Western blots of D1a dopamine receptor. Homogenate of snap-frozen retinas, and protein standards, separated by SDS-PAGE and transferred to nitrocellulose membranes. A, D, Molecular weight (MW) standard proteins, with MW of each indicated in kD by superimposed number. B, Retina proteins run alongside standard proteins in A, and probed with anti-D1a-receptor antibody. A well-focused protein band is seen at migration distance corresponding to an estimated MW of 54 kD. No other proteins are stained over the MW range shown (20–100 kD). C, E, In a different experiment, retina proteins run alongside standard proteins in D. Lane C probed with anti-D1a-receptor antibody that had been pre-incubated overnight with immunogen. Probing of lane E with anti-D1a-receptor antibody shows a well-focused protein band in E at an estimated MW of 54 kD. A faint band is also seen within the MW range reported for glycosylated D1a receptors (here between 55 and 60 kD). Staining of both bands (dark and faint) was blocked completely by immunogen (C).

D1a-receptor-like immunoreactivity in ganglion cells in vertical sections. Ganglion cells identified by retrograde transport of Alexa Fluor 488-coupled dextran introduced into the optic nerve. Labeling with anti-D1a-receptor antibody visualized with Alexa Fluor 555-conjugated secondary antibody. Images are single confocal optical sections obtained with a 40x oil immersion objective. Fluorescence due to each dye obtained individually. A, Direct overlay of fluorescent signals from retrogradely-transported dextran (backfill, in green) and anti-D1a-receptor antibody (D1a, in red) indicate ganglion cells exhibiting D1a-receptor-like immunoreactivity (orange-yellow). Labels along right side indicate position of outer nuclear layer (onl), inner nuclear layer (inl), inner plexiform layer (ipl), and ganglion cell layer (gcl). B, Example of same labeling from a different retina. Immunopositive dendrites emerge from a large soma in right-hand half of panel, and project into distal half of ipl. C, Images of a section obtained under the exact same conditions as B, except that the anti-D1a-receptor antibody was pre-incubated with immunogen before application to section. Scale bar (25 μm) applies to all panels.

Spike timing deviation in absence and presence of SKF-81297. Spiking elicited and recorded using same methods as in Fig. 5. Spike timing was compared moment-by-moment in three 10-sec traces recorded ≤ 4 min before application of SKF-81297 (A), and in three 10-sec traces recorded 12–16 min after application of SKF-81297 (B). For each condition, if spikes occurred at similar times in all three traces (i.e., if spikes were “temporally co-aligned”), the difference between the time of each spike and the average of the times of the co-aligned spikes was measured, and tallied accordingly in A or B. Time was measured from beginning of each current injection to moment of maximum change in slope (dV/dt) along rising phase of each spike. Histograms are binned at 0.2 msec, and include 165 (55 × 3) and 81 (27 × 3) spikes for A and B, respectively. Insets superimpose examples of spikes considered to be co-aligned in traces recorded before (A) and after (B) SKF. All 6 traces (labeled a, b, and c in A and in B) begin at identical times after start of each 10-sec current injection. C, Percentage distribution of spike timing deviation. Spike number in A and B was normalized by respective total spike number and the two histograms were overlaid, with gray signifying overlap of the before (clear) and after SKF (filled) distributions.

Effect of membrane-permeant cAMP analog (8-cpt-cAMP) on spikes elicited by fluctuating current injection. Recording mode, conditions, and figure format as in Fig. 5. A, Current injected (left) and histogram of current amplitude (right). Left and right parts of A superimpose two traces recorded at B and D in E, and their corresponding amplitude histograms, respectively. Inset superimposes current recorded between 8.0 and 8.1 sec of each 10-sec trace. Histograms fit to a Gaussian distribution with mean and standard deviation of approximately 5 pA (arrow) and 8 pA, respectively. B–D, Spiking and sub-threshold membrane voltage changes induced by current in A, ~5 min before (B), ~5 min after (C), and ~13 min after (D) application of 8-cpt-cAMP began (here, 2 μl of 50 mM stock solution was added to 0.9-ml recording bath). Histograms of voltages traversed during each fluctuating current injection are plotted to right. Mean voltages between and during fluctuating current injections were set to −72 mV (dashed horizontal line through voltage traces) and −58 mV (at arrow next to each histogram), respectively. E, Timecourse of 8-cpt-cAMP effect on total spike number. Each point plots total number of spikes elicited by 10-sec injection of fluctuating current (e.g., those in B–D are plotted at correspondingly labeled times in E). F–G, Membrane voltage on expanded time scale. Three traces recorded at times bracketed before and during 8-cpt-cAMP in E are superimposed in F and G, respectively.

Reduction of voltage-gated Na⁺ current and spiking by nM tetrodotoxin. Recording mode, conditions, and figure format as in Fig. 8. A: Voltage-gated Na⁺ current (without leak subtraction) and spikes elicited in a single ganglion cell by depolarizations in voltage- and current-clamp modes, respectively. Current activated by voltage jump from −72 mV to −47 mV as solution superfused over cell is changed from (A₁) control to (A₂) 5 nM TTX and then (A₃) control again. Triangle positioned at zero current level. Dashed horizontal line at peak of control current highlights partial reduction of current amplitude by TTX and full recovery during wash. B: Spikes then elicited in same cell by constant current injections (10 and 30 pA) as solution superfused over cell is changed from (B₁) control to (B₂) 5 nM TTX and (B₃) control. At this concentration, and as seen during the response to dopamine in other cells, TTX reversibly reduced peak current amplitude by 14%, raised spike threshold (viz., abolished spiking elicited by smallest current injections), and curtailed spiking elicited by larger current injections (lower trace, middle column). Triangles positioned at zero voltage level for all traces in each row. C plots mean (solid bar) and SEM (error bar) of peak inward current during microperfusion of control solution, TTX (4–5 nM), and after wash with control solution, for all cells tested (n=3). The means in control and TTX differed significantly (P<0.0001, paired t-test).

Inhibition by dopamine and by SKF-38393, and reversal of these effects by SCH-23390. A, B: Cell-attached, voltage-clamp mode at 33 °C; currents appear as vertical lines due to slow time base, with downward deflections occurring at peak depolarization of each spike. Spikes elicited by stepwise changes in patch electrode voltage (5 mV in A, 10 mV in B). Solution continuously superfused over cell by U-tube microperfusion. Spikes in control solution (left, “control”) are blocked by inclusion of 10 μM dopamine (middle). Loss of spikes is complete within 2.5 min after onset of dopamine application at lower stimulus step size, but only partial at the higher step size. Spikes are completely blocked by 5 min after dopamine first reached the recording bath. Addition of SCH-23390 (so that the superfusate contains 10 μM dopamine and 10 μM SCH) blocks the response to dopamine at both stimulus strengths. C, D: Inhibition by SKF-38393. Ruptured-patch, current-clamp mode at 34 °C; 200-msec injections of constant current (10 pA in C, and 30 pA in D). Solution continuously superfused over cell by U-tube microperfusion. As in cell-attached recordings, spike firing is continuous during both stimulus pulses in control solution (left, “control”) and is inhibited during same stimulus pulses by 10 μM SKF-38393 (middle, “SKF 10 μM”). Spikes are first lost at low stimulus strength and subsequently lost at higher stimulus strength, too. SCH-23390 (i.e., perfusate containing 10 μM SKF and 10 μM SCH) blocks the response to SKF. Triangles at left show reference level for all traces in each row (zero-current in A, B; zero-voltage in C, D).

Block of I_h does not preclude spike inhibition by dopamine. Voltage-gated Na⁺ current, I_h, and spikes elicited in a single ganglion cell in ruptured-patch configuration at 34 °C. A: Currents recorded in voltage-clamp mode without leak subtraction while solution superfused over cell was changed from control (left) to 3 mM Cs⁺ (right). Steps above current traces show stimulus timing and polarity. Holding potential was −72 mV. Test potentials were −47 mV to activate Na⁺ current (upper rows) and −77, −92, −107 mV to activate I_h (lower rows). Cs⁺ blocks I_h (including portion activated at −72 mV; see Lee and Ishida, 2007) without affecting Na⁺ current. Triangles at left show zero current level for each row. Insets show the Na⁺ current on an expanded time scale. B: Spikes then recorded in current-clamp mode in response to sequence of constant current injections (12, 22, and 32 pA) while superfusate was changed (as marked by brackets) from 3 mM Cs⁺ (first two rows of spikes) to 3 mM Cs⁺ and 6 μM dopamine (next nine rows), then 3 mM Cs⁺, 6 μM dopamine, and 5 μM SCH-23390 (last eight rows). Stimulus timing shown at top of B. Voltage traces displayed in sequence they were collected, with each row showing responses to same current injections, and each row initiated at 30-sec intervals. Tick marks along right side show ground level for each row of traces. C plots number of spikes elicited by each current injection in B, showing inhibition of spikes by dopamine and antagonism of this response by SCH-23390.

Reduction of voltage-gated Na⁺ current by D1-type dopamine receptor activation. Ruptured-patch configuration at 34 °C; voltage-clamp mode with no leak subtraction. Currents activated in a single cell by 4-msec depolarizations (A) and 1-sec hyperpolarizations (B). Holding potential was −72 mV. Test potentials were −57 mV and −47 mV to activate voltage-gated Na⁺ current (A, grey and black traces, respectively) and −77, −92, and −107 mV to activate I_h (B). Stimulus timing and polarity shown by steps above current traces. Triangles at left show zero-current level for all traces in each row. As labeled above current traces in A, solution superfused over cell was changed from control (A₁, B₁) to 3 mM Cs⁺ (A₂, B₂), 6 μM dopamine and 3 mM Cs⁺ (A₃, B₃), 6 μM dopamine, 3 mM Cs⁺, and 5 μM SCH-23390 (A₄, B₄), 6 μM dopamine, 3 mM Cs⁺, 5 μM SCH-23390, and 1 μM TTX (A₅, B₅), and control (A₆, B₆). Peak amplitude of depolarization-activated Na⁺ current at −47 mV in dopamine (A₃, black trace) is 10% smaller than in control (A₁). This reduction was reversed by SCH-23390 (A₄). The depolarization-activated current was blocked by 1 μM TTX (A₅), leaving small uncompensated capacitive inward and no outward current. This TTX block, and the block of I_h by Cs⁺, were reversed by washing with control solution (A₆, B₆). The activation threshold and increase in Na⁺ current by the increment in step depolarizations, and the increase in I_h by the increment in step hyperpolarizations, were similar at the beginning and end of this recording. C: Na⁺ current amplitudes of all cells tested (n=6) as in A, B. Na⁺ current peak amplitude normalized to value in Cs⁺ (left) after reduction by dopamine (middle) and recovery in SCH-23390 (right) while I_h was suppressed. The mean in Cs⁺ differed significantly from that in Cs⁺ plus dopamine (P<0.0005, paired t-test).

Effect of SKF-81297 on spikes elicited by fluctuating current injection. Voltage-clamp-controlled current-clamp (VCcCC) mode recording at room temperature. Perforated-patch configuration in low Ca²⁺ bath solution. Spiking elicited by injecting fluctuating current at intervals of at least 20 sec. A, Waveform of current injected (left) and histogram of current amplitude (far right). Traces of current measured at moments labeled “B” and “D” in E, and histograms constructed from these currents, are superimposed in A. Inset superimposes current recorded between 8.0 and 8.1 sec of each 10-sec trace. Traces from B and D plotted in blue and red, respectively. Histograms fit to a Gaussian distribution; mean and standard deviation are approximately 3 pA (indicated by arrow) and 24 pA, respectively. B–D, Spiking and sub-threshold membrane voltage changes induced by current in A, ~4.5 min before (B), ~6.5 min after (C), and ~16 min after (D) SKF-81297 application began (10 μl of a 1-mM stock solution added to 0.9-ml recording bath; see Methods). Histograms of voltages traversed during each fluctuating current injection plotted to right. Mean voltages between and during the fluctuating current injections were set to −68 mV (dashed horizontal line through voltage traces) and −58 mV (at arrow next to each histogram), respectively. E, Timecourse of SKF-81297 effect on total spike number. Each point plots total number of spikes elicited by 10-sec injection of fluctuating current (e.g., those in B–D are plotted in E at times labeled B, C, and D, respectively). F, mean±SEM of number of spikes recorded during three injections of the current shown in A before and during the response to SFK-81297 in all cells tested (n=6). Lines join the control and SKF values for individual cells. Bars plot the mean±SEM of the values from all cells. The means differed significantly (P<0.009, paired t-test). G–H, Membrane voltage changes on expanded time scale. The three traces recorded at times bracketed before SKF-81297 in E are superimposed in G; those recorded at times bracketed during SKF-81297 in E are superimposed in H. The trace and dot colors show when each recording was made and highlight the similarity in spike timing.

D1a-receptor-like immunoreactivity in ganglion cells in flat-mounted retina. A, As in Figure 2, ganglion cell somata identified by retrogradely transported, fluorescein-coupled dextran (green in A, C, D). The same fluorescence identifies fibers in this image as intraretinal segments of ganglion cell axons, extending as fascicles between the top and bottom edges of panels A and C. B, Binding of polyclonal (Chemicon) anti-D1a-receptor antibody visualized with Alexa Fluor 568-conjugated secondary antibody. C, Overlay of A and B. As in Figure 2, yellow/orange indicates regions of overlapping red (Alexa Fluor 568) and green (fluorescein) signal, signifying D1a-receptor-like immunoreactivity in ganglion cells. D, Same field as C masked to highlight dextran-containing somata only. Arrows show some ganglion cells without noticeable D1a-like immunoreactivity. Fluorescent images A–D are maximum intensity z-projection of 5 consecutive optical sections obtained at 1-μm z-intervals with 3-frame Kalman averaging. Scale bar in D (25 μm) applies to panels A–D. E, Side-by-side histograms of apparent size of ganglion cells identified by fluorescent dextran incorporation via retrograde transport (light bars) and the subset of these cells that also showed D1a-like-receptor immunoreactivity (dark bars). Cells were masked, selected and analyzed in ImageJ from five fields similar to (and including) D from three different retinas. Each ImageJ-reported cross-sectional area converted to diameter of an equivalent circle. Diameters are placed into 1-μm bins centered about the indicated values. F–H: Binding of an anti-D1a receptor antibody and an antibody directed against ganglion cell marker Brn3a. F and G are sequentially collected single optical sections of the ganglion cell layer of a retina incubated in anti-Brn3a and anti-goat DL549-conjugated secondary, and monoclonal (Novus) anti-D1a primary antibody and anti-mouse DL649-conjugated secondary, respectively. Fluorescence from the fluorophores is pseudocolored blue and green. H merges the images in F and G. The crisp green outline of each blue cell profile shows that the monoclonal anti-D1a antibody binds to many of the somata identified as ganglion cells in this field. Scale bar in F (15 μm) applies to panels F–H.