Streptavidin alexa fluor 488

Immunohistochemistry and confocal microscopy were performed as described previously^{4 (link), 5 (link)}. Briefly, after intracardial perfusion with 4% paraformaldehyde in PBS, pH 7.4, the brains were post-fixed overnight and coronal midbrain slices (50 or 100 μm) were prepared. The primary antibody used were mouse anti-tyrosine hydroxylase (TH) (1:1000; Millipore, Temecula, CA, USA), rabbit anti-tyrosine hydroxylase (TH) (1:1000; Calbiochem, San Diego, CA, USA), rabbit anti-PHA-L (1:1000; Vector, Burlingame, CA, USA), goat anti-glutamate transporter (EAAC1; 1:1000; Millipore), rabbit anti-ChAT (1:200; Millipore), mouse anti-GAD67 (clone 1G10.2; 1:500; Millipore), rabbit anti-c-fos (1:500, Calbiochem) and rabbit anti-NeuN (1:1000; Millipore). The secondary antibodies used were Alexa Fluor488 anti-rabbit, AlexaFluor546 anti-goat, AlexaFluor546 anti-rabbit, AlexaFluor546 anti-mouse, Alexa Fluor647 anti-rabbit, Alexa Fluor647 anti-mouse (all 1:750), AlexaFluor488 streptavidin (1:1000) (all Molecular Probes, Eugene, OR). Image acquisition was performed with a confocal system (Zeiss LSM510) using 10x, 40x or 63x objectives and on a Zeiss AxioImager M1 upright widefield fluorescence/DIC microscope with CCD camera using 2.5x and 10x objectives. Images were analyzed using the Zeiss LSM Image Browser software and ImageJ software.
For quantification of ChR2-EYFP fluorescence intensity and quantification of c-fos-positive cells, confocal images were acquired using identical pinhole, gain, and laser settings. Images in the medial and lateral VTA as well as the SN from the same tissue sections were acquired at the same focus level. The medial and lateral VTA was defined as the area that corresponds to the anatomical location of distinct DA subpopulations^{4 (link), 5 (link)}. The medial VTA was defined as the region comprising the medial paranigral nucleus (PN) and medial parabrachial pigmented nucleus (PBP), while the lateral VTA was defined as the lateral parabrachial pigmented nucleus (Supplementary Fig. 8c). No additional post-processing was performed on any of the collected images. ChR2 fluorescence intensity was then quantified using a scale from 0 – 255 in ImageJ to determine the mean intensity across the entire image. For retrobead, AAV and PHA-L injections as well as RV injections in the mPFC and NAc lateral shell the injection-sites were confirmed in all animals by preparing coronal sections (100 μm). Counterstaining of injection sites was performed with green or red Nissl (NeuroTrace 500/525 or 530/615, Molecular Probes, Eugene, OR).
We routinely carried out complete serial analyses of the injection sites. Animals with significant contaminations outside target areas were discarded (see Lammel et al., 2008^{4 (link)} for serial analysis of retrobead injection-sites and definition of DA target areas). For RV injections into the VTA we confirmed that all animals had the center of the viral injection located in the caudal VTA (Bregma -3.4 mm). However, quantification of the “spread” of the RV-ChR2 injected into the VTA is difficult because for expression of the transgene, the RV must be taken up by terminals and the transgene must be synthesized in the cytosol and then transported within the axons. Any EYFP within the VTA and adjacent structures will represent axons/terminals of cells that project to the VTA and adjacent structures as well as the cell bodies of neurons (i.e. RMTg) that have local connectivity within the VTA and adjacent structures. Thus transgene expression in structures adjacent to the VTA does not indicate that LHb or LDT neurons project to these structures. Nevertheless, in Supplementary Fig. 15 we present a serial reconstruction for the caudo-rostral extent of the midbrain showing the expression of ChR2-EYFP one week after injection of RV-ChR2 into the VTA (n=5 mice). TH-stained coronal midbrain sections (100 μM) were prepared from the injected mice and reconstructed using Neurolucida software (MicroBrightfield, Colchester, VT). Sections were labeled relative to bregma using landmarks and neuroanatomical nomenclature as described in the Franklin and Paxinos mouse brain atlas (2001). We report all brain areas in which detectable EYFP was observed. The strongest transgene expression was observed in the caudal VTA and several of its distinct subnuclei, most commonly in the interpeduncular nucleus (IPN). We also always detected high transgene expression in the RMTg. Thus when referred to in the text, the VTA includes the RMTg, which was originally termed the “tail of the VTA”^{22 (link)}.
Because the IPN expressed ChR2-EYFP following intra-VTA injections, we conducted additional double retrograde tracing experiments in which we injected small amounts of green Retrobeads (20 nl; LumaFlauor Inc., Naples, FL) into the IPN (bregma -3.9 mm; lateral 0 mm; ventral 4.55 mm) and red Retrobeads (60 nl; LumaFlauor Inc., Naples, FL) into the VTA (bregma -3.4 mm; lateral 0.35 mm; ventral 4.0 mm). Fluorescently-labeled latex Retrobeads were used in these experiments (n=2 mice) because they show very limited diffusion from the injection site even after several weeks in vivo and thus can be highly localized. While a large number of cells in the lateral habenula contained red beads (~84%, 79/94 cells), confirming a projection from this structure to the VTA, only a small proportion of these cells (~12%, 11/94 cells) also contained green beads (Supplementary Fig. 16). In contrast, a large number of medial habenula cells contained green beads (~98%, 214/218 cells) and less than 2% (3/218 cells) of these also contained red beads (Supplementary Fig. 16), demonstrating that the medial habenula preferentially projects to the IPN. In the LDT, many cells (>100) contained red beads and none of these cells contained green beads (Supplementary Fig. 16). These results suggest that LDT cells likely only project to VTA and not the IPN while the proportion of LHb neurons that project to the IPN in addition to the VTA is small.
For quantification of the expression of RV-ChR2-EYFP in the LDT and LHb 50 μm coronal sections from mice which had been injected with RV-ChR2-EYFP in the VTA were stained for NeuN. 66 confocal images from the LDT and 55 confocal images from the LHb were obtained using a 40X objective (n=3 mice). The percent of ChR2-EYFP-positive cells relative to the number of NeuN-positive cells in a 125 μm × 125 μm area was analyzed using the ImageJ software. Approximately 20% of all NeuN-positive LDT and LHb neurons expressed ChR2-EYFP following RV-ChR2 injection into the VTA (Supplementary Fig. 15).