Cue-induced heroin seeking after withdrawal
Rats demonstrated reliable heroin self-administration (Fig. 1a, final n = 85). The mean ± SEM daily heroin intake (infusions per 6 h) over 10 days was 24 ± 1.4. Three rats were excluded because of failure to reach a mean of 10 infusions per day.
Figure 1. Rats self-administer intravenous heroin and show cue-induced heroin seeking after prolonged withdrawal from the drug. (a) Rats learn to self-administer heroin over 10 days of training. Data represent mean ± SEM heroin infusions per day (total n = 85). (b) Cue-induced heroin seeking in the extinction test. During the extinction tests, active lever presses resulted in delivery of light cue previously paired with heroin infusions, but not heroin; inactive lever presses had no consequences. Total number of lever presses during extinction tests (mean ± SEM; *p < 0.05 vs. inactive lever presses for corresponding withdrawal day). (c) Time course of active lever presses during the extinction tests (mean ± SEM; *p<0.05 vs. lever presses at 60-min and 90-min time points for corresponding withdrawal day).
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Cue-induced heroin seeking was operationally defined as the number of non-reinforced active lever presses during the extinction tests after 14 (n = 41) or 30 days (n = 9) of withdrawal. The number of active lever presses during extinction tests were similar for rats withdrawn for 14 and 30 days (Fig. 1b and c), and were significantly higher than inactive lever presses. The anova for active lever responding during the entire 90-min test session (Fig. 1b), indicated a significant effect of Lever (F1,48 = 71.9, p < 0.001), but not Withdrawal Day or an interaction between the two factors (p values > 0.05). Likewise, the anova of the within-session time course active lever presses (Fig. 1c), which included the between-subjects factor of Withdrawal Day and the within-subjects factor of Session Time in 30-min intervals, indicated a significant effect of Session Time (F1,47 = 18.7, p < 0.001), but no effects of Withdrawal Day or interaction between the two factors (p values > 0.05).
Experiment 1: FACS purification and gene expression in activated prefrontal cortex neurons
As behavioral results for the Extinction-test groups and the No-test groups were similar after 14 and 30 withdrawal days, gene expression results from these two withdrawal times were combined. Prefrontal cortex tissue containing OFC and mPFC was obtained from rats immediately after the 90-min extinction tests (Extinction-test group) or immediately after transport from the housing facility (No-test group). The majority of dissociated cortical cells were found in the lower left quadrant of the forward and side light scattergrams (Fig. 2a, left panel), similar to neurons obtained in (Guez-Barber et al. 2011, 2012). Events in this quadrant were selected for further examination using a gate encompassing this quadrant and incubating them with DAPI (Fig. 2b). Microscopy indicated that most events were round DAPI-labeled cell bodies devoid of processes.
Figure 2. Fluorescent-activated cell sorting (FACS) of Fos-labeled neurons from medial prefrontal cortex (mPFC) and orbital prefrontal cortex (OFC) after prolonged withdrawal from heroin self-administration. (a) Left panel: representative light scatter plot in which each dot represents one event (cell or debris). Forward scatter is a measure of size; side scatter is a measure of granularity. The ‘gate’ indicated by the solid-line box encompasses intact cells. Right panel: representative light scatter and fluorescence plot showing only events from the ‘cells’ gate in the left panel scatter plot. Events high on the Y axis represent neurons. A second gate indicated by the solid-line box labeled ‘neurons’ encompasses all events analyzed on subsequent fluorescence plots. These gates allow selective analysis of neurons and exclusion of other cells and debris. (b) Photomicrographs of FACS-purified cells labeled with 4',6-diamidino-2-phenylindole (DAPI). Left panel: Bright-field image of intact cells indicated by arrows. Middle panel: fluorescence image of blue DAPI labeling of intact cells indicated by arrows. Right panel: fluorescence image of red NeuN labeling of neurons indicated by arrows. (c) Representative light scatter and fluorescence plots from the Extinction-test and No-test rats indicate degree of Fos-labeling of neurons selected by event gates in Fig. 2a. Events high and low on the Y-axis represent activated neurons (Fos+) and non-activated neurons (Fos-). (d) Percentage of activated (Fos+) neurons in the Extinction-test and No-test conditions (mean ± SEM, *p < 0.05, n = 3–5 pooled samples (n = 46–60 rats) per experimental condition).
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After selection of cells encompassed by the first gate from forward and side light scattergrams, a second forward gate was applied to include only cells labeled for the neuronal marker NeuN (Fig. 2a, right panel). Cells within this second gated region (neurons) were then sorted for activation state according to their level of immunolabeling with Fos antibodies (Fig. 2c).
Figure 3. Quantitative PCR indicates different gene expression profile for Fos-positive medial prefrontal cortex (mPFC) and orbital prefrontal cortex (OFC) neurons compared with Fos-negative neurons from the same Extinction-test rats, or compared with all neurons from No-test rats. (a) mRNA levels for the immediate early genes arc, fosB, egr1, and egr2 are increased in Fos-positive neurons from the Extinction-test rats. Dotted line represents gene expression level in all neurons from the No-test rats (set at 1); *p < 0.05 versus Fos-negative neurons from same Extinction-test rats; p < 0.05 versus all neurons from No-test rats. (b) mRNA levels for npy and map2k6 are increased in Fos-positive neurons; *p ≤ 0.05 versus Fos-negative neurons from the same Extinction-test rats. p < 0.05 versus all neurons from No-test rats. Data are mean ± SEM; n = 3–5 pooled samples (n = 46–60 rats) per experimental condition.
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From Extinction-test rats, 9.9% of NeuN-labeled neurons had Fos expression levels above threshold (Fig. 2c and d). We obtained approximately 62 500 Fos-labeled neurons from each rat. All cells labeled for Fos also expressed NeuN; thus only neurons expressed Fos. In contrast, only 4.4% of NeuN-labeled neurons had Fos expression levels above threshold. Overall, heroin cue exposure in the extinction tests induced significantly more Fos-labeled cells than in the No-test group (t(11) = 6.43, p < 0.05), which supports Fos immunoreactivity as a marker of neural activation.
We collected Fos-positive and Fos-negative neurons from the Extinction-test group for subsequent qPCR analyses. In contrast, for control samples, we collected all NeuN-labeled cells from the No-test rats for subsequent qPCR analyses, without separating Fos-positive from Fos-negative neurons. Approximately 4.5% of all events from FACS analysis of No-test samples were NeuN labeled, which corresponded to approximately 302 000 cortical neurons per rat. Our rationale for this control condition was that most heroin cue-activated neurons in the extinction tests are not activated prior to testing; thus, any Fos-positive neurons in the No-test neuron population are not related to the extinction test. Thus gene expression in Fos-positive neurons from the No-test group likely do not represent basal gene expression for other neurons that will eventually be activated during the extinction tests. Any gene expression alterations within Fos-expressing neurons in the No-test samples, which are not associated with the extinction tests, would be diluted 20- to 25-fold and thus have a minimal effect on overall gene expression in these control samples.
Overall, we used gene expression in all neurons from the No-test groups to represent a hypothetical baseline gene expression for all neurons prior to heroin cue exposure in the extinction tests. We then calculated gene expression alterations in Fos-positive and Fos-negative neurons from the Extinction-test group relative to gene expression in all neurons from the No-test group.
For qPCR, Kruskal–Wallis non-parametric tests were performed across three groups of samples: (i) Fos-positive and (ii) Fos-negative neurons from the Extinction-test rats and (iii) all neurons from No-test rats (n = 3–5 samples for each condition; each sample represented 10–11 rats for the Extinction-test group and 4–9 rats for the No-test group). IEG expression was increased in only Fos-positive neurons obtained from the Extinction-test group. mRNA levels for arc, fosB, egr1, and egr2 were elevated in Fos-positive neurons compared with Fos-negative neurons obtained from the same rats (Fig. 4, Χ2(2) = 9.6, 9.4, 6.0, and 8.7, respectively, p ≤ 0.05). Levels of arc, fosB, and egr2 mRNAs in Fos-positive neurons, but not Fos-negative neurons, were also elevated relative to all NeuN-labeled neurons obtained from the No-test group (p < 0.05). Expression levels for npy and map2k6 were elevated in Fos-positive neurons compared with Fos-negative neurons obtained from the Extinction-test rats and compared with all neurons obtained from No-test rats (Χ2(2) = 6.5 and 6.4, respectively, p < 0.05). In contrast, npy expression in Fos-negative neurons from Extinction-test rats was decreased relative to all NeuN-labeled neurons from No-test rats (p < 0.05).
Figure 4. Immunohistochemical characterization of Fos-expressing neurons in medial prefrontal cortex (mPFC) and orbital prefrontal cortex (OFC) after cue-induced heroin seeking on withdrawal day 14. (a) Schematic showing regions analyzed using fluorescent immunohistochemistry (Paxinos and Watson 2005). Representative images: (b) Fos+Arc double labeling in dmPFC, scale bar 50 μm; Arc labeling in red, Fos labeling in green, examples of double-labeled neurons in red and green indicated by white arrows. (c) Fos+neuropeptide Y (NPY) double labeling in OFC, scale bar 50 μm; NPY labeling in green, Fos labeling in red, example of double-labeled neuron in red and green indicated by white arrow.
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Experiment 2: fluorescent immunohistochemistry
To confirm increased expression of npy and arc in Fos-positive neurons, we assessed coexpression of NPY and Arc protein with Fos in histochemical sections of dmPFC, vmPFC, and OFC (Fig. 4 and Table 2). We found that 37% of all Fos-labeled neurons coexpressed Arc (Fig. 4b), and 7.5% of all Fos-labeled neurons coexpressed NPY (Fig. 4c). This represents a preferential activation of NPY-containing neurons, because NPY-containing neurons comprise only 10% of all GABA neurons in the cortex (Kubota et al. 2011), and GABA neurons comprise ~25% of cortical neurons (Tamminga et al. 2004); thus, NPY-containing neurons only comprise 2.5% of all cortical neurons, but comprise 7.5% of all Fos-labeled neurons in our Extinction-test rats. This indicates that Fos-positive neurons are three times more likely to contain NPY than the overall distribution of NPY-containing neurons in cortex.
Table 2. Characterization of Fos-positive neurons in PFC of rats exposed to heroin cues in the extinction tests (n = 4–7 rats)
|Percent Fos-positive neurons containing NPY:||3.85 ± 3.4||4.29 ± 4.8||14.38 ± 7.2||7.51 ± 2.7|
|Percent of Arc-positive neurons containing Fos:||53.32 ± 7.2||44.58 ± 10.8||49.86 ± 9.2||49.59 ± 2.3|
|Percent of Fos-positive neurons containing Arc:||37.02 ± 5.1||22.85 ± 6.7||32.47 ± 7.3||31.35 ± 1.7|