Figure 1.
The PPARα agonist WY14643 reduces nicotine-induced seizures.
(A) Graph displaying the dose-dependency of the severity of nicotine-induced seizures in C57BL/6 mice. Nicotine was administered subcutaneously at 5, 7 and 10 mg/kg (n = 4, n = 5, n = 18, respectively). (B) Schematic representation of the experimental protocol of experiments in (C) and (D). (C) The PPARα agonist WY14643 (WY, 80 mg/kg, i.p., n = 23) reduced the severity of nicotine-induced seizures (nicotine dose: 10 mg/kg, s.c) (*P<0.05 vs. vehicle, Dunn’s-test). This effect was abolished by the selective PPARα antagonist MK886 (MK, 3 mg/kg, i.p.). (n = 16, # P<0.001 vs. WY, Dunn’s-test). (D) This graph shows that the percentage of mice undergoing severe nicotine-induced seizures (indexed by scores >3) is significantly attenuated after WY14643 pretreatment (*P<0.05 vs. vehicle, Fisher’s test). MK886 reversed this effect (P>0.05 vs. vehicle, Fisher’s test). Data are expressed as mean±95% C.I.
Figure 2.
The PPARα agonist WY14643 suppresses nicotine-induced spike-wave activity.
(A) Representative traces of EEG recordings from hippocampal (Hip) and sensorimotor cortical (Ctx) electrodes chronically implanted in mice. Following the administration of vehicle (VEH) and 10 mg/kg nicotine, bursts of synchronous spike-wave (SW) activity with high-amplitude and low-frequency (most in the delta rhythm range) were recorded. This activity was suppressed when animals were pretreated with the PPARα agonist WY14643 (WY, 80 mg/kg), which per se did not change baseline EEG activity. The PPARα antagonist MK886 (MK, 3 mg/kg, i.p.) restored nicotine-induced SW discharges. (B) The graph shows the percentage of mice presenting SW discharges following the three treatment protocols. Vehicles treated mice did show SW activity, whereas 100% of nicotine treated mice displayed bursts of SW activity. The effects of nicotine were blocked in the majority of WY treated mice, since SW burst were recorded only in 33% of treated animals (**P<0.01 vs. vehicle, Fisher’s test). Conversely, when MK was administered 15 before WY nicotine-induced SW activity was recorded in 78% of MK+WY treated mice (#P<0.05 vs. WY, Fisher’s test).
Figure 3.
The clinically used PPARα agonist fenofibrate chronically administered with food reduces nicotine-induced seizures and spike-wave activity, and increases the OEA levels in the frontal cortex.
(A) Graph displaying that fenofibrate (n = 8, FBR, 0.3% w/w in the diet for 14 days) reduced the severity of nicotine-induced seizures (nicotine dose: 10 mg/kg, s.c) (***P<0.001 vs. control diet, CTRL DIET, Dunn’s-test). This effect was not abolished by the selective PPARα antagonist MK886 (FBR+MK, 3 mg/kg, i.p.) (n = 8, **P<0.01 vs. CTRL DIET, Dunn’s-test) but by withdrawal of fenofibrate treatment for 14 days (FBR WASH-OUT, n = 8, # P<0.05 vs. FBR and P>0.05 vs. CTRL DIET, Dunn’s test). (B) This graph shows that the percentage of mice undergoing severe nicotine-induced seizures (indexed by scores >3) is significantly attenuated after chronic fenofibrate treatment (***P<0.001 vs. control diet, Fisher’s test) and restored after fenofibrate withdrawal for 14 days (#P>0.05 vs. control diet). MK886 did not reverse this effect (***P<0.001 vs. control diet, Fisher’s test). (C) Chronic activation of PPARα by fenofibrate changes oleoylethanolamide (OEA), but not palmitoylethanolamide, levels within frontal cortex. Concentrations of these endogenous PPARα ligands are expresses as pmol per gram of tissue. Error bars depict S.E.M. (*P<0.05, Student’s t-test). (D) Representative traces of EEG recordings from hippocampal (Hip) and sensorimotor cortical (Ctx) electrodes chronically implanted in mice. In mice fed with control diet 10 mg/kg nicotine elicits bursts of synchronous spike-wave (SW) activity with high-amplitude and low-frequency (most in the delta rhythm range). This activity was suppressed in animals fed with fenofibrate in food pellets. (E) The graph shows the percentage of mice presenting SW discharges following the four treatment protocols. 86% of control diet fed mice (6 out of 7) did show nicotine-induced SW activity. The effects of nicotine were fully blocked in all fenofibrate treated mice, since SW burst were recorded only in none of treated animals (***P<0.01 vs. control diet, Fisher’s test). MK, administered 15 before nicotine, did not restore nicotine-induced SW activity (***P<0.001 vs. control diet, Fisher’s test), whereas fenofibrate washout did (#P>0.05 vs. control diet, Fisher’s test). Data are expressed as mean±95% C.I.
Figure 4.
The PPARα agonists WY14643 and fenofibrate suppress nicotine-induced increase of spontaneous inhibitory postsynaptic currents (sIPSC) in frontal cortex (FCx) pyramidal neurons.
The graphs illustrate that in mouse FCx slices, nicotine (5 µM perfused at arrows for 30 s) increases sIPSCs frequency in layer II/III pyramidal neurons. Acutely, the PPARα agonists WY14643 (1 µM, WY) (A), and fenofibrate (10 µM) (B) fully suppressed nicotine-induced increase in sIPSC frequency. Similarly, chronic fenofibrate (0.2% w/w for 14 days in food) fully suppressed nicotine-induced increase in sIPSC frequency (C). The gray box represents the time of PPARα agonist (+/− antagonist) perfusion. The PPARα antagonist MK886 (0.3 µM) (open symbols) blocked the effects of acute WY (A) and fenofibrate (B), but not the one of chronic fenofibrate (C), and restored nicotine-induced increase in sIPSCs. Representative recordings of the effect of nicotine, and of PPARα agonists and antagonist, on spontaneous IPSCs from pyramidal cells at Vh = 0 mV are depicted on the upper part of panels A and B. Symbols represent the average±S.E.M.
Table 1.
Table showing frequency and amplitude of spontaneous IPSCs recorded from mice and rat pyramidal neurons in layers II/III of the frontal cortex.
Figure 5.
The PPARα agonist WY14643 enhances phosphorylation of β2 subunits of the nicotinic acetylcholine receptors.
Graph and representative immunoblots showing that the PPARα agonist WY (40 mg/kg, i.p.) caused an increase in β2-subunit phosphorylation (*P<0.05) in rat frontal cortex homogenates. Tissue lysates were subjected to immunoprecipitation (IP) with anti-β2 subunit antibody and were immunoblotted (IB) with anti-phosphotyrosine (PY) antibody. The blots were stripped and reprobed with anti-β2 subunit antibody to normalize for protein loading.