METH CPP Preference Score, Locomotor Sensitization and Conditioned Hyperactivity.

<p><b>A)</b> Preference score in WT and mGlu5 KO mice at habituation and test. Preference score is defined as (time spent in the METH-paired compartment – time spent in the saline-paired compartment). <b>B)</b> Locomotor sensitization in WT and mGlu5 KO mice following acute saline or 2 mg/kg METH i.p. injection over 4 consecutive days. <b>C)</b> Conditioned hyperactivity in WT and mGlu5 KO mice during the CPP test. Data (mean±SEM) analysed using two- or three-way RM ANOVA, followed by one-way ANOVA split by the factor ‘genotype’ with a Bonferroni correction. In Figs <b>A)</b> and <b>C)</b>, significant effects of ‘day’ (<i>vs.</i> habituation) are represented by “#” (^###<i>p</i><.001); significant effects of ‘genotype’ (<i>vs</i>. WT on the same day) are represented by ‘*’ (**<i>p</i><.01). In Fig <b>B)</b>, significant effects of ‘day’ (vs. METH day 1) are indicated by ‘$’ for WT mice (^$$$<i>p</i><.001), ‘#’ for mGlu5 KO mice (^###<i>p</i><.001). Significant effects of ‘day’ (vs. saline day 1) are indicated by ‘*’ for WT mice (***<i>p</i><.001) and ‘?’ for mGlu5 KO mice (???<i>p</i><.001). A significant ‘day’ × ‘genotype’ interaction was present in both Fig. A) and C), suggesting <b>A)</b> a greater change degree of change from habituation to test in WT compared to mGlu5 KO mice, and <b>C)</b> a greater change degree of change from habituation to test in mGlu5 KO compared to WT mice. Abbreviations: Saline 1 - day 1 of saline treatment.</p

Extinction and reinstatement of the operant response for sucrose.

<p><b>A)</b> Average active lever presses during Stable FR1 and the final 2 days of extinction. Both genotypes significantly decreased their active lever presses during both days of extinction. <b>B)</b> Average active lever presses during extinction and cue-induced reinstatement. Both genotypes increased their active lever pressing in the reinstatement test following extinction. Data (mean±SEM) analysed using two-way RM ANOVA, followed by one-way ANOVA split by the factor ‘genotype’ with a Bonferroni correction. Significant effects of ‘day’ (<i>vs.</i> Stable FR1) are represented by “#” (^##<i>p</i><.01; ^###<i>p</i><.001). Abbreviations: EXT - final extinction score; FR1– fixed ratio 1; Reinst - reinstatement.</p

Lever Responses and Infusions during METH IVSA FR1 Acquisition, Self-administration, Extinction and Reinstatement.

<p>Stable FR1 infusions averaged over 5 days of stable self-administration; extinction data averaged over the final two days of extinction. Data presented as means±SEM. Data analysed using two- or three-way RM ANOVA, followed by one-way ANOVA split by corresponding factor, where appropriate. Significant effects of ‘lever type’ are indicated by hash symbols (<i>vs</i>. inactive lever; ^##<i>p</i><.01, ^###<i>p</i><.001); significant effects of ‘genotype’ are indicated by asterisks (*<i>p</i><.05); significant effects of ‘day’ are indicated by ‘$’ (^$<i>p</i><.05). Abbreviations: ALP - active lever press; FR1 - fixed ratio 1; ILP - inactive lever press.</p

Saccharin Two-Bottle Free-Choice Drinking.

<p>Rxfp3 gene deletion does not alter taste perception or motivation to consume saccharin in mice. <i>(a)</i> Average daily saccharin consumption, <i>(b)</i> saccharin preference, and <i>(c)</i> total fluid intake of Rxfp3 KO mice (n = 11) and WT littermate controls (n = 14) over the final week of saccharin access. Data are presented as mean ± S.E.M.</p

Opioid neurochemistry in brains of offspring.

<p>Evaluation of [³³P]preproenkephalin (PPE) mRNA binding in the striatum (Str) by <i>in situ</i> hybridisation histochemistry (ISHH), with comparison between offspring groups (placebo; naltrexone) and drug treatments received during behavioural sensitization (saline-saline, SS; saline-morphine, SM; morphine-morphine, MM). A: Representative autoradiograms showing localisation of [³³P]PPE mRNA binding sites within midsagittal sections of whole rat brain at approximately 4.0 mm from the midline. Relative PPE mRNA expression within Str presented in accordance with quantitative ISHH (A), which was performed as described in <i>Materials and Methods</i>. As indicated by the density scale, the highest binding levels appear as black. B: Quantitative data for PPE mRNA binding in Str are expressed in disintegrations per minute per millimetre squared (DPM/mm²) and represent the mean ± SEM. Using generalised linear mixed modelling: * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001, naltrexone versus placebo by SS versus SM versus MM, naltrexone: n = 14; placebo: n = 17; n≈5/column.</p

Instrumental requirement for PR9-4 and PR3-4 schedules.

<p>Lever response requirement values indicate the number of lever presses necessary for the acquisition of each subsequent infusion (infusion step number). Progressive increases vary between the two schedules such that for the 10^th infusion, 4 lever presses will result in an infusion using the PR9-4 schedule <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052812#pone.0052812-Grasing1" target="_blank">[42]</a>, whereas 32 lever presses are required for the 10^th infusion using PR3-4 schedule <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052812#pone.0052812-Roberts1" target="_blank">[43]</a>. Accordingly, the PR3-4 schedule is deemed more difficult as it progressively requires a greater lever response for an infusion than the PR9-4 schedule. Rat responding for morphine was assessed in the current study using both schedules.</p

Comparison of locomotor activity between offspring groups in response to a novel environment.

<p>Locomotor activity is shown on days 1–3, after saline or morphine administration on days 4–8 (10 mg/kg, s.c.), and after a final morphine challenge on day 16 (5 mg/kg, s.c.). A: Total activity scores in the vertical plane and B: in the horizontal plane, reveal that habituation was greater among naltrexone-exposed offspring. C: Total horizontal locomotor activity did not differ between groups treated with saline and challenged with morphine (saline-morphine). D: Total horizontal locomotor activity did not differ between groups treated with morphine, although sensitization was expressed in both groups after challenge with morphine (morphine-morphine). E: Analysis of data expressing total morphine-induced locomotor activity as a function of basal locomotor activity on day 3 revealed significantly greater activity values for naltrexone-exposed offspring, indicating an increased development of sensitization. Data are expressed as vertical plane entries (A) or horizontal distance moved in centimetres (B, C, D and E) and represent the mean ± SEM (one-way ANOVA: * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001; days 1–3 (A and B): placebo: n = 37, naltrexone: n = 53; days 4–16 (C): placebo: n = 12, naltrexone: n = 19; days 4–16 (D and E): placebo: n = 20, naltrexone: n = 26).</p

Alcohol Two-Bottle Free-Choice Drinking.

<p>Rxfp3 gene deletion does not impact baseline intake or preference for various ethanol solutions (5%-20% v/v) in the two-bottle choice paradigm. <i>(a)</i> Average daily alcohol consumption, <i>(b)</i> alcohol preference, and <i>(c)</i> total fluid intake of Rxfp3 KO mice (n = 16) and WT littermate controls (n = 17) over the final week of access to each ethanol solution. Data are presented as mean ± S.E.M.</p

Comparison of naltrexone- and placebo-exposed offspring to progressive ratio morphine self-administration.

<p>Morphine self-administration under a progressive ratio (PR) schedule of 3–4 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052812#pone-0052812-t001" target="_blank">Table 1</a> for response requirement) at a dose of 0.3 mg/kg/infusion. Data from the PR3–4 session are expressed as total active lever presses (A), breakpoint, defined as the final ratio completed within the 2 h session (B), and number of drug infusions (C) and represent the mean ± SEM (unpaired t-test, * <i>p</i><0.05, naltrexone versus placebo (n = 7). D: Cumulative response record for the PR3-4 session divided into 10 minute time bins. Data are expressed as number of active lever presses and represent mean ± SEM (two-way ANOVA, * <i>p</i><0.05, ** <i>p</i><0.01, naltrexone versus placebo (n = 7) for each time bin).</p

MRI results in SVD cases and controls.

<p><i>Values: Mean (Standard Deviation). Quartile descriptives: Minimum, lower quartile, median, upper quartile, maximum; NBV–Normalised Brain Volume, WMH–White Matter Hyperintensity.CMB–Cerebral Microbleed.</i></p