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. 2021 Dec 29;10:e71981. doi: 10.7554/eLife.71981

Figure 6. Cocaine increases branching of Apex2 dopamine axons in cocaine sensitized mice.

(A) Two representative reconstructions of dopaminergic (DA) axons each from saline- (left, blue) and cocaine- (right, red) treated mice. Green circles represent contact points (i.e., spinules) where other neurons interdigitate with the DA axon. Electron microscopy (EM) insets: four examples of contact points identified in 20 nm resolution datasets from control and cocaine datasets. (B) Scatter plot of the number of DA axons branches versus axon length (μm) (+saline: 0.01 ± 0.002 branches/µm length of axon, n = 44 axons, two mice; +cocaine: 0.04 ± 0.005 branches/µm length of axon, n = 41 axons, two mice. p = 3.96e-8. (C) Scatter plot of the number of contact points (i.e., ‘spinules’) versus axon length (µm) + saline: 0.2 ± 0.07 contact points (c.p.)/µm length of axon, n = 240 contact points over 14 axons, two mice; +cocaine: 0.2 ± 0.03 c.p./µm length of axon, n = 142 contact points over nine axons, two mice. p = 0.90). Data: mean ± SEM. p-Values: two-tailed Mann-Whitney U test. Scale bar = (A) reconstructions = 40 µm, EM insets = 1 µm.

Figure 6.

Figure 6—figure supplement 1. Cocaine-treated mice have increased locomotor activity.

Figure 6—figure supplement 1.

(A) Cartoon depicting cocaine sensitization protocol: Mice received a single intraperitoneal (IP) injection of cocaine (10 mg/kg) or equivalent volume of saline, placed in a novel cage environment, and their locomotor activity was monitored for 1 hr. This procedure was repeated every third day for four rounds of injections. Four days following the last injection (e.g., day 13), mice were perfused and brains processed for Apex2/EM staining (see Materials and methods). (B) Scatter plot of the average distance traveled (cm) versus time for the first day of cocaine or saline injection. (C) Total average velocity (cm) of mice immediately following IP cocaine or saline injections. For cocaine and saline, n = 3 mice each; data = mean ± SEM. p-Value: two-tailed Mann-Whitney U test.

Figure 6—figure supplement 2. Contact points from control and cocaine 20 nm resolution datasets.

Figure 6—figure supplement 2.

Two image montages of electron microscopy (EM) serial section (Z1–Z8) from either a saline- (top: 1,2) or cocaine- (bottom: 1,2) treated mice expressing cyto-Apex2 in dopaminergic (DA) neurons, and imaged at 20 nm in plane resolution. Red arrow points to the cyto-Apex2 DA axon, and blue arrow points to the Apex2-negative neurite interdigitating with the DA axon. Note how the Apex2-negative neurite becomes wholly contained within the DA axon. Scale bar = 1 µm.

Figure 6—figure supplement 3. Cocaine does not change the proportion of dopamine targets.

Figure 6—figure supplement 3.

Pie charts showing proportion of contact points (i.e., ‘spinules’) that are with chemical synapse (CS) axons (red) and dendrites (green) in saline- (top) and cocaine- (bottom) treated mice (+saline: 83% (49/59) axo-axonic, 17% (10/59) axo-dendritic, n = 19 dopaminergic (DA) axons with 59 contact points scored; +cocaine: 77% (56/73) axo-axonic, 23% (17/73) axo-dendritic, n = 20 DA axons with 73 contact points scored).