Peroxisome proliferator-activated receptors-alpha modulate dopamine cell activity through nicotinic receptors

Abstract

Background: Modulation of midbrain dopamine neurons by nicotinic acetylcholine receptors (nAChRs) plays an important role in behavior, cognition, motivation, and reward. Specifically, nAChRs containing beta2 subunits (beta2-nAChRs) switch dopamine cells from a resting to an excited state. However, how beta2-nAChRs can be modulated and thereby how dopamine firing activity is affected remains elusive. Because changes in dopamine cell activity are reflected in the dynamics of microcircuits generating altered responses to stimuli and inputs, factors regulating their state are fundamental. Among these, endogenous ligands to the nuclear receptor-transcription factor peroxisome proliferator-activated receptors type-alpha (PPARalpha) have been recently found to suppress nicotine-induced responses of dopamine neurons.

Methods: We used both in vitro and in vivo electrophysiological techniques together with behavioral analysis to investigate on the effects of modulation of PPARalpha in Sprague-Dawley rat and C57BLJ/6 mouse dopamine neurons and their interactions with beta2-nAChRs. To this aim, we took advantage of a selective reexpression of beta2-nAChR exclusively in dopamine cells by stereotaxically injecting a lentiviral vector in the mouse ventral tegmental area.

Results: We found that activation of PPARalpha decreases in vitro both dopamine cell activity and ventral tegmental area net output through negative modulation of beta2-nAChRs. Additionally, PPARalpha activation in vivo reduces both the number of spontaneously active dopamine neurons and nicotine-induced increased locomotion.

Conclusions: Our combined findings suggest PPARalpha ligands as important negative modulators of beta2-nAChRs on dopamine neurons. Thus, PPARalpha ligands might prove beneficial in treating disorders in which dopamine dysfunction plays a prominent role, such as schizophrenia and nicotine addiction.

Figure 1

PPARα blockade activates VTA dopamine neurons in vitro. (A) MK886 application (0.5 µM) increases dopamine neuron spontaneous activity. Current-clamp recording from a dopamine neuron (left panel) and rate histogram depicting MK886 averaged effects (right panel). (B) In voltage-clamp mode MK886 caused an inward current (V_hold= −70 mV) blocked by the PPARα agonist WY14643 (0.3 µM). (C) WY14643 blocked MK886-induced activation of dopamine neurons. (D) Summary of dose-related effects of PPARα antagonists on dopamine neuronal frequency. Numbers above bars indicate n values. Data expressed as mean ± SEM. *p < 0.05; **p < 0.005.

Figure 2

MK886 enhances dopamine neuron activity through α4β2-nAChRs. (A and B) Time course of MK886 effect, alone or together with mecamylamine (MEC) (100 µM), methyllycaconitine (MLA) (5 nM) or dihydro-β-erythroidine (DHBE) (1 µM), on dopamine neuron activity. Grey and black bars represent time of nAChR antagonist or MK886 application, respectively. Insets show representative traces of dopamine neuron frequency. (C) Bar graph illustrating DHBE mean effect on MK886-induced inward current. Inset shows representative DHBE+MK886 effect on dopamine cell. Grey and black bars represent the time of DHBE and MK886 application, respectively. (D) MK886 effects on dopamine neurons in ^β2−/− and ^β2+/+ mice under voltage-clamp (left panel) and current-clamp (right panel) modes. Inset shows a representative MK886 effect. (E) Left panel, Magnitude of currents induced by nicotine plotted as function of those induced by MK886 at membrane potential of -60 mV. Data fit by linear regression with r² = 0.9481 (p < 0.001). Right panel, R² for five cells in f plotted as a function of voltage membrane. Numbers above bars indicate n values. Data expressed as mean ± SEM. *p < 0.05, ***p < 0.0005.

Figure 3

PPARα blockade increases efficacy of nAChR agonists. (A) Under voltage-clamp mode, MK886 effect on dopamine neuron in presence (grey) or absence (black) of sodium-orthovanadate (OVN). (B) Under current-clamp mode, effects of enhanced endogenous ACh levels acting at nAChRs (neostigmine+atropine) in presence or absence of MK886 (0.3 µM). (C) Current-clamp recording of a dopamine neuron (left panel) showing enhanced nicotine response in presence of MK886 (0.3 µM). Dose-response curves depicting averaged effects of nicotine (right panel) on dopamine neurons in presence or absence of MK886. Numbers above bars indicate n values. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.005.

Figure 4

PPARα activation decreases VTA dopamine neuronal activity. (A) Current-clamp traces of a dopamine neuron before, during and after oleoylethanolamide (OEA) application (10 µM). Rate histogram depicting averaged OEA effects on dopamine neuron frequency in absence (B) and presence (C) of MK886 (0.5 µM). (D), Dose-response curves depicting averaged effects of PPARα agonists on dopamine neuron frequency. (E), Under voltage-clamp mode, effects of OEA (10 µM), WY14643 (WY; 1 µM) and fenofibrate (FRB; 10 µM) on dopamine neurons in absence (left panel) and presence (right panel) of MK886 (0.5 µM). (F), Under current-clamp mode, MK886 (0.5 µM) reversed the effects of both WY14643 (left panel) and fenofibrate (right panel) on dopamine neurons. Numbers above bars indicate n values. Data expressed as mean ± SEM. *p < 0.05.

Figure 5

Hydrogen peroxide is downstream effector of PPARα. (A), Current-clamp recording of a dopamine neuron during bath application of OEA (10 µM) in absence or presence of catalase (500 U/I). (B), Time course of averaged effects of OEA (10 µM) on dopamine neuron frequency in the presence (open circles) and absence (grey area) of catalase. (C) Current-clamp recording of a dopamine neuron during nicotine (1 µM) and OEA (3 µM) application in the absence or presence of catalase. (D) Time course of OEA (3 µM) and OEA+nicotine averaged effects on dopamine neuronal activity in the presence (open circles) and absence (grey area) of catalase. The inset shows that nicotine produced an effect on firing rate (FR) in the presence of OEA (3 µM) and catalase. Catalase was applied through the recording pipette. Data expressed as mean ± SEM. *p < 0.05.

Figure 6

PPARα activation reduces VTA output and nicotine induced stimulation of locomotion. (A and B). Typical evoked field potential recordings showing effects of MK886 (0.5 µM, A) and WY14643 (1 µM, B) on field potential amplitude. Bin= 10 s. Traces from typical experiments (top), time-courses of the effects of MK886 and WY14643 on N1 (middle) and N2 (bottom) components, and the mean averaged responses (bar graph in insets) are shown. (C) Averaged N2 amplitude from the VTA of β2^−/−, β2^+/+ and β2-DA-VEC mice in response to WY14643 (1 µM). Bin= 1 min. (D) WY14643 (40 mg/kg i.p.) decreases the number of VTA DA cells encountered during neuronal sampling in anesthetized rats (left panel), but not averaged firing frequency (right panel). (E) Time-course curve of nicotine (0.02 mg/kg, s.c.) effects on locomotor activity in WY14643- and vehicle- treated mice. (F) Time-course curve of nicotine (0.02 mg/kg, s.c.) effects on locomotor activity in WY14643- and vehicle- treated β2-DA-VEC mice compared with β2^−/− mice. Arrows indicate time of nicotine administration. Data expressed as mean ± SEM. *p < 0.05.