PPARα regulates cholinergic-driven activity of midbrain dopamine neurons via a novel mechanism involving α7 nicotinic acetylcholine receptors

Abstract

Ventral tegmental area dopamine neurons control reward-driven learning, and their dysregulation can lead to psychiatric disorders. Tonic and phasic activity of these dopaminergic neurons depends on cholinergic tone and activation of nicotinic acetylcholine receptors (nAChRs), particularly those containing the β2 subunit (β2*-nAChRs). Nuclear peroxisome proliferator-activated receptors type-α (PPARα) tonically regulate β2*-nAChRs and thereby control dopamine neuron firing activity. However, it is unknown how and when PPARα endogenous ligands are synthesized by dopamine cells. Using ex vivo and in vivo electrophysiological techniques combined with biochemical and behavioral analysis, we show that activation of α7-nAChRs increases in the rat VTA both the tyrosine phosphorylation of the β2 subunit of nAChRs and the levels of two PPARα endogenous ligands in a Ca(2+)-dependent manner. Accordingly, in vivo production of endogenous PPARα ligands, triggered by α7-nAChR activation, blocks in rats nicotine-induced increased firing activity of dopamine neurons and displays antidepressant-like properties. These data demonstrate that endogenous PPARα ligands are effectors of α7-nAChRs and that their neuromodulatory properties depend on phosphorylation of β2*-nAChRs on VTA dopamine cells. This reveals an autoinhibitory mechanism aimed at reducing dopamine cell overexcitation engaged during hypercholinergic drive. Our results unveil important physiological functions of nAChR/PPARα signaling in dopamine neurons and how behavioral output can change after modifications of this signaling pathway. Overall, the present study suggests PPARα as new therapeutic targets for disorders associated with unbalanced dopamine-acetylcholine systems.

Figure 1.

Enhanced cholinergic tone increases levels of endogenous PPARα agonists. A, The levels of endogenous PPARα ligands (PEA and OEA), but not of the endocannabinoid anandamide (AEA), are increased by enhancing acetylcholine activation of nAChRs (neo+atro: p = 0.0001 for both PEA and OEA, t test; n = 5 for all groups). B, α7-nAChR activation by PNU enhances PEA (p = 0.007, t test; n = 5) and OEA (p = 0.04, t test; n = 5) levels in Ca²⁺-dependent manner (PNU+EGTA: p = 0.01 and p = 0.02 for PEA and OEA, respectively; t test). C, Representative immunoblots and summarizing bar graph showing that PNU282987 (3 mg/kg, i.p.) causes an increase in β2 subunit phosphorylation in rat VTA homogenates that is blocked by the PPARα antagonist MK886 (1 mg/kg, i.p.). D, Representative immunoblots and summarizing bar graph showing that the PPARα agonist WY (40 mg/kg, i.p.) increases β2 subunit phosphorylation in rat VTA homogenates. For each experiment in C and D, tissue lysates containing the same amount of total proteins were subjected to immunoprecipitation (IP) with anti-β2 AChR antibody and were immunoblotted with anti-PY antibody. The IPs were run on the same immunoblots and analyzed together. The blots were stripped and reprobed with anti-β₂ AChR antibody to normalize for protein loading. Band size, 50 kD. Numbers in bars indicate n values. Data are expressed as mean ± SEM. *p < 0.05; **p < 0.001; ***p < 0.0001; ^#p < 0.05; PNU+MK versus PNU. veh, Vehicle.

Figure 2.

Enhanced cholinergic tone to α7-nAChRs modulates VTA dopamine cell activity via PPARα. A, Ex vivo, the dopamine cell firing rate enhances during increased cholinergic tone at β2*-nAChRs (neo+atro+MLA). Left, Current-clamp traces of a dopamine neuron before (basal) and during enhanced cholinergic tone (neo+atro) at β2*-nAChRs (neo+atro+MLA). Right, the effects of enhanced cholinergic tone at β2*-nAChRs on dopamine neurons are represented in a bar graph form. B, Bar graph depicting that the effects of increased cholinergic input onto the dopamine cell firing rate depend upon activation of β2*-nAChRs because DHβE fully blocks such effects. C, Left, Current-clamp traces of a dopamine neuron before (basal), during enhanced cholinergic tone at β2*-nAChRs (neo+atro+MLA), and once PPARα are blocked (+MK886). Right, Time course of the effect of neo+atro+MLA alone (shaded area) or together with MK886 (black bar) on dopamine neuron activity. D, The synthesis of endogenous PPARα ligands after raises in ACh and their consequent effects on the dopamine firing rate are not mediated by muscarinic receptor activation (neo+MLA). E, Removal of extracellular Ca²⁺ (EGTA+neo+atro) enhances the dopamine cell firing rate, which is further increased by blockade of α7-nAChRs with MLA. Left, Current-clamp traces of a dopamine neuron before (basal) and during enhanced cholinergic tone in the presence of Ca²⁺ chelator EGTA (+EGTA) and antagonist of α7-nAChRs (+MLA). In the central panel, the peak effects are represented in a bar graph form. Right, Time course of EGTA (shaded area) alone or in combination with neo+atro (thick black line) and MLA and MK886 is represented. The black bars represent time of MLA and MK886 application. Numbers in bars indicate n values. Data are expressed as mean ± SEM. *p < 0.05; **p < 0.001.

Figure 3.

PPARα activation downstream α7-nAChRs alters excitability of VTA dopamine neurons. A, Left, α7-nAChR agonist PNU reduces dopamine cell activity through synthesis of PPARα ligands. Right, Time course of the effects of PNU282987 (shaded area) in the absence (thick red line) or presence of the PPARα antagonists MK886 (thick dark gray line) and GW6471 (thick light gray line). B, Left, Effects of α7-nAChR agonist PNU282987 are not enhanced by pharmacological inhibition of FAAH enzyme through application of URB. Right, Time course of the effects of PNU282987 (shaded area) in the absence (thick red line) or presence (thick dark gray line) of FAAH inhibitor URB597 applied through the recording pipette ([URB]i). Numbers in bars indicate n values. Data are expressed as mean ± SEM. Thick and dashed lines represent means and SEM, respectively. *p < 0.05; **p < 0.001. C, Determination of the dopamine phenotype using immunohistochemistry. Ca, Cb, Examples of rat VTA neurons in which relatively long (>20 min) whole-cell recordings were made with a KCl (a) and K-gluconate (b) internal solution. Post hoc immunocytochemical detection revealed that example neuron a (white arrow indicating presumably a false negative) was TH(−) and example neuron b was TH(+). Neurons that were whole-cell patch clamped were backfilled by including biocytin in the recording pipette (red). TH immunohistochemistry routinely labeled the TH-positive neurons (green) when the whole-cell recordings were made with the K-gluconate internal solution. Scale bar, 20 μm.

Figure 4.

Effects of α7-nAChR activation on excitation of VTA dopamine neurons by nicotine. A, Representative firing rate histograms showing effects of intravenous nicotine (NIC; injected at arrowhead) on discharge activity of individual VTA dopamine neurons recorded from anesthetized rats. Top, Typical response to 0.2 mg/kg nicotine in control conditions after intravenous injection of vehicle (veh; 4 min before nicotine). Middle, Lack of effect of α7-nAChR agonist PNU (0.5 mg/kg, i.v.; 4 min before nicotine) on spontaneous firing rate of dopamine neurons. Notably, PNU282987 prevents stimulating effects of nicotine. Bottom, The effect of nicotine is restored in a MK886-pretreated animal (0.1 mg/kg, i.p., 1 h before beginning of recording session). B, Graph illustrating the time course of nicotine effects on firing rate of VTA dopamine neurons. Nicotine-induced excitation of dopamine neurons is abolished by PNU282987 but restored by MK886. Data are expressed as mean ± SEM. n = 5 for all groups. Shaded regions beyond dashed lines indicate time after nicotine administration.

Figure 5.

Effects of α7-nAChR activation on rat forced swim test require PPARα activation. The effect of α7-nAChR agonist PNU282987 (3 mg/kg, i.p.) in the forced swim test are PPARα 32 mediated (***p < 0.001, PNU vs corresponding vehicle, veh+MK886, and PNU+MK886 groups; ***p < 0.001, amitriptyline group vs all groups). Data are expressed as mean ± SEM. Numbers in bars indicate n values.

Figure 6.

Endogenous PPARα ligands act as brake during high cholinergic tone on dopamine cells. Schematic diagram illustrating the proposed mechanism of PPARα-induced regulation of cholinergic transmission. Left, During low cholinergic activity, acetylcholine (ACh) preferentially binds to high-affinity β2*nAChRs. Production of endogenous PPARα ligands, the N-acylethanolamines AEA, OEA, and PEA, by the Ca²⁺-sensitive NAPE-PLD remains low and does not trigger PPARα-mediated modulation of β2*-nAChRs. Right, When acetylcholine transmission is potentiated, i.e., by blocking acetylcholinesterase (AChE) with neostigmine (NEO), Ca²⁺-permeable low-affinity α7-nAChRs are activated. α7-nAChRs are located extrasynaptically on the somatodendritic region of dopamine neurons (Jones and Wonnacott, 2004). α7-nAChRs are also activated by the selective agonist PNU and are blocked by MLA. α7-nAChR-mediated increase in intracellular Ca²⁺ stimulates N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD), leading to the production of OEA and PEA (and AEA). These molecules, in turn, activate PPARα that exerts negative modulation of β2*-nAChRs operated by a tyrosine kinase-mediated phosphorylation (P). Phosphorylation of β2*nAChRs might reduce responses to acetylcholine or promote rapid internalization of these receptors. The effects of endogenous PPARα ligands are mimicked by the synthetic agonist WY and blocked by the PPARα antagonist MK886 (MK). Increases in levels of N-acylethanolmines can also be triggered by blockade of FAAH by URB597. FAAH is the major inactivating enzyme for OEA, PEA and AEA and converts these molecules in ethanolamine and oleic acid (OA), palmitic acid (PA), and arachidonic acid (AA), respectively. However, in this case, AEA might counteract the effects of OEA and PEA by activating TRPV1 (Melis et al., 2008).