AN ANALYSIS OF LIGHT ADAPTATION AND INHIBITORY INPUT TO RETINAL AMACRINE CELLS By AARON JOSIAS SALAZAR A Thesis Submitted to The Honors College In Partial Fulfillment of the Bachelors degree With Honors in Physiology THE UNIVERSITY OF ARIZONA May 2015 Approved by: Erika Eggers, PhD Departments of Physiology and Biomedical Engineering Abstract The retina avoids signal saturation through the use of both dim-light sensing rod and bright-light sensing cone photoreceptor circuits. Photoreceptors are responsible for converting an image into an electrical signal, which is transmitted to bipolar cells and ganglion cells. Amacrine cells modulate the interaction between bipolar cells and ganglion cells through inhibitory signaling, and therefore play a large part in inner retinal processing. However, inhibitory connections between amacrine cells are a little understood signaling component of the retina. Within this study, amacrine cells within the mouse retina were separated into two categories: narrow-field glycinergic and wide-field GABAergic. Amacrine cells were subjected to a light stimuli under dark-adapted, light-adapted, and receptor isolated conditions, and the peak amplitude and charge transfer of the L-IPSC was measured. Wide-field amacrine cells received an increase in the percentage of inhibition from glycine after light adaptation. There is an overall decrease in spontaneous activity with light adaptation, but an increase in the percent of glycinergic spontaneous activity. Spatial inhibition to narrow-field amacrine cells becomes narrower with light adaptation. L-IPSC peak amplitude decreases with light adaptation with application of a full-field light stimulus, and also decreases as the distance between the stimulus and the cell body increases. Keywords: amacrine cell, gamma-aminobutyric acid (GABA), glycine, L-IPSC, sIPSC, light adaptation Introduction: Images are transformed into electrical signals at the level of the rod and cone photoreceptors in the retina. When a light stimulus is recognized by a photoreceptor, the release of glutamate onto postsynaptic bipolar cells stops. This signal is then interpreted across various types of bipolar cells, and is then transmitted to ganglion cells, the axons of which exit the eye through the optic nerve and transmit retinal information to the brain. There are two major categories of interneurons that contribute to inner retinal image processing before the information is relayed through the ganglion cell axons - horizontal cells and amacrine cells, although amacrine cells likely play a larger role in light adaptation of spatial signaling (Dedek et al. 2008; Mazade and Eggers in submission). Inhibitory connections between amacrine cells, the subcategories into which they can be organized and how inhibitory input to these cells changes between the rod and cone pathways is the focus of this thesis. Approximately 30 types of amacrine cells have been identified in the mammalian non-primate retina (Lin & Masland 2006). The most recent work on this topic results from studies in the rat and mouse retina. However, results in the mouse retina dominate because of the powerful molecular tools that can be brought to bear in this species (Zhang & McCall 2012). This is why we are working on the mouse retina. Amacrine cells are generally categorized into one of two categories: wide-field GABAergic amacrine cells or narrow-field glycinergic amacrine cells (Pourcho & Goebel 1983; Menger et al. 1998). GABAergic amacrine cells provide lateral interactions across the inner plexiform layer (IPL), and are implicated in lateral inhibitory signaling within the IPL, typically within a single layer (Lin and Masland 2006; Eggers 2010). This is important for determining the spatial sensitivity of ganglion cells. Glycinergic amacrine cell dendrites are primarily involved in local interactions between the different sublaminas of the IPL. The most prominent and bestcharacterized glycinergic narrow-field amacrine cell is the AII, involved in signal transfer from the rod to cone pathway (Kolb and Famiglietti 1974; Famiglietti and Kolb 1975). Up to 11 further narrow-field amacrine cells have been identified in different species, of which at least 8 are glycine-positive (Pourcho and Goebel 1985; Menger et al. 1998; MacNeil and Masland 1998; Badea and Nathans 2004). This paper encompasses data analysis from two separate studies within the Eggers lab on inhibitory signaling to retinal amacrine cells. While amacrine cells modulate the excitatory bipolar cell to ganglion cell pathway, they also inhibit other amacrine cells through lateral inhibition. Little is known about this or how it changes between rod and cone pathways. The first study focused on the roles that narrow-field and wide-field amacrine cells play in inner retinal processing. This study was conducted by analyzing the sources of inhibitory input to amacrine cells through isolated rod and cone pathways, through application of a full-field light stimulus. Since rod and cone pathways signal at different light intensities, applying a bright background light that causes rod saturation allows the two pathways to be investigated separately (Dacheux and Raviola 1986; Xin and Bloomfield 1999). Application of this background light has allowed for the investigation of inhibitory signaling to bipolar cells to be studied in the Eggers lab, wherein they found that there was a shift from glycinergic to GABAergic inhibition with light adaptation (Eggers 2013). Typically light adaptation causes a decreased sensitivity to light as a result of isolation of the cone pathway, and increased sensitivity to small stimuli. Larger GABAergic input could reflect an adjustment of OFF bipolar cell spatial inhibition, which may be one mechanism that contributes to retinal spatial sensitivity in bright light conditions (Mazade and Eggers 2013). In the first study, GABAergic and glycinergic inhibition were isolated before and after light adaptation in order to determine whether or not there is a change in the relative contributions of inhibitory signaling from the two main inhibitory neurotransmitters within the retina. The second study focused on the degree to which amacrine cells might play a part in this inner retinal processing with light adaptation by studying how spatial inhibition to both narrow-field and wide-field amacrine cells changes with light adaptation. The results of this study might be related to how ganglion cells increase their spatial sensitivity and visual acuity after ambient illumination is increased (Balrow et al. 1957; Dedek et al. 2008; Farrow et al. 2013). Methods: Mouse retinal slice preparation. Animal protocols were approved by the University of Arizona Institutional Animal Care and Use Committee (IACUC). Male mice 35-60 days of age were euthanized with carbon dioxide (Eggers and Lukasiewicz 2006a; Eggers et al. 2013). Their eyes were enucleated, and the cornea and lens were removed. The eyecup was incubated for 20 minutes in cold extracellular solution (see Solutions and drugs) with 800 U/mL of hyaluronidase to dissolve the remaining vitreous humor. The hyaluronidase solution was then replaced with icecold, oxygenated extracellular solution, and the retina was trimmed into one large rectangle by removing the peripheral retina and leaving only the central retina in the optic disk. A nitrocellulose membrane filter paper (0.45-µm pore size, Millipore) was placed on the retina section, which was transferred to a hand chopper. An average of six 250-µm slices were cut, rotated 90°, and mounted onto glass cover-slips with vacuum grease. Cells used from these slices were never more than 700 µm away from the center of the retina, and only cells near the center of each slice were used for recordings. In this way, much of the differential input due to the dorsal-ventral cone opsin gradient reported in mice was mitigated (Applebury et al. 2000; Haverkamp et al. 2005). The tissue was maintained in oxygenated extracellular solution at room temperature. All dissection procedures were performed under infrared illumination to preserve the light sensitivity of the preparations. Solutions and drugs. The extracellular recording solution used for dissection and to examine light-evoked currents contained (in mM) 125 NaCl, 2.5 KCl, 1 MgCl2, 1.25 NaH2PO4, 2 CaCl2, 20 glucose, 20 glucose, and 26 NaHCO3, and was bubbled with 95% O2-5% CO2. For voltage clamp recordings, the intracellular solution contained (in mM) 120 CsOH, 120 gluconic acid, 1 MgCl2, 10 HEPES, 10 TEA-Cl, 10 phosphocreatine-Na2, 4 Mg-ATP, 0.5 Na-GTP, 10 EGTA and 50 µM Alexa Fluor 488 (Invitrogen, Carlsbad, California, USA) and was adjusted to pH 7.2 with CsOH. To isolate the inhibitory receptor (R) inputs, SR-95531 (SR, 20 µM) to block GABAARs, (1,2,5,6-tetrahydropyridine-4yl) methyphosphinic acid (TPMPA, 50 µM) to block GABACRs, and strychnine (1 µM) to block glycine Rs were used. All drug solutions were washed on the slice for 5 min before recordings began using a gravity-driven superfusion system (Cell Microcontrols, Norfolk, VA) at a rate of ~1-2 mL/minute. Unless otherwise indicated, all chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). Whole-cell recordings. Whole-cell patch-clamp recordings, sampled at 10 kHz, were made from amacrine cells in retinal slices. Light-evoked inhibitory post synaptic currents (L-IPSCs) and spontaneous (s)IPSCs were recorded from retinal amacrine cells voltage clamped to 0 mV, the reversal potential of nonselective cation channel currents. Amacrine cell recordings were stable and no rundown of the light response was observed over the recording period. Resting membrane potentials were recorded passively in current clamp (I-clamp) mode. Liquid junction potentials of 20 mV were corrected for at the beginning of each recording. Electrodes were pulled from borosilicate glass (World Precision Instruments, Sarasota, FL) on a P97 Flaming/Brown puller (Sutter Instruments, Novato, California, USA) and had resistances of 5-7 MΩ. Mice were darkadapted overnight, and all recording procedures were performed in the dark under infrared illumination to preserve the light sensitivity of the slices. Recordings were made in extracellular solution heated to 32°C, using thin stage and inline heaters (Cell Microcontrols, Norfolk, VA). Responses were filtered at 6 kHz with the four-pole Bessel filter on a Multi-clamp 700B patchclamp amplifier (Molecular Devices, Sunnyvale, California, USA) and digitized with a Digidata 1140 data acquisition system (Molecular Devices, Sunnyvale, California, USA). Morphological identification of cell subtypes. Alexa Fluor 488 included in the recording pipette was used to label amacrine cell subtypes. The cells were imaged with Nikon Digital Sight camera with Elements software using a Nikon Intensilight C-HGFIE Fluorescent lamp (Nikon Instruments, Tokyo, Japan). These images were opened in Photoshop, marked and flattened in order to produce a new image, as shown in Figure 1A and Figure 1B. They were classified as either narrow-field or wide-field based on their axonal morphologies within the inner plexiform layer. Cells that had a dendritic span of less than 100 µm were considered narrow-field, and those that were wider than 100 µm were considered wide-field amacrine cells. Full-field light stimuli. Full-field light stimuli were evoked with a light-emitting diode (LED; Agilent HLMP-3950, λpeak=525nm, Palo Alto, CA) projected through the camera port of the microscope, which elicited a strong response in both dark- and light-adapted conditions. We focused our recordings on cells located within the regions of mixed green/UV cone opsin input (Applebury et al. 2000; Haverkamp et al. 2005). Thus, 525-nm light should stimulate both rods (peak sensitivity at 500 nm) and cones robustly. The stimulus intensity (max of 9.5 X 105 photons·µm-2/s-1), background intensity (950 photons·µm-2/s-1), and duration (30 ms) were controlled with Clampex software by varying the current through the LED. The background intensity was chosen as it was shown to maximally activate rods (Wang and Kefalov 2009). A rod-saturating background light was applied for 5 minutes to light-adapt the retina slice and was sustained throughout all light-adapted recordings. Spontaneous (s)IPSC data was analyzed with Clampfit software. An sIPSC template was calculated for each data file with the average of ∼10 prototypical events from the recording. The software used this template to automatically detect spontaneous events, which were manually accepted or rejected on the basis of strict criteria: events used to calculate the frequency were rejected if they appeared to be noise, and events used to calculate the average peak amplitude were rejected if they appeared to be noise or consisted of two or more overlapping events. Frequency was calculated by dividing the number of events by the recording time. Peak amplitude and interevent interval histogram distributions were normalized to the number of events. The signal-to-noise ratio (SNR), which gives a measurement of the actual response signaled over the background noise, was calculated by dividing peak amplitude of the L-IPSC by the variance of the baseline. Full-field inhibitory input data analysis and statistics. L-IPSC traces from a given response condition were averaged with Clampfit software (Molecular Devices), and the charge transfer (Q) and peak amplitude measured in each condition. Because of the significant amount of spontaneous activity, it was difficult to measure a peak from amacrine cell L-IPSCs. Therefore, to estimate the peak, average traces were decimated (50 fold) and each point was replaced with the average of those data points to limit variations due to spontaneous activity. To determine changes in total current, the Q was measured, which represents the magnitude of the response. Q was measured in Clampfit over the length of the roughly 1 second response, using the same time parameters in each condition for the same cell. For intensity-response curves, light-evoked responses were normalized to the maximal response in the dark-adapted condition. The normalized data were plotted against the log10 of the stimulus intensity. Bar light stimuli. Bar stimuli (25 µm wide) were used to determine spatial inhibition, and were presented using a white organic light-emitting diode (OLED Microdisplay, eMagin EMA100503 SXGA Monochrome White XL, Bellevue, WA) projected through the camera port of the microscope, which elicited strong responses in both dark- and light-adapted conditions. Recordings were from cells located within the regions of mixed green/UV cone opsin input (Applebury et al. 2000; Haverkamp et al. 2005) which ensured that all possible pathways were present. The stimulus intensity (7.83 x 104 photons/µm2/sec) and the background rod-saturating light (1150 photons/µm2/sec) were controlled with custom MatLab software by controlling the intensity, size, location, and duration of the bar stimuli through the OLED screen. The background intensity was chosen as it was shown to maximally activate rods (Wang and Kefalov, 2009) and produced significant changes in inner retinal inhibition (Eggers and Mazade, 2013). A long light stimulus (1 s) was used to determine the type of inhibition and excitation to all recorded bipolar cells as well as to illicit robust responses with a very small stimulus. An adapting background light was applied for 5 min to light-adapt the retina slice and was sustained throughout all light-adapted recordings. Spatial inhibitory input data analysis and statistics. L-IPSC traces from a given response condition were averaged using Clampfit software (Molecular Devices, Sunnyvale, California, USA) and the charge transfer (Q) and peak amplitude were measured in each condition. Due to the significant amount of spontaneous activity, it was difficult to measure a peak from amacrine cell L-IPSCs. Therefore, to estimate the peak, average traces were decimated (50 fold) and each point was replaced with the average of those data points to limit variations due to spontaneous activity. To determine changes in total current, the Q was measured, which represents the magnitude of the response. Q was measured in Clampfit over the length of the response, typically 1-2 seconds, using the same time parameters in each condition for the same cell. The baseline Q was added to the Q standard deviation which was subtracted from all raw Q measurements to negate any current due to baseline or spontaneous events. For spatial distribution curves, light-evoked Q’s were normalized to the maximal response in the darkadapted condition and peak amplitude raw data was used. The normalized and raw data were plotted against the distance the stimulus was from the cell. In order to compare between the dark and light-adapted conditions at any given stimulus distance, both sides of the spatial distributions at equal distances were averaged and plotted as bar graphs. 2-way Analysis of Variance (ANOVA) with Student-Newman-Keuls (SNK) posthoc test was used to compare spatial distributions before and after light adaptation as well as between response characteristics at each stimulus distance. Student’s t-tests (2-tailed, paired) were used to compare resting membrane potentials and timing parameters before and after light adaptation. Differences were considered significant when p < 0.05 (*). All averaged data are reported as mean ± SEM. Results: Identification of narrow-field and wide-field amacrine cells Within the mouse retina, narrow-field, radially spanning amacrine cells (Fig 1A) were found to have an average dendritic spread of 58 µm, and highly stratified wide-field amacrine cells (Fig 1B) were found to have an average dendritic spread of 124 µm. A Student’s t-Test was conducted to determine whether or not there was a clear difference in dendritic field of the narrow- and wide-field amacrine cells. The calculated p-value was less than 0.05, meaning that the difference in dendritic field for narrow- and wide-field amacrine cells is statistically significant (Fig 1C). Wide field amacrine cells receive more glycinergic inhibition under light adapted conditions In the first study, the L-IPSC in the retinal amacrine cell was measured first after dark adaptation. Strychnine was applied to the cells to blockade inhibition from glycine before light adaptation in order to isolate for GABAergic inhibition in the dark-adapted cell. This was repeated for amacrine cells after light adaptation in order to determine whether or not the percentage of glycinergic and GABAergic inhibition changed under the different conditions. There was no observable difference in the ratio of GABAergic to glycinergic inhibition to narrow-field amacrine cells (Figure 2A). However, after light adaptation of wide field amacrine cells there was a significant decrease in the ratio of GABAergic to glycinergic inhibition. Figures 2E and 2F demonstrate the light response in narrow- and wide-field amacrine cells with isolation of GABAergic inhibition both before and after light adaptation. The difference in inhibitory signaling to wide field amacrine cells before and after light adaptation is statistically significant according to the Student's T-Test, which might suggest that the contribution of glycinergic inhibitoin to amacrine cells increases with light adaptation. There is an increase in sIPSC activity due to glycine receptors with light adaptation As can be seen in Figure 2E and 2F, there is a significant amount of background spontaneous activity, which may affect signal transmission of light-evoked responses. Spontaneous activity was measured under dark-adapted and light-adapted conditions and also after application of sequential receptor antagonists in order to determine the source of spontaneous activity and how it changed under the described conditions. The frequency and peak amplitude of spontaneous inhibitory post synaptic currents (sIPSCs) were measured for both narrow- and wide-field amacrine cells before and after light adaptation with the application of strychnine, as shown in Figure 3. There was no statistically significant difference in either peak amplitude or spontaneous frequency with light adaptation alone (Figure 3A, 3B). However, there was a statistically significant difference in spontaneous peak amplitude with the application of strychnine in the dark for both narrow- and wide-field cells (Figure 3D). This might suggest more glycinergic spontaneous activity in the light-adapted retina in both categories. In addition to that, there was a significant decrease in spontaneous frequency after the application of strychnine to light adapted cells in both subcategories. This suggests that sIPSCs are primarily caused by the activation of glycine receptors through various cone pathways (Figure 3E). Histograms for spontaneous event peak amplitudes and interevent intervals, along with example traces for spontaneous activity were shown in Figures 4, 5, and 6 for the three different methods of treatment: with strychnine before light-adaptation, with light adaptation and with strychnine after light-adaptation. Spatial inhibition to narrow-field amacrine cells becomes narrower with light adaptation In the second study, the spatial extent of inhibition was measured before and after light adaptation. A 25 µm bar of light was applied in 100 µm increments from 600 µm to the left of the cell body to 800 µm to the right. The spatial inhibitory graphs for charge transfer were normalized to the center L-IPSC in the light and in the dark. The normalized charge transfer was plotted before and after light adaptation, and an ANOVA with repeated measures test conducted (Figure 7A). The ANOVA test yielded a p value of 0.002, meaning that the difference in charge transfer before and after light adaptation was statistically significant when paired with increasing distance of the light stimulus from the center of the amacrine cell for narrow-field amacrine cells. One sided analysis of charge transfer for narrow-field amacrine cells with spatial inhibition was conducted by averaging the charge transfer at equal distances from the center of the cell. This is shown in Figure 7C. This confirmed that there was a statistically significant distance in charge transfer at distances greater than or equal to 300 µm from the center of the cell, and suggests that spatial inhibition to narrow-field amacrine cells narrows significantly after light adaptation. Sufficient data was not collected to create a similar graph for wide-field amacrine cells as in Figure 7A. The values for charge transfer when the light stimulus was directly above the cells were averaged over all cells, normalized to the charge transfer in the dark and shown in Figure 7D, in order to show how charge transfer changed with light adaptation and after the application of receptor antagonists. This location was used because the greatest value for charge transfer was observed here. This was repeated before and after light adaptation, after isolation for GABAa receptors and after isolation for glycine receptors (Figure 7D) in both narrow-field and wide-field amacrine cells. A student t-Test showed that there was a significant difference in charge transfer for narrow field amacrine cells with light adaptation and after isolation for GABAa across all amacrine cells. From this, we can conclude that glycinergic inhibition plays a critical role in inhibitory signaling in the rod pathway. Peak amplitude decreases as the distance between the light stimulus and the center of the cell increases, and also decreases with light adaptation Peak amplitude of the L-IPSC was measured as the light stimulus was moved in 100 µm increments across the retinal slice for narrow-field amacrine cells, in order to determine whether or not there was a noticeable change with light adaptation. A one sided analysis bar graph was created, and the difference in peak amplitude was found to be statisically significant according to the Student-Newman-Keuls Method at 700 and 800 µm from the center of the cell. The peak amplitude decreased significantly at long distances away from the cell body with light adaptation (Figure 8A). While the charge transfer and peak amplitude decreased with increasing distance of the light stimulus from the cell body after light adaptation, the signal to noise ratio increased significantly (Figure 8B). This was most likely due to a decrease in sIPSC frequency and peak amplitude with light adaptation. The peak amplitude difference with light adaptation at the center bar for narrow field amacrine cells was also graphed. Application of the student t-Test showed that there was no statistical difference in peak amplitude of the L-IPSC with light adaptation (p>0.05). Discussion In this study we found that there was an increase in the percent of inhibition that is received from GABAergic input after light adaptation in wide-field amacrine cells. There is also a narrowing in spatial inhibition with light adaptation. We also decided to redefine what constituted “narrowfield” and “wide-field” in the mouse retina. Traditionally, amacrine cells have been divided into broad categories of narrow field (30-150 µm), small-field (150-300 µm, medium field (300-500 µm), and wide-field (>500 µm) cells, based on their dendritic span across the IPL (Kolb et al. 1981). Division by size is useful because dendritic spread is an important determinant of a cell’s function in visual processing (Masland, 2012). However, these values were determined in the cat retina. The size might differ due to the difference in retinal size between the mouse and the cat. When measurements were made in the mouse retina, amacrine cells that go between multiple sublamina of the IPL did not normally exceed a dendritic span of more than 100 µm. Similarly, amacrine cells with dendritic spans that exceeded 100 µm had axonal processes that were highly stratified, as shown in Figure 1A. A previous study found that most narrow-field amacrine cells were approximately 40 µm in diameter, although the dendritic tree diameters never exceeded 100 µm (Menger 1998). This study was also conducted in the rat retina, not the mouse. This is why a new classification system for mouse retinal amacrine cells was proposed, where narrow-field was defined as having a dendritic span of less than 100 µm, and wide-field as having a dendritic span of more than 100 µm. It is understood that there are multiple subcategories into which amacrine cells can be further classified, but for the purpose of this paper only the two subcategories were used. In the first study with a full-field light stimulus, a decrease in both the charge transfer and peak amplitude of the L-IPSC was observed with light adaptation. Narrow-field and wide-field amacrtine cells had both GABA and glycine inputs in the dark and light in all cellsAn increase in the proportion of glycinergic signaling contributing to the L-IPSC after light adaptation could have occurred as a result of an increase in glycinergic inhibition with light adaptation, or as a result of a decrease in GABAergic inhibition, or both (Figure 2B). In both cases, there was a clear decrease in charge transfer after light adaptation, application of strychnine, and the application of SR, but not after the application of TPMPA. Therefore, the data would suggest that GABAc receptors do not play a large role in modulating narrow-field or wide-field amacrine cell inhibitory signaling (Figure 2C, 2D). This is expected, since in the mammalian retina GABAC receptors have been described on bipolar cells only (Enz et al. 1996; Euler et al. 1996; Feigenspan and Bormann 1994a; Feigenspan et al. 1993; Pan and Lipton 1995; Yeh et al. 1996). Both light adaptation and increasing distance from the light stimulus to the amacrine cell body caused a decrease in both the total charge transfer and peak amplitude in narrow- and wide-field amacrine cells. The spatial inhibitory response of narrow-field amacrine cells narrowed with light adaptation, which indicates an increase in spatial sensitivity through the cone photoreceptor pathway in comparison to the rod pathway. Similar activity was observed in bipolar cells in a previous study. Total inhibition became significantly narrower and smaller under light adapted conditions, with little to no inhibition remaining at the furthest distances (Mazade and Eggers in submission). This shows that spatial inhibitory input to the inner retina becomes narrower and smaller with increasing background light. Spatial inhibition was studied in order to determine the effect of light adaptatation might have on center surround inhibition modulated by retinal amacrine cells. Both narrow- and wide-field amacrine cells measured in this study had dendritic spans that never exceeded 300 µm across the IPL. However, when a bar of light was applied over 300 µm away from the center of the amacrine cell being measured, a decrease in the inhibitory response was still observed. This may be due to gap junctions beween amacrine cells (Mazade and Eggers in submission). Previous studies have shown that gap junctions become uncoupled between AII amacrine cells, and AII amacrine cell activation is decreased (Mazade and Eggers 2013, Dacheux and Raviola 1986) after light adaptation, which would explain the narrowing in spatial inhibition after the application of a rod saturating background. In addition to this, horizontal cells in the outer retina become uncoupled with light adaptation (Xin and Bloomfield 1999), although amacrine cells likely play a larger role in light adaptation of spatial signaling (Dedek et al. 2008; Mazade and Eggers in submission). Upon application of strychnine in the dark and light, it was found that the percent of glycinergic inhibition to wide-field amacrine cells increased after light adaptation. There is also a decrease in spontaneous frequency after light adaptation and receptor isolation with strychnine. Spontaneous currents are modulated by the various receptors for glycine and GABA. A decrease in spontaneous frequency from might indicate a decrease in activity at GABA receptors, or an increase in activity at glycine receptors. This could be from other amacrine cells and also bipolar cells. This decrease in spontaneous activity could have also contributed to the increase in the signal to noise ratio after light-adaptation in narrow-field amacrine cells. The signal to noise ratio is calculated by dividing the peak amplitude by the variance of the baseline. By decreasing spontaneous activity, the variance decreased, which caused an overall increase in the signal to noise ratio, shown in Figure 8B. Acknowledgements I would like to thank Reece Mazade for providing the data necessary for this study. I would also like to thank Erika Eggers, Reece Mazade and Johnnie Moore-Dotson for guidance on the creation of this thesis, technical assistance, and for providing the tools required to make this research project possible. Grants: - NIH grant EY018131 (EDE) - University of Arizona NIH Graduate Training in Systems and Integrative Physiology grant 5T32GM008400 (REM) - ARCS Foundation (REM) References Applebury ML, Antoch MP, Baxter LC, Chun LL, Falk JD, Farhangfar F, Kage K, Krzystolik MG, Lyass LA, Robbins JT. The murine cone photoreceptor: a single cone type expresses both S and M opsins with retinal spatial patterning. Neuron 27: 513–523, 2000. Badea, T.C., Nathans, J. Quantitative analysis of neuronal morphologies in the mouse retina visualized by using a genetically directed reporter. J. Comp. Neurol 480: 331–351, 2004. Barlow HB, Fitzhugh R, Kuffler SW. 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