Article Figures & Data
Figures
- Figure 1.
Amperometric detection of calcium-dependent vesicular dopamine release from TH+ periglomerular cells. A, Schematic drawing of the coronal section of a rodent brain shows A16 dopamine neurons (green) located in the glomerular layer of the olfactory bulb. TH+ periglomerular neurons are apparent in a confocal image from a region (red square) of the glomerular layer in a TH-GFP mouse. Bottom panels, at 1 and 16 days in culture GFP-labeled putative dopamine neurons (TH+) could be distinguished from other cells (TH−) that were labeled by DAPI (blue). B, Amperometric electrodes were calibrated by sustained puff of dopamine (3 μm–5 mm) from a perfusion pipette (right) onto the carbon fiber (top left). Dopamine perfusion elicited a dose-dependent current on the carbon fiber electrode at a holding potential of +700 mV (right panels). The amperometric current was directly proportional the dopamine concentration with a detection threshold of ≤ 3 μm (bottom left). C, Exemplary amperometric recordings before (left panels) and following whole-cell dialysis with a 20 μm Ca2+-containing internal solution (right panels). The carbon fiber was gently pressed on the surface of a cell (black cylinder, top inset); the whole-cell patch pipette was positioned on the opposite side of the cell (top inset, right). Following the onset of whole-cell recording, TH+ cells at either 1 DIC (top panels) or 7 DIC (middle panels) showed an increase in spike frequency, whereas TH− cells (bottom panels) had no amperometric spikes. TH+ cells at 7 DIC were preincubated with l-DOPA to enhance the sensitivity of the amperometric recording. Plots of the averaged cumulative spike frequency at 1 DIC (8 cells, 689 events) and 7 DIC (7 cells, 2536 events) confirm the increase in amperometric events during stimulation. Amperometric recording was begun 5 s after the onset of whole-cell dialysis.
- Figure 2.
Quantal size of amperometric spikes was affected by l-DOPA and reserpine. A, Schematic representation of vesicles and corresponding exemplary amperometric spikes recorded at 1 DIC (left) following preincubation with l-DOPA (7 DIC, middle) and following incubation with l-DOPA and reserpine (7 DIC, right). At 7 DIC, the quantal charge of amperometric spikes increased dramatically following l-DOPA incubation (l-DOPA: 2628 spikes, 5 cells; control: 0 spikes, 5 cells), but this increase was reduced by subsequent l-DOPA /reserpine cotreatment (1868 spikes, 6 cells). The quantal size following reserpine was comparable to TH+ cells at 1 DIC (1180 spikes, 8 cells). The histogram values represent the average of the median amperometric spike charge for each cell. B, In vivo confocal imaging of acute olfactory bulb slices incubated with a fluorescent analog of dopamine revealed staining within glomerular cell layer, but not the external plexiform layer (left, 20×, Z-projection of 11 images, 30 μm depth). At higher magnification, punctate staining was evident within each glomerulus (middle, 20×, Z-projection of 11 images, 10 μm depth) as well as in the soma and dendrites of individual cells (right, 63×). Large, bright, linear structures represent staining within blood vessels.
- Figure 3.
Electron microscopy of single TH+ periglomerular cells. A, Individual TH+ neurons in cell culture were identified with confocal microscopy for subsequent identification for electron microscopy as described in Materials and Methods. B, Electron micrographs of TH+ periglomerular cells showed predominately small clear vesicles and occasional LDCVs. In the example shown, a single LDCV is present in this image from a TH+ periglomerular cell soma. The LDCVs constituted < 2% of the total vesicles observed in images from 10 cells (29 LDCVs of 1484 total vesicles). C, The size of vesicles in the soma (black, n = 874 from 11 cells) was the same as in dendrites (blue, n = 610 from 5 cells). Gaussian fits gave mean vesicle diameters of 49 ± 8 nm and 50 ± 7mn for soma and dendrites, respectively. D, Amperometric spikes recorded in a chromaffin cell (black trace) were much larger and longer-lasting that spikes recorded in periglomerular cell (red traces). The same recording methods were used for both cell types. Corresponding electron microscopy images of a chromaffin granule (left) and periglomerular dopamine neuron vesicles (right) also revealed the size difference between an LDCV in a chromaffin cell and the small clear vesicles in periglomerular cells. Scale, 50 nm. E, The distribution of the cubic root of charge (proportional to vesicle radius) was also consistent with a uniform vesicle population. The dotted line is a best fit to a single Gaussian. The threshold was set at 4 pA (circa 3× RMS); thus, the left side of the distribution likely missed some small events, whereas the few larger events likely represent multivesicular release. Events recorded in TH+ cells at 1 DIC are shown.
- Figure 4.
Simultaneous detection of GABA and dopamine release from TH+ periglomerular cells. A, Confocal image of periglomerular cells showed colocalization of TH+ fluorescence (green) with the vesicular GABA transporter in the soma and dendrites (VGAT immunoreactivity, red). B, A brief (500μs) depolarization (top) of a voltage-clamped TH+ neuron (Vh −70 mV) in a microisland culture evoked a fast inhibitory postsynaptic current (black trace, average of 10 sweeps). The GABAA receptor antagonist SR95531 entirely blocked the IPSC (gray trace). Peak amplitudes measured at the peak (dashed line) are shown in the inset (n = 5, p = 0.005, paired t test). C, Simultaneous recording of a GABAA-mediated IPSC (top trace, black, overlap of 10 sweeps) and dopamine (bottom trace, red, overlap of 10 sweeps) in a TH+ cell. The GABA-mediated IPSC reached a peak within a few milliseconds after depolarization, whereas there was not an abrupt increase in the frequency of amperometric spikes. Rather, the rate of asynchronous release increased from the baseline and remained increased for many seconds. D, The GABA current and amperometric event frequency were normalized and plotted as a cumulative probability for all cells recorded with the protocol shown in C. As expected, the vast majority of GABA release (black line, average of eight cells) occurred immediately following action potential (AP) stimulation (arrow). The pattern for dopamine release was much different. As shown by the positive slope of the solid red line before stimulation, there were ongoing spontaneous amperometric events (red line). Stimulation caused an increase in spike frequency that persisted for seconds, as indicated by the change in the slope (red line, average of 6 cells). The bin size for amperometric spikes was 10 ms and total spikes = 1431. The extrapolated rate of spontaneous amperometric spikes is shown in the dotted line.
Tables
Gene Sorted TH+ mRNA [ng] HEK293 Adrenal glands Tyrosine hydroxylase 334 ± 41.3 0.2 ± 0.05 246.2 ± 12.9 Dopamine β-hydroxylase 0.03 ± 0.02 ND 226.5 ± 18.1 VMAT1 0.03 ± 0.02 0.06 ± 0.04 301.8 ± 38.2 VMAT2 164.7 ± 52.2 0.07 ± 0.02 186.3 ± 20.3 NET 0.02 ± 0.002 0.2 ± 0.08 327 ± 32.6 DAT 755.3 ± 175 12.7 ± 1.38 8.4 ± 1.3 VGAT 60.3 ± 3.7 0.02 ± 0.01 0.14 ± 0.01 FACS sorting of TH+ periglomerular neurons showed that mRNAs necessary for dopamine synthesis (TH), storage (VMAT2), and reuptake (DAT) were present. However, dopamine-β-hydroxylase, necessary for norepinephrine synthesis, was not detected. HEK293 cell served as a negative control; chromaffin cells in adrenal glands showed the expected pattern for norepinephrine synthesis. Vesicular GABA transporter (VGAT) mRNA was present in TH+ periglomerular neurons, confirming a dual-neurotransmitter phenotype for TH+ periglomerular cells. Data represent average of three different cell sorts. Values in bold type indicate the presence of RNA.
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