Presynaptic recording of quanta from midbrain dopamine neurons and modulation of the quantal size - PubMed (original) (raw)

Presynaptic recording of quanta from midbrain dopamine neurons and modulation of the quantal size

E N Pothos et al. J Neurosci. 1998.

Abstract

The observation of quantal release from central catecholamine neurons has proven elusive because of the absence of evoked rapid postsynaptic currents. We adapted amperometric methods to observe quantal release directly from axonal varicosities of midbrain dopamine neurons that predominantly contain small synaptic vesicles. Quantal events were elicited by high K+ or alpha-latrotoxin, required extracellular Ca2+, and were abolished by reserpine. The events indicated the release of 3000 molecules over 200 microsec, much smaller and faster events than quanta associated with large dense-core vesicles previously recorded in vertebrate preparations. The number of dopamine molecules per quantum increased as a population to 380% of controls after glial-derived neurotrophic factor (GDNF) exposure and to 350% of controls after exposure to the dopamine precursor L-dihydroxyphenylalanine (L-DOPA). These results introduce a means to measure directly the number of transmitter molecules released from small synaptic vesicles of CNS neurons. Moreover, quantal size was not an invariant parameter in CNS neurons but could be modulated by neurotrophic factors and altered neurotransmitter synthesis.

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Figures

Fig. 1.

Fig. 1.

A 1, Ventral midbrain dopamine (DA) neurons in culture were immunostained for tyrosine hydroxylase (TH), using a fluorescein-conjugated secondary antibody. A 2, The same cell pair immunostained for the brain vesicular monoamine transporter (VMAT2) and visualized by a horseradish peroxidase/diaminobenzidine reaction product. The VMAT2-labeled varicosities (examples indicated by_arrows_) measured 0.5–4 μm in diameter at their longest axis and occurred both with and without obvious contact to other neurons. Scale bar, 5 μm.B 1,B 2, Presumed presynaptic compartments (examples indicated at arrows) were labeled by the endocytic tracer FM1-43 (4 μ

m

in the presence of 6 μ

m

nicotine for 1 min; the nicotinic acetylcholine receptor is presynaptic at midbrain DA neurons) (Marshall et al., 1997). The neurons were maintained in Ca2+-free medium for 10 min with little decrement in label. The figures display the label at 5 and 10 min. The perfusion pipette (P) extends from the base and appears_green_ in the pseudocolored images because of the autofluorescence. B 3,B 4, Then these sites were stimulated with 40 m

m

K+ medium (10 sec), decreasing the label at several sites in the path of the perfusion. Three further applications of 40 m

m

K+ medium (10 sec) induced further decrement of FM1-43 label, demonstrating that the sites studied contain organelles that recycle on stimulation. The site indicated by the top left arrow, which is farther from the perfusion pipette, shows less decrement than sites closer to the pipette. Scale bar, 5 μm.C, Quantitation of decrease in mean fluorescence per region of interest (ROI) after stimulation (n = 23 sites). Each box and_whiskers symbol_ corresponds to the exposures as indicated in the figure above. The mean is indicated by the_black horizontal cross_ on the vertical bars, the limits (whiskers) at the ends of the_bars_ indicate the maximum and minimum values for the data set, and the box limits indicate the 5th and 95th percentiles of the data set.

Fig. 2.

Fig. 2.

A 1, A combined fluorescent/incandescent image of a neuron labeled with Lucifer yellow by whole-cell patch clamp. Magnification bar, 10 μm.A 2, Fluorescent image of a detail corresponding to the area indicated by the broken box in_A_ 1. The placement of the 5 μm (vertical axis) electrode is indicated by the_oval_. Two labeled varicosities are apparent under the electrode surface. A 3, The_top trace_ shows amperometric events at the site in_A_ 2 after 3 sec of high K+ in the presence of 1.2 m

m

Ca2+ (the arrow indicates the application of high K+ for each_trace_). The events were not evoked by high K+ in Ca2+-free medium (middle trace). Recovery occurred when high K+ in the presence of 1.2 m

m

Ca2+ was reapplied (bottom trace). Of 14 neurons labeled by Lucifer yellow injection, five showed amperometric spikes from apparent varicosities. For each of three sites so examined, the removal of Ca2+ resulted in the complete cessation of spikes, and spikes resumed after the readministration of high K+ with Ca2+. All sites where quanta were observed had TH+ processes underneath the electrode, although only three of the five dye-filled neurons in which amperometric events were evoked were themselves TH+; therefore, nondye-filled dopaminergic processes were sometimes under the electrode. B, Exemplar amperometric recording contrasting two secretagogues. This culture was exposed to both GDNF and

l

-DOPA (50 μ

m

for 30 min) to promote elevated levels of DA release. Medium containing 40 m

m

K+ was applied for 2 sec (first arrow). Nineteen spikes (amplitudes >4.5 × rms background) were elicited, of which 13 occurred during the perfusion plus 1 sec. α-LTX (20 n

m

) in normal recording medium was applied for 2 sec (second arrow). Then 51 spikes followed, beginning 6.9 sec from the start of the perfusion. A portion of the trace marked by the dashed lines_is shown with increased time resolution in the two lower contiguous traces. The dashed lines extending from the lowest contiguous trace indicate those spikes with amplitudes greater than 4.5 × rms background noise. The_lowest trace shows these spikes at further increased time resolution. The rightmost pair of events that are displayed appear to overlap slightly at the base. Interevent intervals were 1000 ± 492 msec (mean ± SEM) for high K+ and 1935 ± 556 msec for α-LTX. The quantal sizes elicited by the two secretagogues were not significantly different (p = 0.24; KS-Z = 1.0302, Kolmogorov–Smirnov test). The_top trace_ in B was digitally filtered at 1 kHz to provide better signal-to-noise resolution at low temporal resolution; the lower traces were digitally filtered at 10 kHz. Note that the same spikes are visible with both filters.

Fig. 3.

Fig. 3.

Electron micrograph of a rapidly fixed midbrain DA axonal varicosity in postnatal culture. The preparation was exposed to the osmophilic DA analog 5-hydroxydopamine (5-OHDA; 50 μ

m

for 12 hr at 36.7°C) and then fixed with a rapid exposure to glutaraldehyde, which is very effective at preserving intravesicular 5-OHDA. Two hundred and forty-six small synaptic vesicles (40–60 nm in diameter) labeled with 5-OHDA are present (two small vesicles are indicated by single arrows). One large dense-core vesicle is present (160 nm in diameter; double arrow);m indicates a mitochondrion. Ribosomes in a neighboring dendrite are visible at the scale bar. The large dense object in the varicosity could be an endosome, although the quality of the membrane preservation precludes certain identification. Additional micrographs of 5-OHDA-labeled vesicles in midbrain DA culture with the use of conventional fixation methods have been published (Sulzer and Rayport, 1990; Rayport et al., 1992). Scale bar, 1 μm.

Fig. 4.

Fig. 4.

Morphological effect of GDNF on midbrain DA neurons in culture. A, Three weeks after plating, TH immunostaining indicates a dense plexus of neurite outgrowth. The_arrows_ indicate TH-unlabeled cell bodies.B, Cultures exposed to GDNF display twofold more stained cell bodies (see Table 1) and also maintain a dense plexus of neurites.C, VMAT2 immunolabel in a control culture indicates sites containing dopaminergic synaptic vesicles.Arrowheads indicate examples of VMAT-labeled varicosities. D, Shown is a VMAT2 stain of a culture exposed to GDNF. The distribution of VMAT2-labeled varicosities along the axis of the axon and the two-dimensional structure of the varicosities were not altered by GDNF exposure (see Table 1). Scale bar, 50 μm.

Fig. 5.

Fig. 5.

Amperometric recording from a presumed axonal varicosity of a GDNF-exposed neuron before and after exposure to

l

-DOPA. α-LTX (20 n

m

) was perfused for 3 sec (first dotted line); the _trace_displays the period of 45–210 sec that follows (events at this site were not observed until 45 sec after α-LTX stimulation). The mean quantal size was 10,300 ± 1000 molecules (n = 49). Then the culture was exposed to 100 μ

ml

-DOPA for 30 min, and α-LTX was reapplied (second dotted line); the trace displays the period of 45–210 sec that follows. Of the total events elicited (n = 317), the mean size was 39,700 ± 3700 molecules; of these, n = 90 appear to be overlapping events. If apparent overlapping events (e.g.,rightmost pair of Fig. 2_B_,bottom expansion) are removed from consideration, the mean quantal size was increased to 18,300 ± 1600 molecules (p < 0.0001 different from control; KS-Z = 2.3389).

Fig. 6.

Fig. 6.

A, Sample amperometric events evoked from control neurons. B, Examples of events from GDNF-treated preparations. C, Examples of events after exposure to 20 μ

ml

-DOPA (30 min).D, Distributions of the untransformed (molecules/1000) and cubed root transformations (molecules 1/3) of quantal sizes, maximum amplitude (pA), and width (μsec) of the events reported in Table 2. In each case, exposure to

l

-DOPA or GDNF shifted the population parameters to higher values. Note that the control untransformed quantal sizes distribution uses a different _y_-axis scale than the GDNF and

l

-DOPA groups.

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