Optimized Method to Quantify Dopamine Turnover in the Mammalian Retina

Measurement of dopamine (DA) release in the retina allows the interrogation of the complex neural circuits within this tissue. A number of previous methods have been used to quantify this neuromodulator, the most common of which is HPLC with electrochemical detection (HPLC-ECD). However, this technique can produce significant concentration uncertainties. In this present study, we report a sensitive and accurate UHPLC-MS/MS method for the quantification of DA and its primary metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in mouse retina. Internal standards DA-<i>d</i>₄ and DOPAC-<i>d</i>₅ result in standard curve linearity for DA from 0.05–100 ng/mL (LOD = 6 pg/mL) and DOPAC from 0.5–100 ng/mL (LOD = 162 pg/mL). A systematic study of tissue extraction conditions reveals that the use of formic acid (1%), in place of the more commonly used perchloric acid, combined with 0.5 mM ascorbic acid prevents significant oxidation of the analytes. When the method is applied to mouse retinae a significant increase in the DOPAC/DA ratio is observed following in vivo light stimulation. We additionally examined the effect of anesthesia on DA and DOPAC levels in the retina in vivo and find that basal dark-adapted concentrations are not affected. Light caused a similar increase in DOPAC/DA ratio but interindividual variation was significantly reduced. Together, we systematically describe the ideal conditions to accurately and reliably measure DA turnover in the mammalian retina

Fast responses originate from direct activation of Na_V and K_V-channels.

<p>A, Representative averaged traces of the dose response relationship of a wild-type INL cell under CdCl₂ (0.5mM), black traces, and subsequently the same cell under CdCl₂ + TTX (0.5µM), grey traces, show the block of the fast depolarization component. B, Dose response relationship of a wild-type INL cell under CdCl₂ (0.5mM), black traces, and subsequently the same cell under CdCl₂ + TEA (30mM), grey traces, shows the block of the fast hyperpolarization component. Scale bar applies to all traces.</p

Four patterns of response to electrical stimulation in wild-type retinal INL cells.

<p>Representative averaged traces from a variety of INL cell types in the wild-type retina, black traces: A, Slow depolarization classified by the peak of the response occurring >10ms after stimulus onset. B, Oscillation, positive and negative responses continuing >10ms after stimulus onset. C, Fast depolarization (<10ms after stimulus onset). D, Fast hyperpolarization (<10ms after stimulus onset), dotted line depicts baseline. Grey traces: representative averaged traces (not necessarily from the same cell) after treatment with the synaptic blocker CdCl₂ (0.5mM), response amplitudes were greatly reduced and the oscillation response completely blocked. Note different scales for fast responses.</p

Response amplitude and polarity change in a dose dependent manner with stimulus intensity.

<p>A and B, Representative averaged traces from wild-type and <i>rd/rd</i> INL cells, under CdCl₂ (0.5mM), display fast depolarization responses at low stimulus amplitudes that reverse in polarity as stimulus intensity is increased. C, Raw trace from the <i>rd/rd</i> cell shows the existence of a large amplitude membrane oscillation, even under synaptic block, that is larger than the electrically evoked response, shown with arrow.</p

Electrical Stimulation of Inner Retinal Neurons in Wild-Type and Retinally Degenerate (<i>rd/rd</i>) Mice

<div><p>Electrical stimulation of the retina following photoreceptor degeneration in diseases such as retinitis pigmentosa and age-related macular degeneration has become a promising therapeutic strategy for the restoration of vision. Many retinal neurons remain functional following photoreceptor degeneration; however, the responses of the different classes of cells to electrical stimuli have not been fully investigated. Using whole-cell patch clamp electrophysiology in retinal slices we investigated the response to electrical stimulation of cells of the inner nuclear layer (INL), pre-synaptic to retinal ganglion cells, in wild-type and retinally degenerate (<i>rd/rd</i>) mice. The responses of these cells to electrical stimulation were extremely varied, with both extrinsic and intrinsic evoked responses observed. Further examination of the intrinsically evoked responses revealed direct activation of both voltage-gated Na⁺ channels and K⁺ channels. The expression of these channels, which is particularly varied between INL cells, and the stimulus intensity, appears to dictate the polarity of the eventual response. Retinally degenerate animals showed similar responses to electrical stimulation of the retina to those of the wild-type, but the relative representation of each response type differed. The most striking difference between genotypes was the existence of a large amplitude oscillation in the majority of INL cells in <i>rd/rd</i> mice (as previously reported) that impacted on the signal to noise ratio following electrical stimulation. This confounding oscillation may significantly reduce the efficacy of electrical stimulation of the degenerate retina, and a greater understanding of its origin will potentially enable it to be dampened or eliminated. </p> </div

Four patterns of response to electrical stimulation in <i>rd/rd</i> retinal INL cells.

<p>Representative averaged traces from a variety of INL cell types in the <i>rd/rd</i> retina, black traces: A, Slow depolarization (peak occurring >10ms after stimulus onset). B, Oscillation, positive and negative responses continuing >10ms after stimulus onset. C, Fast depolarization (<10ms after stimulus onset). D, Fast hyperpolarization (<10ms after stimulus onset). Grey traces: representative averaged traces (not necessarily from the same cell) after treatment with the synaptic blocker CdCl₂ (0.5mM), response amplitudes were not significantly altered but slow depolarization and oscillation responses were completely blocked. Note different scales for fast responses.</p

Light-driven neural activation in the visual cortex of TKO mice.

<p>Multiple immunostaining for c-fos (green) and SMI-32 (red) reveals a clear pattern of activation in response to light (<b>A</b> and <b>C</b>), relative to darkness (<b>B</b> and <b>D</b>). As shown at low magnification in <b>A</b>, light-driven c-fos induction was found in retrosplenial (RSD), primary (V1) and secondary (V2M/L) divisions of visual cortex. The V1 region from <b>A</b> and <b>B</b> is shown at higher magnification in <b>C</b> and <b>D</b> respectively. Light-driven neural activation, as visualized by c-fos positive nuclei, was seen throughout the different layers of primary visual cortex (I-IV). Scale bars: <b>A</b>–<b>B</b> 500 µm, <b>C</b>–<b>D</b> 200 µm.</p

Impact of a PLC-β antagonist on the TKO ERG.

<p>Effects of the intravitreal injection of the PLC-β inhibitor U73122 on the b-wave amplitude (mean±SEM; <b>A</b>) and the average response waveform following vehicle or 1.0 mM U73122 in TKO mice (<b>B</b>). The same data plotted to show paired amplitudes of b-wave and a-wave before and after 1.0 mM U73122 for each individual (<b>C&D</b>). Effects of 1.0 mM U73122 intravitreal injection on wild type b-wave amplitude (<b>E</b>) and the average response waveform (<b>F</b>)<b>.</b> Sample size for TKO n = 4–6 for Vehicle, 0.1 mM and 0.5 mM U73122, and n = 10 for 1.0 mM U73122; for WT n = 4. Drug concentrations given in mM are for the injected preparation, final tissue concentration will be around 10× lower. Data in <b>A</b> analysed by one-way ANOVA and bonferroni post tests. Data in <b>C, D</b> and <b>E</b> analysed by paired two-tailed t-tests. * p<0.05; **p<0.p01. Scale bars in <b>B</b> and <b>F</b> = 50 ms (x-axis), 25 µV (y-axis).</p

Spectral sensitivity of the TKO flash ERG.

<p><b>A</b>, ERG b-wave amplitude (mean±SEM; n = 7) from TKO mice (expressed as % of maximum response at 458 nm) for 458 nm (blue) and 580 nm (yellow) flash stimuli. <b>B</b>–<b>D</b>, The same data plotted with stimulus irradiance at the two wavelengths normalized according to the spectral sensitivity of rod opsin (<b>B</b>), MWS opsin (<b>C</b>), and melanopsin (<b>D</b>). F test statistic allows the use of a single curve for the two wavelengths (p>0.05) when normalised for rod opsin, but not for MWS opsin (p<0.01), or melanopsin (p<0.0001). <b>E</b>. The relationship between p for the F statistic and the λ_max of the putative pigment used to normalize irradiance across the two wavelengths peaked close to 498 nm, the known spectral sensitivity of mouse rod opsin. <b>F.</b> b-wave amplitude (mean±SEM) for a range of monochromatic stimuli calculated to be isoluminant for rods (filled circles) or MWS-cones (open circles). Lines depict best fit by linear regression analysis, slope significantly different from 0 for the MWS-cone (p<0.05) but not rod (p>0.05) conditions. Note that the 580 nm datapoint contributes to both series.</p

ERG responses in <i>Gnat1^−/−;Cnga3^−/−;Opn4^−/−</i> (TKO) mice.

<p><b>A</b>, Dark adapted flash ERG traces from a representative TKO mouse and representative traces from two <i>rd/rd cl</i> mice; arrow depicts flash onset; scale bar  = 50 ms (x-axis), 25 µV (y-axis); numbers to left are stimulus irradiance in log cd/m². <b>B</b>, Mean (±SEM; n = 5) a- and b-wave amplitudes for flash ERG in TKO mice. <b>C</b>, Representative light-adapted ERG traces in wild type (WT) and TKO mice (Scale bar =   = 50 ms (x-axis), 25 µV (y-axis)). <b>D</b>, b-wave amplitude (mean±SEM) at the brightest flash (3.5 log₁₀ cd/m²) in wild-type (n = 6), TKO (n = 4) <i>Gnat1^−/−</i> mice (n = 5) compared by one-way ANOVA (p<0.001) and Bonferroni's post test. <b>E</b>, Estimated threshold irradiance (box shows median±upper lower quartiles, whiskers range of data) for a reliable ERG response in TKO (n = 5), <i>Gnat1^−/−</i> (n = 3) and wild type mice (n = 6) compared with one-way ANOVA (p<0.0001) and bonferroni post test. *** p<0.001; ** p<0.01; ns p>0.05.</p