) (C57BL6/12SvEV hybrids) (Deans et al., 155 2002) and Trα(-/-) (C57BL6/12SvEV hybrids) (Calvert et al., 2000) aged between 12 – 18 156 weeks were used for experiments. Preliminary evidence suggested that these strains were 157 adequately backcrossed to ensure that strain differences did not affect these recordings 158 (Supplemental Figure 1). 159 160 Electroretinograms. Before testing, mice were allowed to adapt to the dark overnight. D 161 Under dim red light, mice were anesthetized with a solution of ketamine (95 mg/ml) and 162 xylazine (5 mg/ml). The pupils were dilated with a single drop of 1% tropicamide and 163 2.5% phenylephrine. A drop of 0.5% proparacaine hydrochloride was applied for corneal 164 anesthesia. Mice were placed on a heating pad maintained at 39°C, inside a Ganzfeld 165 dome coated with highly reflective white paint (Munsell Paint, New Windsor, NY). A 166 small amount of 2.5% methylcellulose gel was applied to the eye, and a platinum needle 167 electrode was placed in contact with the center of the cornea. Similar platinum reference 168 and ground electrodes were placed in the forehead and tail, respectively. After placement 169 in the dome, mice were allowed to remain in complete darkness for 5 min. Signals were 170 amplified with a Grass P122 amplifier (bandpass 0.1 Hz-1000 Hz; Grass Instruments, 171 West Warwick, RI). Data were acquired with a National Instruments Lab personal 172 computer Data Acquisition board (sampling rate 10,000 Hz; National Instruments, 173 Austin, TX). Traces were averaged and analyzed with custom software written in Matlab 174 (Mathworks, Natick, MA). 175 Flashes were calibrated using a photometer (International Light model IL1700, 176 Newburyport, Massachusetts) fitted with a scotopic filter in integrating mode that gave 177 results as scotopic (sc) cd-s m. In order to convert these units to 178 photoisomerizations/rod, we used the same conversion factors as reported previously 179 (Saszik et al., 2002). Namely, that 1 sc td s = 121.6 Rh* per rod. This calibration was 180 also appropriate for the mouse M-cone with a peak near that of rods (Lyubarsky et al., 181 1999). For the area of the dilated pupil, we use 3.14 mm, as reported in (Pennesi et al., 182 1998). Flashes for scotopic measurements were generated by a Grass PS-33 183 photostimulator (Grass Instruments, West Warwick, RI). Light was spectrally filtered 184 with a 500 nm interference filter (Edmund Industrial Optics, Barrington, NJ). A series of 185 metal plates with holes of varying diameters and glass neutral density filters were used to 186 attenuate the flash. As the strength of the flash increased, the number of trials was 187 decreased and the time between each flash was increased. To remove oscillatory 188 potentials before fitting, the scotopic b wave was digitally filtered using the filtfilt 189 function in Matlab (low-pass filter; Fc =60 Hz). The relationship between b-wave 190 amplitude and flash intensity can be described by a saturating hyperbolic function (Naka– 191 Rushton) with the form (Naka and Rushton, 1966): 192 b = (bmax.scot • I)/(I0.5 +I) (1) 193 where bmax,scot is the saturated scotopic b-wave amplitude and I0.5 is the intensity that 194 provides half saturation. The baseline (from the trough of the unfiltered a-wave) and peak 195 of each filtered trace were measured, and the data for multiple intensities were fitted to 196 the above equation. For purposes of finding the local maximum, a time window of 10 to 197 200 ms after the flash was used. To find the negative scotopic threshold response 198 (nSTR), a local minimum was found in a time window 150 to 250 ms after the flash was 199 used. For purposes of illustrating the positive scotopic threshold response (pSTR) and 200 the nSTR, a digital 60 Hz notch filter was used, as well as a Gaussian smoothing filter 201 (Igor Pro, WaveMetrics, Lake Oswego OR). 202 For stronger stimuli, 1500W Novatron (Dallas, TX) xenon flash lamps provided 203 intense illumination. For these light levels, time between flashes was increased to up to 2 204 minutes to allow the retina to dark adapt. 205 206 Immunohistochemistry. Immunohistochemistry was performed using the indirect 207 antibody method. For vibratome immunolabeling experiments, free-floating 40 μm 208 vibratome sections were blocked with 10% normal donkey serum in PBS with 0.5% 209 Triton X-100 and 0.1% sodium azide for 2 hr to overnight to reduce nonspecific labeling. 210 The tissues were incubated in primary antibodies in the presence of 3% donkey serum 211 and PBS with 0.5% Triton X-100 and 0.1% sodium azide for 1–5 d at 4°C. Tissues were 212 then washed with PBS containing 0.5% Triton X-100 and 0.1% sodium azide; tissue 213 immunoreactivity was visualized by incubating with fluorescent secondary antibodies 214 overnight. After extensive rinsing, the tissue was mounted with Vectashield (Vector 215 Laboratories, Burlingame, CA). Mounted slides were observed with a confocal laser 216 scanning microscope (LSM) with a krypton–argon laser (LSM 510; Zeiss, Thornwood, 217 NY). Images `were acquired using 40X and 63X oil immersion objectives and processed 218 with Zeiss LSM personal computer software and Adobe Photoshop 6.0 (Adobe Systems, 219 San Jose, CA). Mouse monoclonal antibody used was Connexin36 (Chemicon Int., 220 Temecula, CA) (1:500). Rabbit polyclonal antibodies used were PKCα (Sigma-Aldrich, 221 St. Louis, MO) (1:500). Secondary antibodies used were Cy3 Donkey anti-mouse IgG 222 (Jackson ImmunoResearch, West Grove, PA) (1:200), Alexa Fluor 488 Donkey anti223 rabbit IgG (Molecular Probes, Eugene, OR). 224 225