Table_1_Amphetamine and the Smart Drug 3,4-Methylenedioxypyrovalerone (MDPV) Induce Generalization of Fear Memory in Rats.docx

Human studies have consistently shown that drugs of abuse affect memory function. The psychostimulants amphetamine and the “bath salt” 3,4-methylenedioxypyrovalerone (MDPV) increase brain monoamine levels through a similar, yet not identical, mechanism of action. Findings indicate that amphetamine enhances the consolidation of memory for emotional experiences, but still MDPV effects on memory function are underinvestigated. Here, we tested the effects induced by these two drugs on generalization of fear memory and their relative neurobiological underpinnings. To this aim, we used a modified version of the classical inhibitory avoidance task, termed inhibitory avoidance discrimination task. According to such procedure, adult male Sprague–Dawley rats were first exposed to one inhibitory avoidance apparatus and, with a 1-min delay, to a second apparatus where they received an inescapable footshock. Forty-eight hours later, retention latencies were tested, in a randomized order, in the two training apparatuses as well as in a novel contextually modified apparatus to assess both strength and generalization of memory. Our results indicated that both amphetamine and MDPV induced generalization of fear memory, whereas only amphetamine enhanced memory strength. Co-administration of the β-adrenoceptor antagonist propranolol prevented the effects of both amphetamine and MDPV on the strength and generalization of memory. The dopaminergic receptor blocker cis-flupenthixol selectively reversed the amphetamine effect on memory generalization. These findings indicate that amphetamine and MDPV induce generalization of fear memory through different modulations of noradrenergic and dopaminergic neurotransmission.</p

Western blot analysis of Kv 2.1 and Kv4.2 subunit expression in cerebral cortex and hippocampus.

<p>Representative immunoblot of cerebral cortex and hippocampus enriched membrane proteins (50 µg/lane) from (Ctr), Aβ_25–35, Aβ_25–35+SP and SP treated rats. Protein markers are shown at right (in kDa). The immunoreactive signals for <b>a</b>) Kv2.1 and <b>b</b>) Kv4.2 were quantified and normalized against β-actin and expressed as a percentage of control (CTR). Data represent mean (±SEM) from 3 independent experiments. Statistically significant differences were calculated by one-way analysis of variance (ANOVA) for repeated measures followed by Tukey's test for multiple comparisons (**p<0.01 versus Ctr value).</p

Immunofluorescence analysis of Kv1.4 subunit expression in hippocampus and cerebral cortex.

<p>Upper panel. Representative immunofluorescence photomicrographs showing Kv1.4 expression in <b>a</b>) hippocampus and <b>b</b>) frontal cortex after memory tests in the four experimental treatments: Control (Saline), Aβ_25–35-i.c.v. treated rats (Abeta), Aβ_25–35-i.c.v. and SP-i.p. treated rats (Abeta+SP), SP-i.p. treated rats (SP). Brain sections were labeled with the neuronal marker NeuN (green) and with the anti Kv1.4 antibody (red). As shown by the merge channel all neurons are Kv1.4 positive. Note the diffuse increase in Kv1.4 fluorescence intensity in the Abeta group and the decrease in the Abeta+SP group compared to the Control. Scale bar: a) 20 µm; b) 60 µm. Lower panel. Histograms showing image analysis performed on neuronal cytoplasm (first row) and the surrounding neuropil (second row). The indexes used were: total fluorescence intensity, vesicles diameters, and vesicles fluorescence intensity. Data represent means (±S.E.M.) obtained from three independent experiments. Statistically significant differences were calculated by one-way analysis of variance (ANOVA) for repeated measures followed by Tukey's test for multiple comparisons (**p<0.01 versus Saline; #p<0.05, ##p<0.01 versus Aβ_25–35treatment).</p

Subanalysis of plasma concentrations of ECs and related NAEs after excluding individuals of African descent (n = 7).

<p><i>Panel A</i>: significantly higher ANA plasma concentrations in PTSD patients as compared to healthy controls (diff. of means = 0.136, t = 2.64, *p = 0.035; ANOVA with Holm-Sidak post hoc test). <i>Panel C</i>: significantly higher SEA plasma levels compared to controls (diff. of ranks = 12.9, Q = 2.96, p<0.05; Kruskal-Wallis ANOVA on Ranks with Dunn's post hoc test). <i>Panel D</i>: significantly increased OEA concentrations in PTSD patients in relation to healthy controls (diff. of ranks = 11.9, Q = 2.47, p<0.05; Kruskal-Wallis ANOVA on Ranks with Dunn's post hoc test). <i>Panel F</i>: OLDA concentrations across the 3 study subgroups (Kruskal-Wallis ANOVA on Ranks indicated a significant difference among the groups (⁺p = 0.02) but significance was lost after correction for multiple comparisons (diff. of ranks 11.7, Q = 2.23, p>0.05). The differences between groups shown in <i>Panel B</i> (2-AG) and <i>Panel E</i> (PEA) were not statistically significant in the subanalysis. Data are mean ± SEM.</p

Plasma level comparisons of ECs and related NAEs between non-traumatized control subjects, trauma-exposed subjects and patients with PTSD.

<p><i>Panel A</i>: ANA plasma concentrations; *significantly higher ANA concentrations in PTSD patients as compared to healthy controls (diff. of means = 0.120, t = 2.64, *p = 0.012; ANOVA with Holm-Sidak post hoc test). <i>Panel B</i>: 2-AG plasma levels; *significant difference compared to controls (diff. of means = 2.68, t = 3.12, unadjusted p = 0.003, critical level p = 0.017). <i>Panel C</i>: SEA levels: *significant difference compared to controls (Diff. of means = 1.87, t = 2.77, unadjusted p = 0.008, critical level p = 0.017). <i>Panel D</i>: *significantly higher OEA plasma levels in individuals with PTSD (diff. of means = 2.01, t = 3.03, unadjusted p = 0.004, critical level p = 0.017) compared to controls. <i>Panel E</i>: significant differences in PEA plasma concentrations compared to individuals after trauma exposure who did not develop PTSD (^#p<0.05, diff. of ranks = 16.6, Q = 2.64, Kruskal-Wallis ANOVA on Ranks with Dunn’s post-hoc test). <i>Panel F</i>: significantly lower OLDA plasma concentrations compared to the control group (*p<0.05, diff. of ranks = 13.8, Q = 2.45) and in comparison to individuals after trauma exposure without PTSD (^#p = 0.05, diff. of ranks = 12.40, Q = 2.75, Kruskal-Wallis ANOVA on Ranks with Dunn’s post-hoc test). Data are mean ± SEM.</p

SP reduced Aβ25–35-induced overexpression of Kv1.4 subunit in rat hippocampal neurons.

<p><b>a</b>) Example of Western blot obtained from hippocampal cultures exposed to 20 µM Aβ_25–35 (Aβ alone or in the presence of SP (100 nM) and analyzed 48 h later using a polyclonal antibody against Kv1.4 subunit. The same blots were stripped and reprobed with an antibody against β-actin as internal control (lower panels). Quantitative analysis is depicted below the blots and was determined by band densitometry analysis considering the values found in CTR cells as 100. Data represent means (±S.E.M.) obtained from 4 independent experiments run in duplicate. (**p<0.001 versus CTR, #p<0.05 versus Aβ_25–35 treatment). <b>b</b>) Representative immunofluorescence photomicrographs showing Kv1.4 expression in primary hippocampal cultures. Note the increase in immunofluorescence in the Aβ_25–35 neurons, as compared to control neurons, reversed by SP treatment. Images were obtained from three independent experiments. Scale bar: 20 µm.</p

Correlation between the number of CAPS traumatic event types and OLDA plasma concentrations (r = −0.50, p = 0.03, n = 19).

<p>The solid line represents the regression line and dotted lines 95% confidence intervals.</p

Relationship between OLDA plasma concentrations and CAPS scores.

<p>Panel A: CAPS sum score (r = −0.68, p<0.01, n = 19); Panel B: CAPS – intrusion subscore (r = −0.65, p<0.01); Panel C: CAPS – avoidance subscore (r = −0.59, p<0.01); Panel D: CAPS – hyperarousal subscore (r = −0.66, p<0.01). Solid line indicates regression line and dotted lines 95% confidence intervals.</p

Correlation between CAPS scores and PEA plasma levels in individuals after trauma exposure (n = 19).

<p>Panel A: CAPS sum score (r = 0.54, p = 0.02); Panel B: CAPS – intrusion subscore (r = 0.65, p<0.01); Panel C: CAPS – avoidance subscore (r = 0.21, p = 0.40); Panel D: CAPS - hyperarousal subscore (r = 0.29, p = 0.23). Solid line indicates regression line and dotted lines 95% confidence intervals.*marks significant correlations.</p

Demographic and clinical data of the study groups.

a<p>Data are mean±SD; (y) =  years; (f) =  female; (m) =  male.</p>b<p>Trauma-exposed patients received amitriptylin (n = 2), PTSD patients mirtazapin (n = 2) and amitriptylin+mirtazapin (n = 1).</p>c<p>These scores were only available for ethnically matched controls recruited by the Trauma Center of University of Konstanz (n = 9).</p>d<p>Trauma-exposed individuals without PTSD were of Caucasian origin (n = 6) (two from Iran and two from Turkey, one from Bosnia and one from Afghanistan) and 3 were Black-Africans (one from Gambia, one from Eritrea and one from Senegal).</p>e<p>Trauma- exposed individuals with PTSD were Caucasians (n = 8) (two from Turkey, two from Iran, one from Afghanistan, one from Syria, one from Kosovo and one from Bosnia) and two were Black-Africans (one from Nigeria and one from Togo).</p>f<p>Controls were Caucasians (twenty were Germans, one each were from Turkey, Armenia, Israel, two from Romania and two were Russians) and 2 were from Africa (Eritrea and Sudan).</p>*<p>Significantly higher values compared to trauma-exposed individuals <i>without</i> PTSD and to healthy controls.</p>#<p>Significantly higher values compared to healthy controls.</p