-80 Figure 1. Identification of dopaminergic neurons in. VTA slices (a) Micrographs demonstrate the location of the VTA with
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1 Potential (mv) A B C Potential (mv) D E F Biocytin TH Merge R G H Supplemental -8-8 Figure 1. Identification of dopaminergic neurons in -1 VTA slices. -1 (a) Micrographs demonstrate the location of the VTA with -12 the landmark -12 of the substantia nigra compacta (SNc) in a brain slice Time (s) Time (ms) Scale bar, 5 μm, (b) where the stimulating electrode (S) was placed rostral to the recording microelectrode (R). Scale bar, 2 μm, (c) when recording from a VTA neuron. Scale bar, 25 μm. (d) Micrographs of a recorded VTA neuron filled with biocytin and (e) expressing tyrosine hydroxylase (TH), and (f) a superimposed image. Scale bar, 4 μm. Inset in (f): Membrane currents elicited by voltage steps from -7 mv to -5 to -14 mv, in -1 mv-increments in this dopaminergic neuron. Scale bars, 2 ms and 4 pa. Besides, (g) dopaminergic neurons have lower basal firing rates. Scale bars, 1s and 2 mv, (h) wider action potentials, Scale bars, 5 ms and 2 mv. and stronger firing adaptation than GABA neurons. Note that robust Ih currents were elicited by hyperpolarizing voltage steps. VTA: Ventral Tegmental Area, SNc: Substantia Nigra compacta, SNr: Substantia Nigra reticulate.
2 Te mv Te mv Current (pa) Current (pa) IPSC (% of Baseline) 14 1 nm Orexin A Time (ms) Time (ms) Sw 122/123 Time Sw (ms) 121/ Time 2 3 Supplemental Figure 2. Long term treatment with orexin A did not cause receptor desensitization. Time courses of effects of orexin A (1 nm) on IPSCs (n=4). Upper panels are the representative IPSC traces taken at time point 1, 2 and 3, respectively, from a neuron. Scale bars, 5 ms and 2 pa.
3 Current (pa) Te pa EPSC (% of Baseline) nm Orexin A Time (ms) Sw 151 Time (ms) Time (ms) Sw 153/153 Sw 152/ Time (min) Supplemental Figure 3. Orexin A did not depress evoked EPSCs Time courses of effects of orexin A (1 nm) on EPSCs (n=5). Upper panels are the representative IPSC traces taken at time point 1, 2 and 3, respectively, from a neuron. Scale bars, 5 ms and 2 pa. 3
4 A B IPSC (% of Baseline) 3 M AM nm Orexin A Time (min) IPSC (% of Baseline) ** (3) Orexin A Orexin A + AM251 # Supplemental Figure 4. Orexin A depressed IPSCs in dopaminergic neurons of mouse VTA slices in a manner reversed by a CB1R antagonist. (a) The time course of the effect of orexin A (1 nm) on the IPSC amplitude before and after treatment with AM 251 (3 μm). (b) The effect of orexin A (n=3) (black bar) alone or in combination with AM 251 (right slashed bar) on the IPSC amplitude. Data analysis and presentation are the same as Fig. 1. **p<.1 (one sample t test); # p<.5 vs. orexin A alone (paired t test)
5 PAG fr mm VTA -3.8 mm MM SNc mm SNr mm Supplemental Figure 5. Reconstructed bilateral injection sites in the VTA are shown in drawn midbrain coronal sections. Distance from bregma is shown to the right of each section (in millimeters) 1. Injection sites from mice used in the stress induced reinstatement experiment, on site (closed circles); off site (open circles). fr: fasciculus retroflexus, MM: Medial Mammill, PAG: Periaqueductal gray, SNc: Substantia Nigra compacta, SNr: Substantia Nigra reticulate,vta: Ventral Tegmental Area.
6 Supplementary Table 1. Endocannabinoid levels in the VTA of extinguished mice with and without restraint stress. No Stress Stress Endocannabinoids (pmol/g tissue) 2-AG ± ± 42.73* 2-OG ± ± 32.8 P-Gly ± ± PEA ± ± SEA ± ± OEA ± ± 1.11 LEA ± ± 3.79 Mice were randomly divided into restrained and non-restrained groups. Both groups received the same cocaine-conditioning and extinction training in the CPP test. After extinction, the restrained, but not the non-restrained, group received a 3-min restraint stress. Brain tissue containing the VTA was quickly isolated after the restraint stress. Endocannabinoids were extracted and measured by LC/MS/MS as described in Methods. Data are presented as means ± SE. *p<.5, n=8 for each group. 2-AG, 2-arachidonyl glycerol; 2-OG, 2-oleoylglycerol; P-Gly, N-palmitoleoyl glycine; PEA, palmitoylethanolamine; SEA, stearoylethanolamine; OEA, oleoylethanolamine
7 and LEA, dihomo-γ-linolenoylethanolamine.
8 Supplementary References 1. Paxinos G, Franklin K. The Mouse Brain in Stereotaxic Coordinates (Deluxe Edition), Second edn. Academic Press, California, USA (21).
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