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1.
Figure 4

Figure 4. From: Nicotine-Mediated Activation of Dopaminergic Neurons in Distinct Regions of the Ventral Tegmental Area.

Differential expression of nAChR subunit genes in DAergic neurons within the aVTA and pVTA. TH-ir neurons from each region were collected by LCM, and nAChR expression was analyzed by qRT-PCR. A total of 23 104 and 23 886 of TH-ir neurons were collected from the aVTA and pVTA, respectively (n=6 mice per brain region). The line within each box plot represents the median difference of mRNA levels. The upper and lower edges of each box represent the 75th and the 25th percentile, respectively, whereas the upper and lower bars represent the maximum and minimum value, respectively. Expression of each subunit gene in the pVTA was compared with that in the aVTA using the 2−ΔΔCt method. *p<0.05.

Rubing Zhao-Shea, et al. Neuropsychopharmacology. 2011 April;36(5):1021-1032.
2.
Figure 5

Figure 5. From: Nicotine-Mediated Activation of Dopaminergic Neurons in Distinct Regions of the Ventral Tegmental Area.

Activation of α4* nAChRs is necessary and sufficient for nicotine activation of pVTA DAergic neurons. (a) Average number of TH-ir, c-Fos-ir neurons per slice from the aVTA or the pVTA (averaged regions as described in Figure 1) taken from WT (left) or α4 KO mice receiving two saline injections (white bars) or saline, followed by a 0.5 mg/kg nicotine injection (black bars). (b) Average number of TH-ir, c-Fos-ir neurons per slice from the aVTA or the pVTA in WT or Leu9′Ala mice receiving two saline injections (white bars) or saline, followed by a 0.01 mg/kg nicotine injection (black bars). Cells were counted from 26 to 36 VTA slices per mouse. n=3 mice per treatment. ***p<0.0001. Bonferroni's post-test.

Rubing Zhao-Shea, et al. Neuropsychopharmacology. 2011 April;36(5):1021-1032.
3.
Figure 6

Figure 6. From: Nicotine-Mediated Activation of Dopaminergic Neurons in Distinct Regions of the Ventral Tegmental Area.

Response to nicotine in DAergic neurons under voltage clamp. Representative whole-cell responses to nicotine in (a) aVTA or (b) pVTA DAergic neurons from α4 KO (left), WT (middle), and Leu9′Ala mice. Neurons were held at −60 mV, and 1 μM nicotine was bath applied for 4 min. (c) Average peak responses to 1 μM nicotine in the aVTA (white bar) or pVTA (black bar) DAergic neurons from each mouse line. Peak responses typically occurred 2–3 min after initial nicotine application. The gray bar illustrates the average peak response to nicotine in pVTA DAergic neurons after 10 min application of 100 nM α-conotoxin MII (E11A). (n=7 neurons per region per genotype). *p<0.05.

Rubing Zhao-Shea, et al. Neuropsychopharmacology. 2011 April;36(5):1021-1032.
4.
Figure 7

Figure 7. From: Nicotine-Mediated Activation of Dopaminergic Neurons in Distinct Regions of the Ventral Tegmental Area.

Effect of VTA infusion of an α6* nAChR antagonist on nicotine-induced VTA DAergic neuron activation. (a) Average number of TH-ir, c-Fos-ir neurons in the aVTA and the pVTA (averaged regions as described in Figure 1) from WT mice receiving VTA infusion of vehicle (black bar) or α-conotoxin MII (E11A) (gray bar), followed by a 0.5 mg/kg nicotine injection. Control mice received infusion of 100 nM conotoxin MII (E11A), followed by a saline injection. (b) Average number of TH-ir, c-Fos-ir neurons in the aVTA and pVTA (averaged regions as described in Figure 1) from Leu9′Ala mice receiving VTA infusion of vehicle (black bar), or 100 nM α-conotoxin MII (E11A) infusion (gray bar), followed by a 0.01 mg/kg nicotine injection. Control mice received infusion of conotoxin MII (E11A), followed by a saline injection. Cells were counted from 25 to 34 VTA slices per mouse (n=3–4 mice per treatment). ***p<0.001.

Rubing Zhao-Shea, et al. Neuropsychopharmacology. 2011 April;36(5):1021-1032.
5.
Figure 2

Figure 2. From: Nicotine-Mediated Activation of Dopaminergic Neurons in Distinct Regions of the Ventral Tegmental Area.

Nicotine selectively activates DAergic neurons within the pVTA. (a) Photomicrographs illustrating representative midbrain sections that include the aVTA (top), pVTA (middle), or tVTA (bottom) from C57Bl/6J mice injected with 0.5 mg/kg nicotine. Sections are immunolabeled for TH (red) and c-Fos (green). White boxes delineate slice regions that are magnified in the adjacent photomicrographs. White arrowheads point to neurons which are TH-ir, c-Fos-ir. Here, 40 × magnification images that illustrate labeling of TH (left), c-Fos (middle), and both (right), indicating nuclear localization of c-Fos in TH-expressing neurons are shown. Scale bar=100 μm. (b) Number of TH-ir, c-Fos-ir neurons per VTA slice taken from mice administered two saline injections (Sal/Sal), a saline injection 15 min before a 0.5 mg/kg nicotine injection (Sal/NIC) or 3 mg/kg mecamylamine 15 min before nicotine (MEC/NIC). Distance from bregma is indicated on the x axis in millimeters. In all, 48 slices per treatment per mouse were analyzed (n=3 mice per treatment). ***p<0.001, compared with saline and mecamylamine treatment using two-way ANOVA and Bonferroni's post-test.

Rubing Zhao-Shea, et al. Neuropsychopharmacology. 2011 April;36(5):1021-1032.
6.
Figure 3

Figure 3. From: Nicotine-Mediated Activation of Dopaminergic Neurons in Distinct Regions of the Ventral Tegmental Area.

Direct activation of VTA DAergic neurons by nicotine. (a) Diagram of a mesocortical slice that includes the pVTA (red circle). Recordings were made from both pVTA and aVTA neurons. (b) Current-clamp (I=0) trace illustrating the baseline firing frequency of a VTA DAergic neuron. (c) Whole-cell voltage clamp recording from a VTA neuron that expresses the hyperpolarization-activated cation current, Ih. The neuron was voltage clamped at −60 mV and hyperpolarized to −120 mV for 1.0 s before returning to the holding potential (Top). (d) Representative current-clamp (I=0) recording from an aVTA DAergic neuron at baseline (ACSF, left), during application of 1 μM nicotine (middle, 3 min application), and after washout of nicotine (right). (e) Representative current-clamp (I=0) recording from a pVTA DAergic neuron at baseline (ACSF, left), during application of 1 μM nicotine (middle, 3 min application), and after washout of nicotine (right). Burst firing upon washout (3–7 spikes per burst) was seen in 5 of 7 neurons with robust nicotine responses. (f) Summary of responses to nicotine in DAergic neurons from either the aVTA (white bar, n=8/8) or the pVTA (black bar, n=7/16). Each bar graph represents the fold change in action potential number produced by nicotine normalized to the baseline firing frequency. The response to nicotine in pVTA DAergic neurons was blocked by pre-exposure to 10 μM mecamylamine (gray bar). (g) Average response to nicotine as described in panel f (n=5 neurons per region). Responses were recorded in cell-attached mode at 30–32°C. **p<0.01.

Rubing Zhao-Shea, et al. Neuropsychopharmacology. 2011 April;36(5):1021-1032.
7.
Figure 1

Figure 1. From: Nicotine-Mediated Activation of Dopaminergic Neurons in Distinct Regions of the Ventral Tegmental Area.

Definition, size, and density of DAergic neurons within the aVTA, the pVTA, and the tVTA. (a–c) Definitions of VTA subregions (Ikemoto, 2007; Kaufling et al, 2009; Paxinos and Franklin, 2000; Perrotti et al, 2005; Shabat-Simon et al, 2008). Representative midbrain slices containing the aVTA, pVTA, or tVTA. TH-ir neurons are labeled red. Brain region borders are outlined in white. (Panel a) aVTA (bregma from −2.92 to −3.28 mm): defined as the region dorsal to the medial mammillary nucleus and medial to the substantia nigra pars compacta (SNC). It contains two subregions: the ventral tegmental area rostral (VTAR) and the parabrachial pigmental area (PBP). The aVTA does not include midline nuclei such as the interfascicular nucleus (IF) and the rostral linear nucleus (RLi). A10 DAergic neurons located in the supramammillary nucleus described by Hokfelt et al (1984a, 1984b) also are not included in the aVTA. (Panel b) pVTA (bregma from −3.28 to −3.80 mm): defined as the region dorsal to the interpeduncular nucleus (IPR), medial to the SNC and ventral to the red nucleus (RPC and/or RMC). It contains three subregions: the PBP, ventral tegmental area caudal (VTAC), and the paranigral nucleus (PN). The pVTA does not include the midline nuclei, such as the IF and the RLi and/or the caudal linear nucleus (CLi). A10 DAergic neurons located in the dorsal raphe nucleus described by Hokfelt et al (1984a, 1984b) are not included in the pVTA. (Panel c) tVTA (bregma from −3.80 to −4.04 mm): defined as the most caudal extent of the VTA. Rostrally, the tVTA is limited to a subregion posterior to the PN and dorsolateral to the IP. More caudally, the position of the tVTA shifts dorsally and slightly laterally to become embedded within the superior cerebellar peduncle decussation (SCP). The tVTA has a low density of DAergic neurons and a high density of GABAergic neurons. The tVTA does not include the midline nuclei RLi or CLi. (Panel d) Quantification of DAergic neuron soma size in the aVTA (open bar), the pVTA (filled bar), and the tVTA (gray bar). Each bar represents the percentage of DAergic neurons with the indicated soma diameter (x axis in μm). (Panel e) Bar graph representation of DAergic neuron density in the aVTA, the pVTA, and the tVTA. In all, 2508–2864 DAergic neurons were measured per mouse (n=3). **p<0.01, ***p<0.001 compared with that of aVTA. #p<0.001 compared with that of aVTA and pVTA.

Rubing Zhao-Shea, et al. Neuropsychopharmacology. 2011 April;36(5):1021-1032.

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