The FASEB Journal • Review

Specific needs of dopamine neurons for stimulation inorder to survive: implication for Parkinson disease

Patrick P. Michel,*,†,‡,1 Damien Toulorge,*,†,‡ Serge Guerreiro,*,†,‡

and Etienne C. Hirsch*,†,‡

*Université Pierre et Marie Curie-Paris 6, Centre de Recherche de l’Institut du Cerveau et de laMoelle Epinière, Unité Mixte de Recherche (UMR) S975, Paris, France; †Institut National de laSanté et de la Recherche Médicale, UMR 975, Paris, France; and ‡Centre National de la RechercheScientifique, UMR 7225, Paris, France

ABSTRACT Parkinson disease (PD) is a degenerativebrain disorder characterized by motor symptoms thatare unequivocally associated with the loss of dopami-nergic (DA) neurons in the substantia nigra (SN).Although our knowledge of the mechanisms that con-tribute to DA cell death in both hereditary and sporadicforms of the disease has advanced significantly, thenature of the pathogenic process remains poorly under-stood. In this review, we present evidence that neuro-degeneration occurs when the electrical activity andexcitability of these neurons is reduced. In particular,we will focus on the specific need these neurons mayhave for stimulation in order to survive and on themolecular and cellular mechanisms that may be com-promised when this need is no longer met in PD.—Michel, P. P., Toulorge, D., Guerreiro, S., Hirsch, E. C.Specific needs of dopamine neurons for stimulation inorder to survive: implication for Parkinson disease.FASEB J. 27, 000–000 (2013). www.fasebj.org

Key Words: calcium � electrical activity � neurodegeneration

Parkinson disease (PD) is a progressive, hypokinetic,neurodegenerative brain disorder characterized by mo-tor symptoms that include bradykinesia, rigidity, akine-sia, abnormal posture, and resting tremor. Althoughclear signs of the pathology are observed in several

brain regions, the motor symptoms are unequivocallyassociated with the loss of dopaminergic (DA) neuronsin the substantia nigra (SN). Significant advances havebeen made in our understanding of the mechanismsthat contribute to disease onset and progression inboth hereditary and sporadic forms of PD (1, 2).Current evidence suggests that alterations in severalmetabolic pathways converge to produce chronic neu-ronal suffering, mainly manifested by suboptimal en-ergy metabolism, oxidative damage, endoplasmic retic-ulum (ER) stress, misfolding and aggregation of theprotein �-synuclein, and synaptic dysfunction (3). How-ever, the sequence of the pathogenic events leading tothe loss of DA neurons in the course of the disease isnot completely understood.

SN DA neurons are remarkable in that they possessintrinsic membrane properties that allow them to dis-charge in a single-spike pacemaker mode; i.e., in a cellautonomous manner that does not depend on afferentinput (4–6). Afferent input, however, modulates thisactivity, inducing random firing and burst-firing pat-terns (7, 8). Changes in the firing rate and, moreimportant, the firing pattern of DA neurons are trans-lated into changes in the amounts of dopamine re-leased either in the terminal axonal fields in thestriatum (9) or in the somatodendritic compartment inthe SN (10). A number of studies suggest that themechanisms that control the activity of SN DA neuronsare not only crucial for the control of dopamine releasebut also for the survival of these neurons. More specif-ically, evidence indicates that DA neurons degeneratewhen they become electrically less active. This reviewfocuses on the specific need of DA neurons for stimu-lation in order to survive and the molecular mecha-nisms that are compromised, under pathological con-ditions, when this need is no longer met.

1 Correspondence: Institut du Cerveau et de la MoelleÉpinière, Hôpital de la Salpêtrière, 47, bd de l’hôpital, 75013Paris, France. E-mail: patrick-pierre.michel@upmc.fr

doi: 10.1096/fj.12-220418

Abbreviations: 4-AP, 4-aminopyridine; APA, apamin; Ca2�cyt,

cytosolic calcium; CTX, charybdotoxin; DA, dopaminergic;ER, endoplasmic reticulum; GALR1, galanin receptor 1;GIRK, protein-gated inwardly rectifying K�; IP3R, inositol1,4,5-trisphosphate receptor; MPP�, 1-methyl-4-phenylpyri-dinium; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine;nAChR, nicotinic acetylcholine receptor; Nav, voltage-gatedNa�; NIC, nicotine; NK1R, neurokinin 1 receptor; N/OFQ,nociceptin/orphanin-FQ; NOP, N/OFQ peptide receptor;PD, Parkinson disease; PI3K, phosphatidylinositol 3-kinase;PPN, pedunculopontine nucleus; PX, paraxanthine; P2XR,purinergic type 2X receptor; ROS, reactive oxygen species;RyR, ryanodine receptor; SK, small-conductance Ca2�-acti-vated K�; SN, substantia nigra; SP, substance P; TEA, tetra-ethyl ammonium; TH, tyrosine hydroxylase; TRCP1, transientreceptor potential canonical type 1; TTX, tetrodotoxin;VDCC, voltage-dependent calcium channel; VTA, ventraltegmental area

10892-6638/13/0027-0001 © FASEB

The FASEB Journal article fj.12-220418. Published online May 22, 2013.

SN DA NEURONS ARE EXQUISITELYDEPENDENT ON DEPOLARIZING STIMULI FORTHEIR SURVIVAL DURING DEVELOPMENT

The idea that DA neurons develop a specific need forstimulation in order to survive came initially fromexperimental studies in dissociated primary cell cul-tures or slice cultures from rodent mesencephalon.The selective death of DA neurons, which occursspontaneously by apoptosis in rat or mouse primarymidbrain cultures, was prevented by chronic applica-tion of mild depolarizing stimuli (11–13). In particular,low-level activation of voltage-gated sodium (Nav) chan-nels by the alkaloid veratridine or �-scorpion toxinwere highly effective in preventing the loss of DAneurons (12). Pharmacological blockade of excitatoryglutamatergic or cholinergic neurotransmission (13)did not affect survival maintained by Nav channelactivation, however, which suggests that depolarizationwas not mediated by other populations of neuronspresent in the cultures. As expected, tetrodotoxin(TTX), a selective blocker of Nav channels, preventedrescue by both veratridine and the �-scorpion toxin(12, 13). TTX per se failed, however, to reduce thenumber of DA neurons below control levels at all stagesof maturation of the cultures, suggesting that the needfor electrical stimuli emerged progressively and selec-tively in the subpopulation of DA neurons that isspontaneously vulnerable to degeneration. Interest-ingly, non-DA neurons in the cultures, mostly GABA-ergic, did not die spontaneously in this preparation andremained insensitive to the effect of TTX. The death ofthe DA neurons results, therefore, from a processspecific to these cells.

The requirement for stimulation was probably un-masked by the isolation of DA neurons in dissociatedcultures that precluded excitatory input (12); in embry-onic or postnatal organotypic mesencephalic slice cul-tures; i.e., model systems that partially preserve thetissue-specific organization of synaptic connections,spontaneous loss of DA neurons was less evident (14–16). The death of DA neurons was extensive, however,in postnatal midbrain slices treated chronically withTTX (14), indicating that basal Nav channel activationwas necessary for the survival of these neurons underthese conditions.

Depolarization produced by blockade of K� channelsalso promoted the survival of DA neurons in midbraincultures. In particular, two small peptide toxins fromanimals, apamin (APA) and charybdotoxin (CTX),protected the DA neurons in this model system (12,13). APA is a bee venom derivative that depolarizes DAneurons by blocking small-conductance Ca2�-activatedK� (SK) channels that modulate the amplitude ofafterhyperpolarization (17); it plays a major role in theswitch between tonic and burst firing of DA neuronsunder physiological conditions (18). Consistent with itspharmacological profile, APA had a protective effectthat was blocked by TTX and potentiated by suboptimalconcentrations of the Nav channel agonist veratridine(12). CTX a scorpion venom peptide that blocks large-conductance Ca2�-activated K� channels (19, 20) alsoprotected DA neurons in midbrain cultures (13). Of

interest, these data further reinforce the biologicalrelevance of the culture model, since the presence offunctional Ca2� activated K� channels is an index ofthe maturation of DA neurons (20). Tertiapin-Q, a beevenom component that depolarizes DA neurons byspecifically blocking protein-gated inwardly rectifyingK� (GIRK) channels (21), increased the number ofneurons expressing the rate-limiting enzyme of dopa-mine synthesis, tyrosine hydroxylase (TH), in midbraincultures (22). The effect of tertiapin-Q on GIRK chan-nels was interpreted, however, to result from the recov-ery of the DA phenotype in neurons in which theexpression of TH was repressed (22). Finally, due to thelarge spectrum of K� channels that they block (23),tetraethyl ammonium (TEA) and 4-aminopyridine (4-AP) proved to be more efficacious in protecting DAneurons than APA or CTX used separately (13).

INTRINSIC DEFICITS THAT REDUCE THEACTIVITY OF SN DA NEURONS RENDERTHESE NEURONS HIGHLY VULNERABLE TODEGENERATION IN THE ADULT BRAIN

Several lines of evidence suggest that DA neurons in theadult brain need to maintain a certain level of activitynot only to carry out their normal physiological func-tions but also to ensure their survival. For example, Lisset al. (24) showed that the destruction of DA neuronsinduced by chronic treatment with the mitochondrial-neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP) could be prevented by maintaining their normalpacemaking activity through genetic knockout of thechannel pore-forming subunit Kir6.2 of ATP-sensitivepotassium (KATP) channels, a type of channel thatnormally opens in response to intracellular ATP deple-tion. Patch-clamp recordings in midbrain slice culturesshowed that the active metabolite of MPTP, the mito-chondrial complex I inhibitor 1-methyl-4-phenylpyri-dinium (MPP�), caused the activation of KATP channelsin SN DA neurons from control mice, silencing them.However, SN DA neurons in midbrain slices fromKir6.2�/� mice maintained normal activity in the pres-ence of the same concentration of toxin (24). One mayconclude, then, that when DA neurons become lessactive due to mitochondrial dysfunction, they alsobecome vulnerable to degeneration. Consistent withthis view, the concentration-dependent increase inKATP channel conductance induced by MPP� inKir6.2�/� SN DA neurons from midbrain slices (24)correlated with its ability to kill these neurons selec-tively in midbrain cultures (25). The concentrations ofcomplex I inhibitors that activated KATP channels in SNDA neurons had no effect on neighboring DA neuronsin the ventral tegmental area (VTA), which are muchless vulnerable to degeneration (24), further linkingDA cell death to activity-dependent mechanisms. Thedeleterious effects of MPTP on SN DA neurons ap-peared, however, to be amplified in Kir6.2�/� micethat received a single acute administration of MPTP(24); this finding indicates that the consequences ofKATP channel activation might be diametrically op-posed, depending on the duration and severity of the

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metabolic insult. Consistent with this view, hippocam-pal neurons in Kir6.2�/� mice were more vulnerable toacute ischemic attacks than wild-type mice (26).

Liss et al. (24) established that the opening of KATPchannels in DA neurons in which mitochondria aredysfunctional and the ensuing changes in activity didnot primarily result from ATP depletion but ratherfrom the production of reactive oxygen species (ROS)by mitochondria. Bao et al. (27) also showed that partialmitochondrial inhibition produced by the plant toxinrotenone causes functional DA denervation throughH2O2 elevation and KATP channel activation, leading tothe speculation that ROS might become detrimental inthe context of PD, not just because they damage cellcomponents but also indirectly, because they progres-sively reduce the activity of DA neurons (28). Consis-tent with this hypothesis, we observed that severalantioxidants acted cooperatively with a mild depolariz-ing stimulus to rescue DA neurons in a model system ofrat midbrain cultures in which low-level oxidative stressdevelops spontaneously (29).

Another model of PD that links the death of DAneurons to activity-dependent mechanisms is the Mi-topark mouse, in which the nuclear genome-encodedmitochondrial gene Tfam can be conditionally deletedin DA neurons (30). This mouse model was developedbecause genes that cause familial Parkinsonism arelinked to mitochondrial function, and a mitochondrialcomplex I deficiency was observed in PD SN (31). Asalready mentioned, DA neurons are also particularlyvulnerable to toxins that impair mitochondrial func-tion (32). Patch-clamp recordings in Mitopark micerevealed that the patterns of activity of DA neuronswere profoundly altered long before the onset ofdegeneration (33). In particular, a large number ofneurons was significantly hyperpolarized and showedno spontaneous firing (33). Silencing resulted from adecrease in currents transported by hyperpolarization-activated cyclic nucleotide-gated ion channels, a cationchannel that helps to maintain the rate and regularityof neuronal discharge (33). The frequency of sponta-neous firing increased, however, in a subset of DAneurons, which suggests that impairments caused byTfam deletion are heterogeneous in SN DA neurons.Interestingly, with adequate stimulation, the silent neu-rons fired in a normal pacemaker fashion, suggestingthat these neurons might be in the early phase ofsuffering but not yet irreversibly compromised, whereasoveractive neurons may be prone to die more rapidly(33). In any case, DA neurons from MitoPark miceappeared to be hypofunctional, since vesicular dopa-mine release, measured by microdialysis, was selectivelyimpaired before the onset of locomotor deficits. Inter-estingly, striatal dopamine release was also reduced inmice with a mutation in LRRK2 (34), a PD gene thataffects mitochondrial function in DA neurons (2).

Altogether, these results support the hypothesis thatDA neurons depend critically on mitochondrial func-tion to establish membrane excitability (35), whichallows them to control their own survival. It should benoted that TH, the expression of which is regulatedelectrically (36, 37), disappears entirely from a largefraction of vulnerable but still viable neuromelanized

DA neurons in PD brains (38), which might signify thatthese neurons also become progressively less activelong before dying. Of interest, a bee venom prepara-tion that contains the brain-penetrant SK channelblocker APA, susceptible to maintain DA neurons elec-trically active and/or phenotypically functional (17),afforded protection to DA neurons in acute andchronic MPTP mouse models of PD (39, 40) and alsoprovided symptomatic relief to patients with PD in anexploratory clinical trial (41). APA itself significantlyreduced DA cell demise in mice undergoing a chronicexposure to MPTP, thus confirming indirectly that SKchannel blockade may account for at least part of theneuroprotective effects of bee venom (40).

EFFECT OF AFFERENT EXCITATORY INPUTSON THE SURVIVAL OF SN DA NEURONS

A number of studies have shown that afferent excitatoryinput converging on DA neurons might contribute tothe survival of these neurons by preserving their activ-ity. More specifically, stimulation by neurotransmitterssuch as acetylcholine (ACh), substance P (SP), andextracellular ATP might promote the survival of DAneurons during cell suffering.

Cholinergic nicotinic neurotransmission and DA cellsurvival

Postmortem studies in patients with PD have revealed thatpedunculopontine nucleus (PPN) cholinergic neurons,which exert excitatory nicotinic control over SN DAneurons, are largely affected in PD (42–44), and thattheir loss correlates with the level of DA denervation (45).This finding raises the possibility that excitatory cholin-ergic input might contribute to the survival of SN DAneurons in the adult brain and that this effect mightprogressively disappear in PD. Consistent with this view,the depolarizing alkaloid nicotine (NIC), the preferentialagonist of nicotinic acetylcholine receptors (nAChRs),was protective in several animal models of PD, includingmice or monkeys injected repeatedly with MPTP or ratsthat received stereotactic injections of 6-OHDA (46–48).In line with these observations, prospective cohort andcase-control studies have identified tobacco smoking asthe most significant protective factor against the risk ofdeveloping PD (49–51), presumably through self-admin-istration of NIC, although the implication of other com-pounds in tobacco cannot be not totally excluded; therelative risk of developing PD has been estimated to bereduced by 60% among active cigarette smokers com-pared to those who never smoked (49). This findingindicates that NIC might protect tobacco smokers fromdeveloping PD by mimicking or reinforcing cholinergicstimulation of SN DA neurons by the PPN, which isprogressively impaired in PD.

The molecular mechanisms underlying the protectiveeffects of NIC on DA neurons remain poorly understood,in particular concerning the type of receptor involved.Conventional pharmacological studies in midbrain cul-tures suggested that �7 and non-�7 nicotinic receptorsmight be implicated in NIC-mediated neuroprotection

3DA NEURONS NEED STIMULATION TO SURVIVE

(13, 52). Studies in genetically engineered mice con-firmed that the �7 nicotinic receptor subtype played amajor role (13). It should be noted, however, that �7nAChRs were detectable in only 40% of SN DA neurons(53). This might explain why a similar percentage of theseneurons were protected by NIC in midbrain cultures.Interestingly, the selective �7 nAChR antagonist methyl-lycaconitine abrogated (54) whereas the selective �7agonist GTS-21 reproduced (55) the protective effects ofNIC in rodent models of PD. It has been proposed thatneuroprotection by NIC may require the activation of �7nAChRs on DA neurons (13, 55), but an antiinflamma-tory effect involving �7 nAChRs on astrocytes and/ormicroglial cells is also conceivable (54, 56); the twomechanisms are not mutually exclusive (55).

It is important to note that the protective effects ofNIC were detectable in midbrain cultures only whenthe DA neurons were stimulated concurrently withother depolarizing agents, such as K� channel blockers(ref. 13 and Fig. 1). The protection of DA neurons byNIC in the presence of TEA, 4-AP, APA, or CTX wascorrelated with the effect produced by the same treat-ments in the absence of the alkaloid. This findingsuggests that, under control conditions, DA neuronssuffer from an intrinsic deficit in excitability in additionto the absence of excitatory input. Under pathologicalconditions in which DA neurons have intrinsic deficits(mitochondrial dysfunction, for example), survival pro-motion by acetylcholine released by PPN nerve termi-nals might be compromised. Since PPN cholinergicneurons are also affected in the course of the disease(42, 43), neurodegeneration would become self-perpet-uating as the disease progresses.

Other excitatory neuromodulators and DA cell survival

SP is a small neuropeptide that functions as an excitatoryneurotransmitter or neuromodulator by activating a G-protein-coupled receptor, the neurokinin 1 receptor(NK1R; ref. 57). SP is released onto SN DA neurons ortheir dendritic arbors via GABA-ergic input from thestriatum (58) and glutamatergic and cholinergic inputfrom the PPN (59). In the mesencephalon, SP exerts atonic facilitatory influence on DA neurons (60), suggest-

ing that lack of proper stimulation by the peptide mightpossibly play a role in the death of these neurons in PD. Anumber of arguments support this hypothesis. SP� neu-rons from the PPN that project onto DA neurons havebeen reported to be severely affected in PD (61). Inmidbrain cultures, both SP and synthetic agonists ofNK1Rs provided long-term protection for DA neurons,whereas NK1R antagonists suppressed these effects (62).In a rat model of PD, activation of NK1Rs by septide, asynthetic fragment of substance P, protected DA neuronsand improved motor deficits associated with their degen-eration (63). Interestingly, the protective effects of SP inmidbrain cultures were reversed by blockade of Navchannels with TTX, confirming that depolarization by SPwas necessary for the neuroprotection (62).

Purinergic type 2X receptors (P2XRs) are nonselec-tive cation channels that open in response to extracel-lular ATP. In the mesencephalon, extracellular ATPreleased as a cotransmitter has been reported to mod-ulate the firing of SN DA neurons via different subtypesof P2XR (64). Interestingly, the nonhydrolyzable P2XRagonist �,�-methylene-ATP and ATP itself promotedthe survival of DA neurons in midbrain cultures (65),suggesting that excitatory purinergic transmissionmight contribute to the survival of these neurons.Loss-of-function mutations in Parkin, which are respon-sible for familial forms of PD, were reported to reducedepolarizing currents generated through ATP-gatedP2XRs (66). The level of activation of these receptorsmight therefore influence the survival and function ofDA neurons under pathological conditions. Altogetherthese data confirm the view that excitatory input mightmodulate not only the activity of DA neurons but alsotheir ability to survive in unfavorable circumstances.

EFFECT OF INHIBITORY NEUROMODULATORSON THE SURVIVAL OF SN DA NEURONS

In that SN DA neurons need stimulation for survival,inappropriate inhibitory signals might increase theirvulnerability and/or affect their normal function. Ex-perimental studies with two inhibitory neuropeptides

Figure 1. NIC-mediated neuroprotection of DAneurons in midbrain cultures is unmasked by K�

channel blockers. A) TH� neurons degeneratespontaneously and progressively as they mature invitro. Separate treatments with NIC (1 �M) orTEA (1 mM) provide no or partial protection,respectively, to vulnerable TH� neurons, whereascombined treatment with NIC and TEA com-pletely rescues these neurons. B) NIC (1 �M)-mediated increases in TH� neurons in 10-DIVcultures exposed concomitantly to optimal con-centrations of TEA (1 mM), 4-AP (500 �M), APA(1 �M), or CTX (10 nM) plotted against corre-sponding increases in TH� neurons in matchedcultures exposed to K� channel blockers only.DIV, days in vitro. *P � 0.05 vs. correspondingcontrol; **P � 0.05 vs. corresponding treatmentwith TEA. Adapted from Toulorge et al. (13) withpermission.

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suggest that they could exacerbate neurodegenerativechanges under pathological conditions.

One of these inhibitory peptides is the opioid-likenociceptin/orphanin-FQ (N/OFQ) peptide that actson a Gi-protein-coupled receptor (67). Injection ofN/OFQ into the SN reticulata prevented the localrelease of dopamine, an effect that was reversed bypharmacological blockade of the N/OFQ peptide re-ceptor (NOP; ref. 68). Interestingly, Marti et al. (69)showed that genetic knockout of this receptor partiallyprotected SN DA cell bodies and striatal nerve termi-nals against MPTP. This finding indicates that tonicinhibition through NOP was permissive for MPTP-in-duced cell death; MPP�, the active metabolite of MPTP,itself induces functional inactivation of DA neurons (24),suggesting that NOP activation by endogenous N/OFQamplified this effect. The expression N/OFQ was en-hanced in the SN of 6-OHDA-hemilesioned rats and inthe cerebrospinal fluid of patients with PD undergoingsurgery for deep brain stimulation compared to non-Parkinsonian neurological controls (70), supporting theview that N/OFQ could play a role in PD, possibly byreducing the excitability of DA neurons.

Galanin, a neuropeptide hormone, slows the firingrate of rodent DA neurons and, as a consequence,reduces the release of dopamine in target areas (71). Itis believed that inhibition of midbrain dopamine activ-ity by galanin is mediated via the galanin receptorsubtype 1 (GALR1; ref. 22), which activates GIRKchannels in SN DA neurons (72). In midbrain cultures,galanin was reported to cause a dramatic reduction inthe number of TH� cells. This effect was only observedin cultures that had been previously exposed for severaldays to a lipophilic analog of cAMP (22), which stimu-lates TH expression (73). It may be concluded thatgalanin did not affect the survival of DA neurons but

rather prevented the induction of the enzyme by thecyclic nucleotide in a population of DA neurons hyper-polarized by the peptide (22). This is of interest since,as already mentioned TH disappears from a populationof vulnerable but still viable SN DA neurons in PD (38).To our knowledge, however, innervation of the SN bygalaninergic fibers has not been demonstrated. It ap-pears that persistent and inappropriate inhibitory sig-nals might affect the phenotypic features and/or theviability of DA neurons under pathological conditionsin which cells suffer. The mechanisms by which afferentexcitatory and inhibitory input might modulate thesurvival of SN DA neurons are illustrated in Fig. 2.

IS ELECTRICAL STIMULATION CRITICAL FORTHE INITIATION OF CA2�-RELATED EVENTSINVOLVED IN THE SURVIVAL OF DANEURONS?

Neuronal stimulation is associated with Ca2� entry viavoltage-and/or store-operated calcium channels butalso with Ca2� mobilization from the ER via intracellu-lar calcium release channels (35). The flow of calciumions through these channels or receptor/channelsmight, therefore, convert stimulatory events into signalsthat keep DA neurons functional and healthy. Argu-ments supporting this hypothesis are presented here.

Voltage-operated calcium channels and DA cell survival

The implication of voltage-dependent Ca2� channels(VDCCs) in the survival of DA neurons is illustratedprimarily by experiments in midbrain cultures. Protec-tion of DA neurons by Nav channel agonists and K�

+ -1

2

deathsurvival rescue

3+ - + -

A B C

toxic insultintrinsic deficit

toxic insultintrinsic deficit

Figure 2. How stimulatory and inhibitory input might modulate the survival of SN DA neurons. A) Normal condition in which thespontaneous activity of healthy DA neurons is modulated through the combined influence of excitatory and inhibitory input.B) Condition in which toxic insults or intrinsic deficits lead progressively to functional silencing of DA neurons, and ultimately to theirdeath. C) DA neurons can be rescued 1) by preserving cell autonomous mechanisms that control their spontaneous activity, 2) bymimicking stimulation by physiological excitatory input, or 3) by preventing the effects of inhibitory input.

5DA NEURONS NEED STIMULATION TO SURVIVE

channel blockers, such as 4-AP, APA, and CTX, but alsoby NIC, was prevented by flunarizine (12, 13), presum-ably due to blockade of low-threshold T-type VDCCs(74). Notably, the ability of flunarizine to preventrescue by depolarizing signals was unrelated to itsability to block D2 DA receptors since sulpiride, aspecific blocker of these receptors, had no effect on DAcell survival under the same experimental conditions(12). That T-type Ca2� currents might influence thefunction and survival of DA neurons in the adult brainis also suggested by the observation that flunarizine andits parent compound cinnarizine, used to treat mi-graine, caused PD symptoms in a few patients thatpersisted after treatment was discontinued (75, 76).

The role of high-threshold N-type or L-type VDCCswas also demonstrated in midbrain cultures. The pro-tective action of the neuropeptide SP on DA neuronswas attributed to a rise in resting cytosolic calcium(Ca2�

cyt) levels through NK1R modulation of N-typecalcium channels (62). These effects were sensitive to aspecific inhibitor of N-type VDCCs, the cone snail toxin�-conotoxin MVII-A (77), and resistant to L-type orT-type VDCC blockers (62). The effects of SP on DAneurons were also prevented by TTX, which indicatesthat the opening of N-type VDCCs probably occurredafter sequential activation of NK1Rs and Nav channels(62). It is of note that tonic activation of NOP, thereceptor for the N/OFQ peptide, indirectly activatedN-type VDCCs (78). This might explain why overstimu-lation of this receptor exacerbated DA cell death in theMPTP model of PD (69). Finally, treatment of midbraincultures with the wide-spectrum K� channel blockerTEA or with high concentrations of potassium ionsincreased the survival of DA neurons mediated byL-type calcium currents (11–13). The protective effectof TEA required, however, concomitant activation ofL-type and T-type Ca2� channels (13), whereas protec-tion by high K� was mediated exclusively by L-typechannels (11, 12). Of importance, the protective effectsof high K� were detected only if N-methyl-d-aspartateglutamate receptors were blocked to prevent concomi-tant excitotoxic stress and the calcium overload thatoccurs under these conditions (11, 12). Membranedepolarization induced by high K� was also reported tostimulate the phenotypic differentiation of DA neuronsin cultures of ventral midbrain neuronal precursorcells, but the underlying mechanism, which involvedepigenetic histone modification, was resistant to L-typeVDCC blockade (79).

Regardless of the conditions, the range of Ca2�cyt

levels required for optimal protection of DA neuronsappeared relatively narrow, consistent with the ideathat the relationship between Ca2�

cyt levels and DA cellsurvival follows an inverted U-shaped curve (11, 12). Ithas indeed been suggested that sustained elevation ofintracellular calcium levels might become deleteriousfor DA neurons (11, 80). In particular, it was reportedthat activity-dependent calcium entry through L-typechannels (probably a subset of them having a Cav1.3pore) can elevate mitochondrial oxidant stress in SNDA neurons, thus increasing their sensitivity to toxinsand genetic mutations associated with early onset formsof PD (81). In line with these observations, the L-type

channel blocker isradipine was protective in a rodentmodel of PD (82). Epidemiological studies of dihydro-pyridine class L-type calcium channel blockers forassociation with PD yielded, however, conflicting re-sults. A case-control study (83) and a historical cohortstudy (84) suggested that L-type calcium channel block-ers were protective. No delay in the progression of PDwas observed, however, in a large cohort of patientstaking brain-penetrant L-type calcium channel blockersfor hypertension (85).

The idea that Ca2�has to be maintained within alimited range of concentrations for optimal survival ofDA neurons is perhaps best illustrated by NIC protec-tion. We have shown that NIC protects DA neurons inmidbrain cultures through �7nAChRs, but only ifCa2�

cyt levels were already increased by concurrentdepolarization (13). This effect of Ca2�was not medi-ated by an increase in the density of �7nAChRs at theplasma membrane as initially suspected but most likelyby the switch of these receptors to an active conforma-tion state (86) that persisted as long as calcium levelsremained elevated. Under depolarizing conditions,NIC itself increased Ca2�

cyt levels indirectly through amechanism that required the successive activation of�7nAChRs, Nav channels, and T-type VDCCs, consis-tent with observations in slice preparations (87). Nota-bly, T-type calcium channel blockade prevented NIC-mediated protection of DA neurons (13), underliningthe key role played by calcium in this mechanism.

The increase in Ca2�cyt evoked by NIC and concom-

itant depolarizing treatments activated sequentially thecalcium effector protein calmodulin and the phospha-tidylinositol 3-kinase (PI3K)/Akt-dependent signalingpathway (13). Of importance, this pathway was alsoreported to control the survival of DA neurons in thedeveloping and adult rodent brain (88). In line withthese observations, genetic knock out of the tumorsuppressor PTEN, a negative regulator of PI3K-depen-dent signaling, protected DA neurons in an animalmodel of the disease (89). We might speculate thatdeficits in activity and in resting calcium levels are notrestricted to SN DA neurons in PD, and may also occurin the less vulnerable DA neurons of the VTA. It isprobable, however, that these deficits are less intense inVTA neurons and principally impair their response toNIC and other reward drugs, as suggested earlier (13,90). This would fit with the observation that patientswith PD generally do not engage in impulsive oraddictive behaviors (91). A mechanistic relation be-tween neuronal survival and drug reward mechanismsis also suggested by the observation that T-type VDCCs,essential for NIC-mediated neuroprotection, were alsocrucial for addiction to the alkaloid mediated by VTADA neurons (92).

Ca2� release channels and DA cell survival

Ryanodine receptors (RyRs) and inositol 1,4,5-trispho-sphate receptors (IP3Rs), which are ER Ca2�-releasechannels, translate membrane depolarization signalsinto local and rapid increases in Ca2�

cyt (93). One oftheir functions is to facilitate somatodendritic release ofdopamine (10, 94). These channels might also play a

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role in the control of DA cell survival. Evidence for theimplication of IP3Rs in DA cell survival is limited to datashowing that chronic inhibition of ER calcium releasewith 2-aminoethoxydiphenyl borate and xestospongin-C de-creased DA cell survival in midbrain cultures by pre-venting the transfer of calcium from ER to mitochondria(95). Interestingly, the synaptic protein �-synuclein, which isa major component of Lewy body aggregates in PD andis mutated in rare familial forms of the disease, facili-tated mitochondrial Ca2� transients elicited by activa-tion of IP3Rs (96). These results suggest that calciumrelease through these receptors might possibly influ-ence the survival of DA neurons via the control ofcellular bioenergetics.

Stronger evidence points to the role of RyRs in thecontrol of DA cell survival. Notably, stimulation of RyRsby two specific agonists—the alkaloid ryanodine andthe dimethylxanthine paraxanthine (PX)—the use ofwhich bypasses activation of VDCCs normally requiredto stimulate RyRs via Ca2�-induced calcium release,robustly protected DA neurons in paradigms in whichneurodegeneration was either spontaneous or inducedby trophic factor deprivation or MPP� intoxication(97). Protection by these treatments was accompaniedby moderate and sustained increases in Ca2�

cyt levels.Blockade of RyRs with dantrolene abolished the in-crease in survival due to the increase in calcium elicitedby ryanodine or PX, confirming that ER Ca2� mobili-zation was crucial for DA cell rescue. Protection by sucha mechanism is coherent with data showing that thepreservation of DA cell excitability by genetic (24, 69)or pharmacological manipulations (40, 46) protects DAneurons in MPTP-intoxicated mice. Interestingly, PX

and its prodrug caffeine protected DA neurons in arodent model of PD (98). Finally, genome-wide associ-ation studies have established that the CD157 gene(originally named BST-1) is a susceptibility gene for PD(99, 100); it encodes an ectoenzyme that catalyzes thesynthesis of cyclic ADP-ribose, a cyclic nucleotide deriv-ative that sensitizes ER calcium mobilization throughRyRs. An effect of susceptibility variants on RyR func-tion and calcium-induced calcium release has, however,not yet been reported.

Finally, the potential role of ER-mediated calciumrelease in DA cell survival needs to be considered inrelation to data showing that the type 1 transientreceptor potential cation channel (TRPC1), the mainfunction of which is to restore calcium levels in the ERafter depletion (101), might contribute actively to thesurvival of DA neurons. Indeed, Selvaraj et al. (102)showed that when MPP�, the active metabolite ofMPTP, was applied to neuroblastoma cells in culture,TRPC1-mediated Ca2� entry and ER Ca2� stores de-creased prior to cell degeneration. Interestingly, resto-ration of ER calcium concentrations through overex-pression of TRPC1 protected neuroblastoma cells inculture from exposure to MPP� and midbrain DAneurons in adult mice from repeated injections ofMPTP, confirming that ER Ca2� mobilization might becrucial for DA cell survival. It was suggested that therestoration of ER Ca2� served to maintain the activa-tion of PI3K/Akt-dependent signaling (102). ER Ca2�

depletion was also reported to favor the accumulationof unfolded/misfolded proteins in the ER lumen caus-ing ER stress (102). This might contribute to theprotein aggregation characteristic of PD. A summary of

VDCC

+ +

TRPC1

RyR ?

survival

mit

Cyt

+

*

?

K+ Ca2+ α7nAChR NK1 NOP

GalR1

APA, CTX, …bee venom?

geneticknock out(KATP)

Ca2+

ER

phenotypiccontrol

PI3K/Akt IP3R

α-Synuclein

Figure 3. How membrane depolarization mightreduce the vulnerability of DA neurons throughthe modulation of Ca2�-dependent signalingevents. The blockade of K� channels by APA,CTX, tertiapin-Q, 4-AP, and TEA preserves theactivity and excitability of DA neurons, allowingthe opening of VDCCs and thus the preservationof Ca2�

cyt in the range of concentrations re-quired for survival of DA neurons and/or themaintenance of their neurotransmitter pheno-type. The protective effects of bee venom arepossibly mediated by APA, a brain-penetrantblocker of SK channels. Genetic knockout of thepore-forming unit of KATP channels also protectsDA neurons from death by maintaining theirelectrical activity. ACh and SP acting through�7nAChRs and NK1Rs, respectively, help main-tain intracellular calcium levels in a protectiverange of concentrations. The alkaloid NIC mim-

ics stimulation of SN DA neurons by cholinergic neurons in the PPN. Notably, engagement of �7nAChRs is observed only if basalCa2�

cyt concentrations reach a threshold level. Increasing calcium levels through ATP-gated P2XRs may also improve DA cellsurvival (not shown for simplification). Aberrant stimulation of the Gi-protein-coupled receptors NOP and GalR1 by theirphysiological ligands N/OFQ and galanin, respectively, reduce the activity of DA neurons, thus increasing their vulnerability.GALR1 activation might only prevent the expression of TH without causing degeneration. ER calcium mobilization via RyRsmight also protect DA neurons by increasing Ca2�

cyt. RyRs can be activated with PX and ryanodine, but under physiologicalconditions this might result from activation of VDCCs through Ca2�-induced calcium release. Calcium release via IP3Rs alsoprotects DA neurons by stimulating calcium uptake into mitochondria. The synaptic protein �-synuclein, a major componentof Lewy body aggregates in PD that is mutated in rare familial forms of the disease, facilitates mitochondrial Ca2� transientselicited by activation of IP3Rs, a mechanism that presumably improves cellular bioenergetics. The store-operated TRPC1channels might contribute to the survival of DA neurons by restoring ER calcium levels after depletion under stressfulconditions. The PI3K/Akt-dependent signaling pathway might mediate some of the survival promoting effects of Ca2�

cyt.

7DA NEURONS NEED STIMULATION TO SURVIVE

how activity-dependent mechanisms might reduce thevulnerability of DA neurons through the modulation ofCa2�-dependent signaling events is presented in Fig. 3.

In summary, the data presented in this review showthat DA neurons need to be stimulated in a tightlyregulated manner in order to survive, probably throughthe control of calcium homeostasis to keep Ca2�

cyt inan optimal range of concentrations. The cellular struc-tures and molecular components that regulate activity-dependent processes are thus susceptible to be affectedin PD. Note, however, that PD pathogenesis is complexand a combination of multiple genetic and environ-mental factors. This signifies that even if a reduction inneuronal activity augments the vulnerability of DAneurons to degeneration, a number of other mecha-nisms are likely to contribute as well.

Recent work by the authors mentioned in this reviewreceived financial support from Institut National de la Santéet de la Recherche Médicale and Fondation pour la Recher-che Médicale (to D.T.). P.P.M. gratefully acknowledges sup-port from the program Investissements d’Avenir (ANR-10-IAIHU-06) and Association France Parkinson.

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Received for publication August 31, 2012.Accepted for publication May 14, 2013.

10 Vol. 27 September 2013 MICHEL ET AL.The FASEB Journal � www.fasebj.org