New method to visualize neurons with DAT in slices of rat VTA using fluorescent substrate for DAT, ASP+

Abstract

The ventral tegmental area (VTA), and in particular dopamine (DA) neurons in this region of midbrain, has been shown to play an important role in motivation (goal-directed behavior), reward, and drug addiction. Most evidence that implicates VTA DA neurons in these functions are based on widely accepted but indirect electrophysiological characterization, including the hyperpolarization activated non-specific cation current (Ih), spike frequency, and inhibition by D2 receptor agonists. In this study, we used a known neuronal dopamine transporter (DAT) fluorescent substrate [4-(4- (dimethylamino) styryl)-N-methylpyridinium iodide] (ASP+) to visualize DAT-containing cell bodies of DA neurons in VTA region in rat brain slices. Uptake of 100 nM of ASP+ in brain slices of rat VTA region marked 38% of visible neurons, while other neurons from this region and 100% neurons from hippocampus slices were not fluorescent. Using patch-clamp techniques, we have found that pronounced Ih current was present in all fluorescent neurons from VTA area, also spike frequency was similar to the widely accepted values for DA neurons. Furthermore, additional study has shown that there are 84% coincidence of ASP+ fluorescence in neuronal cell bodies and Falck-Hillarp labeling of DA cells. Electrophysiological recordings during ASP+ application have confirmed that low concentrations (100 nM) of ASP+ have no visible effect on neuronal activity during 1-2 hours after staining. Thus, uptake of fluorescent monoamine analog ASP+ by DAT can be an additional criterion for identification of DAT-containing neurons in slices.

Keywords: DAT fluorescent substrate; dopamine neurons; ventral tegmental area.

Fig.1

A. Whole cell patch-clamp recording from fluorescent cells after slice being stained with 100 nM ASP+. Upper trace: Stimulation step protocol (see. Methods), Middle trace: Recording of fluorescent DA neuron currents as a response to series of 10 (1 sec) 10 mV voltage step stimulations, from −30 to −120 mV. Lower trace: Recording of DA neuron voltage responses to series of 10 (1 sec duration) 200 pA current step stimulations, from +200 pA from holding level to −1400 pA (−45 mV). B. Fluorescent (left) and infra-red (IR) image (right) of path-clamp recording from fluorescent neuron in VTA. Electrode is positioned to record from fluorescent neuron (yellow arrow) that coincides with infra-red image of neuronal cell body, while another neuronal cell body(red arrow) that have no florescence can be seen in IR image as well. Scale:100 µm. C. Double staining of VTA neurons with Falck-Hillarp method and ASP+. From left to right: (1) F-H fluorescence (green); (2) ASP+ fluorescence (red); and (1+2) their colocalization (see Methods). White arrows point to fluorescent neuronal cell bodies that coincide in 1 and 2. Yellow arrow point on neuronal cell body that have F-H fluorescence but have no ASP+ fluorescence. Blue arrows point to blood vessels. Scale:100 µm. (See also web content for animated picture). D. External application of 20 µM of dopamine produced visible inhibition of fluorescent neuron activity.

Fig.2

Confocal images of live brain slices stained with 100 nM ASP+. A - Control staining of hippocampus CA3 zone observed with 10× objective. B. The same CA3 zone with 40× oil objective. Stratum radiatum (with fluorescent astrocytes) and pyramidal cells layer with low fluorescent neurons can be seen (see also Web content Fig 2 for 3D image of this zone). C- Low magnification (10 × objective) of VT midbrain slice, showing overall layout of fluorescent cells in that zone. D - Fluorescent neurons and blood vessels in VTA at higher magnification (40× oil objective), while some of neurons have fluorescence lower, than background (see also Web content Fig 2 for 3D image of this zone).