Because of its similarity to endogenous catecholamines, 6-OHDA produces a combined degeneration of dopamine and noradrenaline neurons. This has been described in initial studies in rats (Breese and Traylor, 1971) and observed also in the mouse model of partial striatal lesion (Bonito-Oliva et al., 2014). In addition, both 6-OHDA and MPTP have been shown to decrease serotonin levels in the hippocampus (Reader and Gauthier, 1984; Santiago et al., 2010). The ability of these toxins to affect multiple neurotransmitter systems altered in PD, is especially important for the study of non-motor symptoms.

#### **Sleep Disturbances in the 6-Hydroxydopamine Model of Parkinson's Disease**

Rats with a unilateral 6-OHDA lesion of the MFB show decreased sleep time during the light-on, inactive phase of the 24-h light-dark cycle, and increased total wake time assessed during a 24-h recording (Vo et al., 2014). When compared to naïve animals, these animals also show increased muscle activity during REM sleep, which is indicative of RBD-like abnormalities (Vo et al., 2014). Sleep analysis has recently been performed in rats injected unilaterally with 6-OHDA in the SNc, which results in a substantial loss of dopamine neurons (Ciric et al., 2019). This intervention leads to increased number and mean duration of wake episodes assessed by cortical and hippocampal EEG during 6 h within the inactive, light-on period (Ciric et al., 2019). Increased wake time, particularly evident during the 12-h dark period, has also been observed in rats with a selective 6-OHDA lesion of the SNc (Qiu et al., 2016). Conversely, cortical EEG recording in rats injected with 6-OHDA in the lateral ventricle showed decreased wakefulness and augmented SWS (Monti et al., 1999). This difference is likely explained by the more generalized effect on multiple components of the catecholamine system exerted by intracerebroventricular (i.c.v.) administration of 6-OHDA, in comparison to selective stereotactic injections in MFB or SNc (Vo et al., 2014; Ciric et al., 2019).

Sakata et al. investigated the effect of bilateral 6-OHDA lesion of the ventral tegmental area (VTA) on sleep-wake cycles (Sakata et al., 2002). They found reduced REM sleep during the light-on period, in comparison with control rats. The 6-OHDA-lesion rats also displayed reduced wake duration and spontaneous activity accompanied by a significant increase of both REM and SWS (NREM) during the dark phase (Sakata et al., 2002). These findings are in line with some of the disturbances observed in PD patients, who are affected by insomnia at night and daytime sleepiness.

Further studies in rats showed that injection of 6-OHDA in the DS or MFB decreases the number of orexin neurons in the LH (Cui et al., 2010; Oliveira et al., 2018), thereby reproducing a similar damage described in PD (Maeda et al., 2006; Thannickal et al., 2007). This loss was paralleled by reduced baseline respiratory frequency during sleep in the light-on period (Oliveira et al., 2018), a condition reminiscent of breathing disturbances and sleep apnea observed in PD patients (Mery et al., 2017; Videnovic, 2017). The same 6-OHDA lesion did not affect the sleep-wake cycle (Oliveira et al., 2018). This contrasts with previous work in which LH orexinergic neurons were preferentially targeted using a neurotoxin acting through the ribosome-inactivating protein saporin (hypocretin2-saporin) (Gerashchenko et al., 2001). In this case, the authors found increased NREM and REM sleep during the dark, active phase, and sleep fragmentation leading to reduced number of REM sleep episodes during the light-on phase (Gerashchenko et al., 2001). Similar changes of REM and NREM sleep in the light-off period, were observed using a cycad toxin-based PD model (see below), which leads to a significant reduction in orexin neurons (McDowell et al., 2010). It should be noted however that in the 6-OHDA study EEG recordings were performed during the last 3 h of the light period and the first 3 h of the dark period (Oliveira et al., 2018), instead of the more appropriate 24-h continuous recordings, as in the previous studies using saporin or cycad (Gerashchenko et al., 2001; McDowell et al., 2010).

Urethane anesthesia is characterized by alternations of oscillatory states which resemble REM-NREM cycling during natural sleep. Despite the obvious limitation of this approach, a number of studies have performed electrophysiological recordings in rodents under urethane to test mechanistic and functional hypothesis related to sleep-like states (Clement et al., 2008; Gonzalez-Rueda et al., 2018; Hauer et al., 2019). Resting-state functional magnetic resonance has been recently employed to examine modifications of functional connectivity in urethaneanesthetized rats with a unilateral partial striatal 6-OHDA-lesion (Zhurakovskaya et al., 2019). In this study, REM- and NREMlike sleep states were identified based on their association with high and low breathing rates, respectively (Pagliardini et al., 2012; Zhurakovskaya et al., 2019). Rats with a 6-OHDA lesion showed diminished intra-cortical, cortico-hippocampal, and striato-cortical functional connectivity, in comparison to naïve, or sham-lesion rats. Notably, these abnormalities were observed only during REM-like sleep (i.e., during states associated with high breathing rate), and may point toward changes in muscle tone suggestive of RBD (Zhurakovskaya et al., 2019). However, it remains to be established whether these alterations of connectivity can also be observed during sleep.

#### **Sleep Disturbances in the 1-Methyl-4-Phenyl-1,2,3,6- Tetrahydropyridine Model of Parkinson's Disease**

Sleep disruptions have also been observed in the MPTP model of PD. However, these alterations are transitory, which could be ascribed to limited or reversible neuronal loss, as well as to parallel compensatory effects (Johannessen et al., 1985; Saitoh et al., 1987; Bezard et al., 2000). Mice treated with five daily injections of MPTP (25 mg/kg) showed increased REM sleep during the 24-h light/dark cycle, in comparison to control. However, this effect was observed only 20 days after toxin injection and disappeared 40 days post MPTP (Monaca et al., 2004; Laloux et al., 2008). Interestingly, the reduction of dopamine neurons produced by MPTP, which was restricted to the SNc and amounted to approximately 30%, remained constant for up to 60 days, suggesting the existence of mechanisms correcting for the neuronal loss produced by the toxin (Laloux et al., 2008). In contrast to the aforementioned work, EEG analysis performed in mice 14 days after a single injection of MPTP (40 mg/kg), reported increased wake, and decreased NREM sleep during the light-off period, but no change in REM sleep. In this study, however, MPTP produced a large reduction of dopamine neurons in both SNc (78%) and VTA (54%) (Revishchin et al., 2016).

A study in the rat, examined the effect on sleep patterns of local injection of MPTP in the SNc, leading to a temporary (24 h) 50% decrease of tyrosine hydroxylase (a marker of dopamine neurons) (Lima et al., 2007). The authors observed a decrease in the latency to SWS onset during both the light-on and light-off periods, which lasted for up to 5 days following MPTP administration. This change was accompanied by increased sleep efficiency and may represent a surrogate marker of daytime sleepiness in PD. The same study also reported a significant increase in the latency to REM sleep in the MPTP rats compared to control animals. However, this disturbance was transient and disappeared 3 days after the administration of MPTP (Lima et al., 2007).

#### **Sleep Disturbances in Other Toxin-Based Models of Parkinson's Disease**

Chronic administration of rotenone, a natural compound commonly used as a pesticide, leads to a significant degeneration of SNc dopamine neurons accompanied by α-synuclein inclusions (Betarbet et al., 2000; Alam and Schmidt, 2002). Working with a rotenone-based rat model of PD, Yi et al. reported increased SWS and REM sleep during the light-off active period and decreased SWS during the light-on period (Yi et al., 2007). These modifications, which are suggestive of PD-related daytime sleepiness and nighttime insomnia, were largely refractory to L-DOPA. Notably, the effect of rotenone was accompanied by increased hypothalamic levels of interleukin-1β, a cytokine previously shown to promote REM and NREM sleep (Krueger, 2008). In line with this observation, i.c.v. administration of the endogenous interleukin-1 receptor antagonist (IL-1RA) counteracted the changes in SWS produced by rotenone during both the active and inactive 12-h periods (Yi et al., 2007). A word of caution should be spent concerning the solution (1:1 DMSO/PEG) commonly used to dissolve rotenone. In one study, this vehicle precluded a conclusive interpretation of the results, since it produced sleep anomalies that occluded those possibly caused by the neurotoxin (Garcia-Garcia et al., 2005); but see (Yi et al., 2007). For this reason, particular care should be taken to include appropriate controls when employing the rotenone model.

In the rat, consumption of seeds from the plant *Cycas micronesica* (cycad) results in degeneration and accumulation of α-synuclein aggregates in dopaminergic and noradrenergic neurons of the SNc and LC (Shen et al., 2010). Sleep analysis performed in this model reported increased NREM and REM sleep during the active 12-h phase. These changes were associated with increased average duration of NREM sleep and increased number of REM episodes (McDowell et al., 2010). Notably, the same study also reported a reduction in the number of orexin neurons in the LH of cycad-fed rats (McDowell et al., 2010), a change which is in line with previous work in orexin depleted rats (Gerashchenko et al., 2001) and may contribute to the observed sleep abnormalities.

Gerashchenko et al. utilized the hypocretin2-saporin neurotoxin (see *Sleep Disturbances in the 6-Hydroxydopamine Model of Parkinson's Disease*) (Gerashchenko et al., 2001) to lesion substantia nigra and VTA, thereby eliminating a large proportion of neurons (dopaminergic and non-dopaminergic) in both regions (Gerashchenko et al., 2006). In the substantia nigra, but not in the VTA, this intervention led to increased wakefulness and reduced amounts of NREM and REM sleep during light-on and light-off periods. These results are indicative of insomnia and are in part consistent with findings in rats with a selective dopamine lesion of SNc (see *Sleep*  *Disturbances in the 6-Hydroxydopamine Model of Parkinson's Disease* and *Sleep Disturbances in the 1-Methyl-4-Phenyl-1,2,3,6- Tetrahydropyridine Model of Parkinson's Disease*) (Lima et al., 2007; Ciric et al., 2019).

#### Genetic Models of Parkinson's Disease

Genetic models are commonly obtained by overexpressing genes linked to PD, or mutations implicated in autosomaldominant or recessive forms of the disease. Additional approaches include interventions disrupting mitochondrial function or specific transcription factors (Dawson et al., 2010; Chesselet and Richter, 2011). These strategies allow the study of effects produced by molecular modifications implicated in familial forms of PD, and to reproduce the accumulation of α-synuclein, which is a hallmark of PD absent in 6-OHDAor acute MPTP-based models (Dawson et al., 2010; Tieu, 2011). The neuronal loss observed in genetic models of PD is generally less pronounced in comparison to that achieved with neurotoxins, particularly 6-OHDA (Dawson et al., 2010; Chesselet and Richter, 2011). However, this does not necessarily represent a limitation to their use, since PD related sleep disorders are often manifested during the early phase of the disease.

In the mouse, overexpression of human α-synuclein driven by the Thy-1 promoter (Thy1-αSyn) leads to a progressive disruption of the nigrostriatal pathway, resulting in a 40% decrease of striatal dopamine (Chesselet et al., 2012). McDowell et al. examined sleep structure in Thy1-αSyn mice and found reduced REM sleep during the 24-h sleep-wake cycle, accompanied by increased NREM sleep during the light-on, inactive period (McDowell et al., 2014). The same authors also observed increased activity during the light-off, wake phase (McDowell et al., 2014). However, analysis of circadian restactivity performed in Thy1-αSyn mice reported reduced wheelrunning particularly pronounced during dark (Kudo et al., 2011). This discrepancy may be accounted for by the different parameters employed to assess the active state of the animals, i.e., whole-body movements detected by telemetry (McDowell et al., 2014) *vs.* wheel-running (Kudo et al., 2011). Notably, both EEG (McDowell et al., 2014) and visual (Kudo et al., 2011) analysis of Thy1-αSyn mice revealed a delay in sleep onset, although during different phases of the 24-h light-dark period.

Circadian rhythm has been also examined in the MitoPark mouse model of PD, in which dopamine neurons are progressively eliminated by selective impairment of mitochondrial function (Ekstrand and Galter, 2009). MitoPark mice showed a general reduction of locomotor activity in concomitance with the gradual loss of dopamine neurons, accompanied by a severe disruption of circadian rhythm in constant darkness (Fifel and Cooper, 2014). A similar loss of endogenous rhythmicity has been observed in neurotoxin models of PD based on striatal (Masini et al., 2017) or ventricular (Gravotta et al., 2011) injection of 6-OHDA. Further studies will be necessary to understand the mechanisms at the basis of these abnormalities as well as their impact on sleep function and architecture.

Mice with reduced (95%) levels of the vesicular monoamine transporter 2 (VMAT2) undergo nigrostriatal dysfunctions affecting the dopaminergic system, which replicate key pathologic features of PD (Caudle et al., 2007). This model displays a number of non-motor symptoms associated with PD, including anomalies of circadian and sleep function (Taylor et al., 2009). VMAT2-depleted mice show reduced ambulation during the active phase of the circadian period (Taylor et al., 2009). They also show a reduced latency to behavioral signs of sleep (Taylor et al., 2009), which contrasts with the delayed sleep onset observed in Thy1-αSyn mice (Kudo et al., 2011; McDowell et al., 2014). Notably, VMAT2-deficient mice show a concomitant reduction of dopamine, noradrenaline, and serotonin in striatum, hippocampus, and cortex (Taylor et al., 2009), which collectively may contribute to the wide range of non-motor symptoms observed in this model.

Altogether, the different findings reported in genetic models of PD fail to provide a coherent representation of one or more specific sleep alterations associated with PD. Aside from the contrasting effects on activity state observed in Thy1-αSyn mice, which may be attributable to different methodological approaches (see above), these inconsistencies are possibly a consequence of distinct genetic strategies (overexpression of α-synuclein as opposed to downregulation of VAMT2), which may produce unique alterations besides those affecting the dopaminergic system.

# CONCLUSIONS AND PERSPECTIVES

Rodent models represent a simple and versatile tool to study not only the classic motor symptoms and complications (i.e., dyskinesia) of PD, but also a number of non-motor co-morbidities. Nonetheless, the development of rat and mouse models of PD-related sleep disturbances has lagged behind in comparison, for instance, to affective and cognitive symptoms. The reason for this lies in part in the complexity of sleep-related disorders, both with regard to their behavioral manifestation and their relationship to distinct cellular and anatomical substrates. The difference in sleep architecture between rodents and humans represents an additional obstacle when interpreting results. In this regard, modeling PD in non-human primates constitutes a clear advantage, since in these animals, sleep structure and EEG patterns closely reproduce the consolidated, monophasic organization observed in humans (Hsieh et al., 2008), which contrasts with the more dispersed distribution of sleep states in rodents (Fifel et al., 2016).

A critical issue related to rat and mouse models, and more in general to all current animal models, is the difficulty to recapitulate the diversified nature of the degenerative processes at the basis of PD, thereby offering the possibility of examining the concomitant involvement of multiple neurotransmitter systems in sleep alterations. Thus, it is not surprising that rodent models of PD do not reproduce the full spectrum of sleep abnormalities associated with PD. However, in spite of these limitations some of these ailments have been reproduced in rats (**Table 1**). For instance, decreased SWS during the quiescent period, suggestive of insomnia, has been described after 6-OHDA-lesion of the MFB (Vo et al., 2014) and in the rotenone

(Continued)

#### TABLE 1 | Continued

NI, not investigated; i.p., intraperitoneal; s.c., subcutaneous; SN, substantia nigra; DA, dopamine; NE, noradrenaline; 5-HT, serotonin; α-syn, α-synuclein; ↑, increased; ↓, decreased. See text for other abbreviations.

model (Yi et al., 2007). Moreover, 6-OHDA-lesion of the VTA (Sakata et al., 2002), as well as administration of rotenone (Yi et al., 2007), or cycad toxin (Shen et al., 2010) increase NREM and REM sleep during the active phase of the circadian cycle, which is indicative of EDS. These results are in line with the analysis of rest-wake circadian rhythm in mice with a bilateral striatal 6-OHDA-lesion, which show decreased activity during the active 12-h period (Masini et al., 2017).

Modifications in sleep architecture have also been observed in mice and rats intoxicated with MPTP (Monaca et al., 2004; Lima et al., 2007; Laloux et al., 2008; Revishchin et al., 2016) (**Table 1**). However, MPTP-based approaches have failed to provide consistent results and are often observed only during a limited period of time following administration of the toxin (Lima et al., 2007; Laloux et al., 2008). This limited efficacy is in line with the lack of alterations of circadian locomotor activity rhythm reported in mice following acute or chronic treatment with MPTP (Fifel et al., 2013).

How can rodent modeling of sleep disorders in PD be improved? Overall, a general effort should be made to refine and harmonize the different approaches used to generate neurotoxinbased models. In the case of 6-OHDA, for instance, rats have been treated with i.c.v. injection, unilateral and bilateral injections in MFB, distinct midbrain dopaminergic nuclei (i.e., SNc and VTA), or in the striatum (see **Table 1**). Moreover, some studies (Sakata et al., 2002; Vo et al., 2014) have used pretreatment with desipramine, a selective inhibitor of noradrenaline reuptake. The different degree of dopamine and noradrenaline depletion produced by these interventions may cause distinct effects in sleep behavior, thereby contributing to discrepancies and confounding interpretations.

Combined loss of dopamine, noradrenaline, and to some extent functional inactivation of serotonin has been achieved with neurotoxins (Breese and Traylor, 1971; Reader and Gauthier, 1984; Santiago et al., 2010; Bonito-Oliva et al., 2013). In addition, 6-OHDA has been found to reduce the number of orexin/hypocretin neurons in the LH (Cui et al., 2010; Oliveira et al., 2018). Therefore, some of the sleep abnormalities observed in rodent models of PD may depend on the loss, or impairment, of one or more of these components of the arousal system. However, several key structures implicated in sleep regulation, including cholinergic and histaminergic nuclei, are spared by neurotoxin or genetic interventions. Therefore it will be important to strengthen construct validity of rodent models by establishing a more stringent link between the observed sleep abnormalities and PD-related biological dysfunctions.

The limitation posed by the relatively restricted neurodegenerative effects observed in most experimental models has been in part circumvented by acting on specific targets. For instance, bilateral injection of ibotenic acid in the PPN has been proposed to represent a model of PD cholinopathy (Ciric et al., 2018). In the rat, this intervention results in altered microstructure of both NREM and REM sleep during the inactive phase (Ciric et al., 2018). However, the neurotoxic effect exerted by ibotenic acid in the PPN is likely to affect not only cholinergic neurons, but also glutamatergic and GABAergic cells.

Chemo- and optogenetics techniques offer a particularly attractive approach to parse the involvement of neuronal networks in PD-related sleep-wake disturbances. For instance, using selective expression of designer receptor exclusively activated by designer drugs (DREADD) (Alexander et al., 2009), Kroeger et al. showed that distinct neuronal populations in the PPN regulate different aspects of sleep-wake behavior (Kroeger et al., 2017). Similar approaches, including optogenetic manipulation, showed that the dopamine neurons of the VTA are required for the initiation and maintenance of wakefulness (Eban-Rothschild et al., 2016; Oishi et al., 2017). Notably, these findings are in line with results obtained in rats with a selective 6-OHDA lesion of the VTA, which display decreased wakefulness during the active, light-off phase (Sakata et al., 2002).

Opto- and chemogenetic interrogation has also been employed to investigate the role in sleep of a population of dopamine neurons located in the dorsal raphe. These cells degenerate in PD (Halliday et al., 1990), but their involvement in non-motor symptoms remains to be in large part assessed. Cho et al. showed that ontogenetic stimulation of these dopamine population induces and maintains wakefulness and that vice versa their chemogenetic inhibition counteracts wakefulness and increases NREM sleep (Cho et al., 2017). In another study, Qiu et al. clarified the contribution to sleep of the external globus pallidus, whose activity is reduced in PD. It was found that, in the rat, activation of this structure resulted in enhanced sleep, possibly mediated *via* inhibition of cortical regions involved in the regulation of arousal nuclei (Qiu et al., 2016).

Recent evidence provides an attractive approach to reproduce the path of PD progression described by Braak et al. (2003). Injection of α-synuclein pathological fibrils in the gut muscle layer, leads to the spread of Lewy bodies from the peripheral nervous system, through the vagus nerve, to the brain, followed by loss of dopamine neurons (Kim et al., 2019). These effects are accompanied by motor impairment and by non-motor symptoms, including cognitive and affective deficits (Kim et al., 2019). The widespread distribution of α-synuclein fibrils, which are observed in several structures including the LC, suggests the potential use in the future of this or similar models for the study of sleep-related co-morbidities in PD.

One particularly interesting aspect of sleep disorders in PD is their potential impact on other frequent non-motor symptoms, such as cognitive and affective disorders. Indeed, cycling and coordinated transition of NREM and REM sleep are implicated in learning and memory processes (Moroni et al., 2014; Boyce et al., 2016). The synchronous activation of hippocampal pyramidal neurons during sleep results in the propagation of information throughout the neocortex, thereby leading to memory consolidation (Buzsaki, 1989). Depression has also been correlated to reduced SWS and increased duration and intensity of REM sleep (Riemann et al., 2001). The existence of a causative

#### REFERENCES

link between PD-related sleep disruptions and psychiatric co-morbidities remain to be further investigated. In this regard, it should be mentioned that memory deficit, depression, and anxiety are commonly observed in rodent models of PD, which may therefore represent valuable tools to help clarifying some of these questions.

#### AUTHOR CONTRIBUTIONS

GF conceived the presented idea and supervised the project. DM and GF wrote the manuscript. CA and MM provided critical feedback and helped shape the manuscript. All authors approved the final version for submission.

# FUNDING

This work was supported by the Swedish Research Council (grant 2015-02886), the Swedish Brain Foundation and the Swedish Parkinson Foundation (GF). This work is part of a joint Brazilian-Swedish Research Collaboration supported by the Swedish Foundation for International Cooperation in Research and Higher Education (STINT) and by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil.

to neurodegenerative disease. *Brain* 130, 2770–2788. doi: 10.1093/brain/ awm056

Jones, B. E. (2003). Arousal systems. *Front. Biosci.* 8, s438–s451. doi: 10.2741/1074

preoptic/anterior hypothalamic neuronal activity in the control of behavioral state. *Behav. Brain Res.* 115, 183–204. doi: 10.1016/s0166-4328(00)00258-8

**Conflict of Interest:** The authors declare that the submitted work was carried out in the absence of any personal, professional or financial relationships that could potentially be construed as a conflict of interest.

*Copyright © 2019 Medeiros, Lopes Aguiar, Moraes and Fisone. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Striatal Cholinergic Interneurons: How to Elucidate Their Function in Health and Disease

Nicolas Mallet 1,2, Arthur Leblois 1,2, Nicolas Maurice<sup>3</sup> and Corinne Beurrier 3\*

<sup>1</sup> Université de Bordeaux, Institut des Maladies Neurodégénératives, Bordeaux, France, <sup>2</sup> CNRS UMR 5293, Institut des Maladies Neurodégénératives, Bordeaux, France, <sup>3</sup> Aix Marseille Univ, CNRS, IBDM, Marseille, France

Striatal cholinergic interneurons (CINs) are the main source of acetylcholine in the striatum and are believed to play an important role in basal ganglia physiology and pathophysiology. The role of CINs in striatal function is known mostly from extracellular recordings of tonically active striatal neurons in monkeys, which are believed to correspond to CINs. Because these neurons transiently respond to motivationally cues with brief pauses, flanked by bursts of increased activity, they are classically viewed as key players in reward-related learning. However, CIN modulatory function within the striatal network has been mainly inferred from the action of acetylcholine agonists/antagonists or through CIN activation. These manipulations are far from recapitulating CIN activity in response to behaviorally-relevant stimuli. New technical tools such as optogenetics allow researchers to specifically manipulate this sparse neuronal population and to mimic their typical pause response. For example, it is now possible to investigate how short inhibition of CIN activity shapes striatal properties. Here, we review the most recent literature and show how these new techniques have brought considerable insights into the functional role of CINs in normal and pathological states, raising several interesting and novel questions. To continue moving forward, it is crucial to determine in detail CIN activity changes during behavior, particularly in rodents. We will also discuss how computational approaches combined with optogenetics will contribute to further our understanding of the CIN role in striatal circuits.

 Cholinergic interneurons (CINs) are the main source of ACh in the striatum [but see (Dautan, 2014)]. Despite their small numbers (1–3% of all striatal cells) and scattered distribution throughout the striatum, CINs have dense terminal fields that overlap those of dopaminergic projections coming from the substantia

#### Edited by:

Véronique Sgambato, Institut National de la Santé et de la Recherche Médicale (INSERM), France

#### Reviewed by:

Jill Crittenden, Massachusetts Institute of Technology, United States John N. J. Reynolds, University of Otago, New Zealand

> \*Correspondence: Corinne Beurrier<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neuropharmacology, a section of the journal Frontiers in Pharmacology

Received: 10 September 2019 Accepted: 15 November 2019 Published: 13 December 2019

#### Citation:

Mallet N, Leblois A, Maurice N and Beurrier C (2019) Striatal Cholinergic Interneurons: How to Elucidate Their Function in Health and Disease. Front. Pharmacol. 10:1488. doi: 10.3389/fphar.2019.01488

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nigra pars compacta (Bolam et al., 1984). CINs contact the two populations of striatal output neurons (also called medium spiny neurons, MSNs) that express either the dopamine D1 or D2 receptors. While most striatal neurons are not autonomously active, CINs exhibit a regular spiking activity in absence of any synaptic inputs (Bennett et al., 2000). Extracellular recordings performed in vivo in the striatum of monkeys also reveal the presence of tonically active neurons (TANs), which are thought to correspond to CINs (Aosaki et al., 1995). Hence, the morphofunctional features of CINs—mainly their extensive arborization primarily directed to MSNs and their tonic activity—place them as potent modulators of striatal output. Striatal output regulation is a fundamental process of the basal ganglia functioning, as a balanced activity between D1 and D2 MSNs is required to ensure correct motor and cognitive behaviors.

The improvement of parkinsonian tremor by both dopaminergic agonists and anticholinergic drugs led to the dopamine (DA)-ACh balance hypothesis, where DA and ACh are believed to play opposite roles in the striatum (Barbeau, 1962). Even though the prescription of anticholinergic drugs has been phased out due to their side-effects, this long standing clinical observation underlines the functional impact of ACh as the level of DA falls and has often led to the consideration of Parkinson's disease (PD) as a hypercholinergic disorder [but see (McKinley, 2019)]. There is indeed compelling evidence showing that DA depletion triggers complex alterations in striatal cholinergic signaling, activity, and connectivity (Aosaki, 1994; Raz, 2001; Ding, 2006; Salin, 2009). However, there is no consensual view explaining how CINs contribute to motor symptoms and abnormal network dynamic in PD.

At the cellular level, CIN modulation of the striatal network has been mainly inferred from the action of ACh agonists or through CIN activation. While nicotinic receptors (nAChRs) are expressed by interneurons and extrinsic afferent terminals, MSNs respond to ACh exclusively via muscarinic receptors (mAChRs): M1 receptors are present on D1 and D2 MSNs and M4 receptors are preferentially expressed by D1 MSNs. Activation of mAChRs modulates an array of voltage-gated channels and intracellular pathways in MSNs. Determining the combinatorial effect of these actions, potentially even opposing each other, is highly challenging and has recently been covered at length by excellent recent reviews (Tanimura, 2018; Ztaou and Amalric, 2019; Abudukeyoumu et al., 2019). A hallmark of CINs is their continuous tonic activity, which is expected to lead to a high level of ACh in the striatum, and the stereotypical bursts and pauses activity that they acquire during sensorimotor learning (Apicella, 2007). We can assume that a drop in ACh release, as expected to happen after a brief decrease in firing, conveys meaningful information to the striatal network. A recent hypothesis proposes that the pause would open a permissive temporal window during which corticostriatal synaptic plasticity occurs (Deffains and Bergman, 2015). However, it is still unclear how inhibition of CIN activity shapes striatal properties. Here, we review the related literature and show how optogenetic and computational approaches may contribute to further our understanding of this topic.

# CONSEQUENCES OF CHOLINERGIC INTERNEURON INHIBITION ON STRIATAL PROPERTIES

The widespread excitatory input from the cortex targeting D1 and D2 MSNs sets the activity of the direct and indirect striatofugal pathways which play a fundamental role in movement planning and action selection. Understanding how CINs modulate the dynamics of corticostriatal processing and MSN activity is therefore essential to uncover basal ganglia function. Cholinergic modulation of long-term corticostriatal plasticity has been addressed in excellent reviews (Lovinger, 2010; Lerner and Kreitzer, 2011) and will not be further discussed here.

The effects of cholinergic antagonists on corticostriatal transmission might provide interesting insights to predict how a pause in CIN firing impacts striatal output. It was reported that atropine, a broad mAChRs antagonist, or methoctramine, at a concentration that blocks M2 and M3 mAChRs, lead to a modest increase in corticostriatal transmission via the inhibition of mAChRs located on the glutamatergic terminals, suggesting the existence of tonic cholinergic presynaptic inhibition (Pakhotin and Bracci, 2007). On the other hand, pirenzepine, a blocker of M1 mAChRs, reduces corticostriatal transmission (Wang, 2006; Tozzi, 2011). In these last two studies, the authors suggest that lowering M1 mAChR activity in MSNs leads to the opening of L-type Ca2+ channels, which then triggers endocannabinoids release. Endocannabinoids are then able to reduce glutamate transmission by activating presynaptic CB1 receptors. Hence, mAChRs inhibition could exert opposite actions on basal corticostriatal transmission depending on their pre- or postsynaptic localization. Nicotinic a7 receptors have been described on cortical glutamatergic terminals but whether these receptors directly modulate corticostriatal transmission is still unclear (Howe et al., 2016).

One of the caveats of pharmacological experiments is that they do not allow to assess the effects of endogenous ACh that depends on the temporal dynamic of CIN firing. Moreover, CINs might co-release glutamate and GABA along with ACh, with effects that cannot be apprehended through this approach (Higley et al., 2009; Lozovaya, 2018). Optogenetic manipulations, enabling control of electrical activity in specific cell types with high temporal accuracy, can provide substantial insights into these issues. Here, we review the few studies showing the impact of optogenetic inhibition of CIN activity in the dorsal striatum. In vitro, we and others have shown that inhibition of CIN firing with halorhodopsin (eNpHR) is associated with a decrease in D1 and D2 MSN excitability that might involve a lowering of M1 mAChRs activation (Maurice, 2015; Zucca et al., 2018). In anaesthetized mice, opto-inhibition of eNpHR-expressing CINs was also reported to decrease MSN activity by hyperpolarizing their membrane potential and increasing the duration of down states (Zucca et al., 2018). In contrast, eNpHR-induced inhibition of CIN firing in freely moving mice did not alter MSN activity (English, 2011). In this last study, it is the rebound of action potentials occurring at the end of the eNpHR-induced hyperpolarization that triggered a decrease in MSN firing. Interestingly, despite the cellular effect induced by this pause-rebound, it was not followed by any detectable behavioral responses (English, 2011). This is in agreement with other works showing that opto-inhibition of CIN firing does not affect locomotion, anxiety‐like behavior, social memory recognition, and visuospatial object recognition (Maurice, 2015; Ztaou et al., 2018). In contrast, the behavior of PD mice, which perform poorly in all these tests, is improved by CIN silencing (Maurice, 2015). Restoring the balance between the striatofugal pathways at the level of the substantia nigra pars reticulata might be one component of the positive effect of CIN inhibition in parkinsonian condition (Maurice, 2015).

What conclusions can we draw from this brief overview? Obviously, more work is needed to understand how CIN inhibition shapes striatal output and modulates basal gangliarelated behavior. The conflicting effects of CIN firing inhibition on MSN activity and the lack of clear behavioral response in normal mice can be interpreted in different ways: (i) the light parameters (i.e. light duration, pattern or timing delivery) used to manipulate CIN firing are not physiologically relevant, (ii) CINs do not impact basal ganglia-related behaviors in physiological conditions and/or (iii) the behavioral tasks used are not appropriate to reveal CIN functions in rodents. What we know about the function of CINs comes from studies carried out in primates, describing the correlative changes in electrical activity of presumed-CINs during behavior. Optogenetics, mainly applied in rodents for technical reasons, is perfectly suited to go beyond correlational analysis and to investigate the causal implication of CINs in behavior. However, we first need to accurately describe the firing properties of these cells in rodents to be able to manipulate their activity in an appropriate way.

# ACTIVITY OF CHOLINERGIC INTERNEURONS DURING BEHAVIOR

The identification of CINs in behaving animals is usually based on their unique in vivo extracellular firing activity (i.e. tonically active at 5 spikes/s, sometimes in a burst mode) and broad spike waveform (i.e. spike duration > 2 ms). These electrical properties are easily distinguishable from all the other striatal cell populations and represent a good signature of CINs as confirmed later by in vivo juxtacellular labelling (Inokawa et al., 2010; Sharott et al., 2012). Using these classification criteria, early studies first defined the pattern of CINs activity during classical conditioning. In these experiments, animals have to learn the association between a neutral stimulus (i.e. often a tone) and an unexpected reward. In this context, CINs classically respond with a pause in firing that occurs shortly after the conditioned stimulus and lasts around 200 ms. This pause can also be preceded and/or followed by excitatory burst responses (Kimura et al., 1984; Aosaki, 1994; Apicella, 2017). Interestingly, this stereotypical pause appears during learning (Aosaki, 1994) and is time-locked to the response of nigral dopaminergic neurons (Morris et al., 2004). It is also dependent on the integrity of both dopaminergic neurons (Aosaki et al., 1994; Raz et al., 1996) and glutamatergic inputs coming from the intralaminar thalamus (Matsumoto et al., 2001).

What is not yet clear is whether the pause and burst components carry different signals used to underlie specific functions (Apicella, 2002; Apicella, 2007). Importantly, these activity patterns are mostly synchronized in the CINs population (Raz et al., 1996) such that they might efficiently translate into global change of striatal ACh level, providing a temporal window for complex pre- and post-synaptic modifications of striatal network and plastic changes (Deffains and Bergman, 2015; Cox and Witten, 2019). As a consequence, the pause response of CINs is considered as a key cellular substrate for reward-based learning, and may be particularly important for stimulusresponse and action-outcome associations. The exact cellular and network explanations underlying the generation of the pause/burst firing responses are not known precisely. Multiple mechanisms have been proposed to generate these responses. They all have in common the capacity to broadcast efficiently the information to spatially-distributed CINs (Goldberg and Reynolds, 2011; Schulz and Reynolds, 2013; Zhang and Cragg, 2017). Such broadcast mechanisms include:

Also, external sources such as GABAergic neurons from the midbrain, or from the globus pallidus (GP), or from unknown origin might also synchronize CIN population (Zhang and Cragg, 2017). Among these GABAergic sources, the inhibitory inputs coming from GP neurons appear to be functionally efficient at inducing a pause in CINs (Klug et al., 2018). However, it is important to mention that the pallido-striatal inputs could originate from two main populations of GP neurons, namely the prototypic and the arkypallidal neurons (Mallet, 2012), and that the respective contribution of arkypallidal or prototypic neurons in the CIN pause response have not yet been assessed. That being said, anatomical evidences would argue that arkypallidal neurons represent a good cellular substrate to generate a synchronized pause in CIN firing. Indeed, arkypallidal neurons provide widespread striatal GABAergic inhibition (Mallet, 2012; Mallet, 2016) that densely target, with "basket-like" perisomatic contacts, the soma and proximal dendrites of CINs (Mallet, 2012). It should also be noted that CINs represent preferential targets for arkypallidal neurons, as suggested by the larger number of apposition that a single-labeled arkypallidal cells make onto CINs (Mallet, 2012). Altogether, we propose that GABAergic arkypallidal neurons constitute a powerful mechanism to generate synchronized inhibitory responses in CINs population. Whether this arkypallidal-CINs circuit is part of a feed-back or a feed-forward loop is not known but should be addressed in future studies.

Apart from the classical conditioning experiments, the contribution of CIN activity has also been tested during operant tasks. In these experiments, the animal has to execute an action to obtain a reward. These studies have revealed the involvement of CINs in more complex behavioral aspects such as contextual (Apicella, 2007), temporal (Morris et al., 2004), goaldirected action (Bradfield et al., 2013), sensori-motor gating (Ding et al., 2010), movement control/modulation (Yarom and Cohen, 2011; Nougaret and Ravel, 2015; Lee et al., 2006), and action inhibition (Lee et al., 2006). Interestingly, the expression of the pause in CIN firing is largely dependent on the behavioral task paradigm (Benhamou et al., 2014). This might actually explain some of the discrepancies originally found between monkey and rodent recordings.

In addition, recent works have taken advantage of transgenic ChAT-Cre mice to genetically identify CINs and record their activity with two-photon calcium imaging and fiber photometry, during spontaneous locomotion in head-fixed animals (Gritton, 2019; Howe, 2019; Rehani, 2019). In doing so, novel features of CIN contributions to global locomotion control have been described. In particular, one study found that CINs increase their activity during behavioral state transition, and could thus favor the transition from one behavioral state to another (Howe, 2019). Alternatively, CINs activation can reduce ongoing movement while synchronizing the activity of MSNs (Gritton, 2019). This synchronizing effect on striatal neurons is a remarkable feature especially considering that excessive expression of synchronized oscillatory activity in the beta frequency band (12–30 Hz) is a hallmark associated with PD and possibly linked to akinesia/bradykinesia in PD patients (Brown, 2007) [but see (Nambu et al., 2015; McGregor and Nelson, 2019)]. This further adds to the view that CIN dysfunctional activity contributes to the pathophysiology of PD. Indeed, there is good evidence to suggest that the loss of DA in the striatum modifies the cholinergic signalling (Tanimura, 2018; McKinley, 2019; Ztaou and Amalric, 2019) and increases the correlated activity between CINs (Raz, 2001). Although the minimal neuronal circuit generating the parkinsonian beta synchronizations in basal ganglia circuits are not known, it is possible that CIN activity represents a good candidate to promote synchronized activity in these neuronal networks. Indeed, cholinergic agonist infusion in the striatum (McCarthy, 2011) and optogenetic excitation of CINs (Kondabolu, 2016) can induce an increase in the expression of beta oscillations. In addition, CINs opto-excitation in normal animals generates parkinsonianlike motor deficits (Kondabolu, 2016) while CINs opto-inhibition in PD mice decreases motor symptoms (Maurice, 2015).

# EXPLORING CHOLINERGIC INTERNEURONS FUNCTIONS IN BASAL GANGLIA NETWORK: CONTRIBUTION OF COMPUTATIONAL MODELING

CINs modulate striatal activity during behavior. A theoretical study of the putativefunction of these neurons inmotor learning and their possible role in pathophysiology through modeling could drive experimentally testable predictions and thereby guide further experimental investigation. Previous modeling efforts involving CINs remain relatively sparse. They range from simulating intracellular and ion-channel dynamics linked to cholinergic signaling to the effect of CIN activity modulation on behavior.

On the microscopic scale, two modeling studies have highlighted the tight coupling between DA neurons and CINs due to the dopaminergic modulation of both the intrinsic currents generating tonic firing (Aosaki et al., 1998; Maurice, 2004; Wilson and Goldberg, 2006; Deng et al., 2007) and the external inputs to CINs (Nicola et al., 2000; Pisani et al., 2000). Szalisznyó and Müller (2009) analyzed conductance-based changes in CIN subthreshold oscillations induced by DA and predicted that DA can switch CINs between stable oscillatory and fixed-point behaviors, with opposing effects of D1- and D2 type dopamine receptors. Tan and Bullock (2008) have shown that DA inputs robustly cooperate with thalamic inputs to control cue-dependent CIN pauses. Thereby, DA strongly affects performance- and learning-related dynamics in the striatum. The DA-CINs coupling could explain the adaptively scaled DA burst and the CIN burst and pause observed experimentally in response to reward-predicting cues. These changes would thus not necessarily require a modification in the weight of synapses onto CINs.

On the macroscopic scale, the influence of CINs on behavior can be either immediate, due to the modulation of striatal output by CINs, or delayed/persistent, due to ACh-dependent plasticity in the striatal network that leads to long-term changes in striatal response to its external inputs. A recent study by Vogt and Hofmann (2012) modeled the modulation in the activity of DA neurons and CINs in relation to external reward delivery and its internal expectation. They show that activity changes and their effect on learning outcome can be explained by a direct effect of neuromodulators (DA and ACh) on postsynaptic activity, even with unmodulated, two-factor spike timing-dependent plasticity (STDP). Obviously, it does not prohibit joint operation together with three-factor STDP rules. Interestingly, CIN pause could represent a time window to gate phasic DA release and "bracket" the plasticity window, while DA variations reciprocally modulate the CIN pause duration to adjust this window (Kim, 2019). In the context of reward-based motor adaptation, phasic DA release could thereby deliver reward information for reinforcement learning in a timely manner. Changes in CIN-DA interactions due to DA depletion would then produce poor performance of motor adaptation. Alternatively, CINs could act mainly on MSNs to suppress their firing and regulate local inhibitory network (Ashby and Crossley, 2011; Franklin and Frank, 2015).

To go beyond this current state of theoretical investigations, one may ask the following questions. What are the respective/ specific roles of DA and ACh during learning? How redundant are these signals? Are they separable in time or space? What is the specific motor impairment expected due to the abnormal CIN activity following DA depletion and could some PD symptoms be linked to CIN signaling dysfunction? These questions may be answered by integrating current experimental evidence and DA-ACh interactions and its effect on striatal dynamics revealed by previous theoretical work in a circuit model of the basal ganglia-thalamo-cortical loop. This model may display action selection properties and DA-driven reinforcement learning (Leblois et al., 2006; Guthrie et al., 2013), as well as PD-related dysfunction under DA depletion. The computational advantages brought by CINs and the neural mechanisms can be investigated in such a theoretical framework. Specific predictions can then be derived from model concerning the effects of manipulating CIN activity in a reinforcement learning protocol. These predictions could eventually be tested experimentally with physiological recordings performed in an operant conditioning task to ensure that the suggested mechanisms are indeed at play in the striatum during motor learning.

#### REFERENCES

#### CONCLUDING REMARKS

Our current understanding of the role of CINs in striatal function derives mostly from extracellular recordings of TANs in monkeys. Because these neurons transiently respond to motivationally relevant cues with brief pauses, flanked by bursts of increased activity, they are classically viewed as key players in reward related learning. However, how CINs, and particularly the pause in their tonic firing, modulate striatal output has yet to be demonstrated. It is also undisputable that CINs play a key role in relation to dysfunctional aspect of basal ganglia information processing such as in PD and it seems important that future works keep dissecting the causal role of CINs in striatal circuits.

### AUTHOR CONTRIBUTIONS

NMal, AL, NMau, and CB drafted, provided critical revision of the article, wrote, and approved the final version of the review.

#### ACKNOWLEDGMENTS

This work was supported by grants from the French Agence Nationale de la Recherche (ANR-2010-1416, ANR-14-CE13- 0024-01, ANR-15-CE37-0006 and ANR-16-CE37-0020-01), Fondation de France (# 146280), France Parkinson nonprofit organization (OPE-2018-0459), and the LABEX BRAIN (ANR-10-LABX-43). We thank Nuno Miguel Luis for the manuscript proofreading.

Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Mallet, Leblois, Maurice and Beurrier. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Pedunculopontine Nucleus Degeneration Contributes to Both Motor and Non-Motor Symptoms of Parkinson's Disease

Nicole Elaine Chambers, Kathryn Lanza and Christopher Bishop\*

Department of Psychology, Binghamton University, Binghamton, NY, United States

Parkinson's disease (PD) is a neurodegenerative disorder characterized by hypokinetic motor features; however, patients also display non-motor symptoms like sleep disorders. The standard treatment for PD is dopamine replacement with L-DOPA; however, symptoms including gait deficits and sleep disorders are unresponsive to L-DOPA. Notably, these symptoms have been linked to aberrant activity in the pedunculopontine nucleus (PPN). Of late, clinical trials involving PPN deep brain stimulation (DBS) have been employed to alleviate gait deficits. Although preclinical evidence implicating PPN cholinergic neurons in gait dysfunction was initially promising, DBS trials fell short of expected outcomes. One reason for the failure of DBS may be that the PPN is a heterogenous nucleus that consists of GABAergic, cholinergic, and glutamatergic neurons that project to a diverse array of brain structures. Second, DBS trials may have been unsuccessful because PPN neurons are susceptible to mitochondrial dysfunction, Lewy body pathology, and degeneration in PD. Therefore, pharmaceutical or gene-therapy strategies targeting specific PPN neuronal populations or projections could better alleviate intractable PD symptoms. Unfortunately, how PPN neuronal populations and their respective projections influence PD motor and non-motor symptoms remains enigmatic. Herein, we discuss normal cellular and neuroanatomical features of the PPN, the differential susceptibility of PPN neurons to PD-related insults, and we give an overview of literature suggesting a role for PPN neurons in motor and sleep deficits in PD. Finally, we identify future approaches directed towards the PPN for the treatment of PD motor and sleep symptoms.

Keywords: Parkinson's disease, pedunculopontine nucleus, neurodegeneration, gait deficits, non-motor symptoms, REM sleep behavior disorder, pharmacology

# INTRODUCTION

#### Cellular, Molecular, and Neuroanatomical Properties of Pedunculopontine Tegmental Nucleus

The pedunculopontine tegmental nucleus (PPN), a heterogeneous brainstem structure, consists of glutamatergic, cholinergic, GABAergic, and glycinergic neurons (Mineff et al., 1998; Wang and Morales, 2009; Pienaar and van de Berg, 2013), which co-express neuropeptides including nitric oxide, substance P, atriopeptin, NADPH diaphorase, calcium binding proteins, and corticotropin-releasing factor

#### Edited by:

Cristina Miguelez, University of the Basque Country, Spain

#### Reviewed by:

Sulev Kõks, University of Tartu, Estonia David Grabli, Sorbonne Universités, France

\*Correspondence: Christopher Bishop<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neuropharmacology, a section of the journal Frontiers in Pharmacology

Received: 21 July 2019 Accepted: 19 November 2019 Published: 15 January 2020

#### Citation:

Chambers NE, Lanza K and Bishop C (2020) Pedunculopontine Nucleus Degeneration Contributes to Both Motor and Non-Motor Symptoms of Parkinson's Disease. Front. Pharmacol. 10:1494. doi: 10.3389/fphar.2019.01494 (Vincent et al., 1983; Standaert et al., 1986; Austin et al., 1995; Martinez-Gonzalez et al., 2012; D'Onofrio et al., 2015). Coexpression of acetylcholine (ACh) with GABA or glutamate has also been reported (Lavoie and Parent, 1994; Wang and Morales, 2009). The PPN is functionally divided into two components, a rostral portion containing GABAergic, glutamatergic, and sparse cholinergic neurons sending projections primarily to motor structures including the substantia nigra and thalamus, and a caudal portion consisting mainly of cholinergic and glutamatergic neurons projecting to structures involved in reward such as the nucleus accumbens (Dautan et al., 2016; Xiao et al., 2016). PPN neurons also send descending projections to cerebellar nuclei, brainstem structures like the pontinus oralis, gigantocellular nucleus, and the spinal cord to modulate movement and muscle tone (Rye et al., 1988; Spann and Grofova, 1989; Martinez-Gonzalez et al., 2014). See Figures 1A, B for PPN efferent projections and their involvement in Parkinson's disease (PD) symptoms.

#### PPN in PD

PD is associated with degeneration of nigrostriatal dopamine (DA) neurons; however, PPN cholinergic, glutamatergic, substance P, GABAergic, and glycinergic neurons also degenerate in PD (Hirsch et al., 1987; Jellinger, 1988; Gomez-Gallego et al., 2006; Rinne et al., 2008; Pienaar and van de Berg, 2013). Microglial activation and inflammatory responses occurring in PD likely contribute to PPN neurodegeneration (Elson et al., 2016). Cholinergic loss, in particular, is linked to PD symptom severity, especially akinesia and gait deficits (Pahapill and Lozano, 2000; Rinne et al., 2008; Bohnen et al., 2013). Multiple factors likely contribute to death of cholinergic PPN neurons in PD. For instance, mitochondrial DNA deletion occurs more often in PPN cholinergic neurons than in other PPN neurons (Bury et al., 2017). If substantial, this may inhibit protein translation and energy production. In addition, alpha synuclein aggregates may differentially affect PPN neuronal subtypes, as glutamatergic and cholinergic PPN neurons show more effects of alpha synuclein aggregates than GABAergic neurons (Heinrich et al., as cited by Surmeier, 2018).

 Past

contacting brain areas involved in PD-related sleep disturbances. STR, Striatum; THAL, Thalamus; STN, Sub-thalamic nucleus; GPi, Globus Pallidus Interna; DRN, Dorsal Raphe Nucleus; Med, Medulla; SNr, Substantia Nigra Pars Reticulata; SNc, Substantia Nigra Pars Compacta; H-Thal, Hypothalamus; (Images modified from serviermedical art https://smart.servier.com).

research in preclinical models indicates that both processes may occur. Additionally, lesioning both nigrostriatal dopamine and PPN cholinergic systems simultaneously results in more severe degeneration (Bensaid et al., 2016). These results imply that the reciprocal connections shared by PPN cholinergic and nigrostriatal DA neurons may be critical to their survival, and that simultaneous lesion alters cellular activity, potentially contributing to neurodegeneration. Accordingly, following nigrostriatal DA lesion, compensatory increases in activity occur in PPN neurons (Palombo et al., 1990; Carlson et al., 1999; Breit et al., 2001; Zhang et al., 2008). Initial or sustained hyperactivity of PPN glutamatergic or subthalamic nucleus glutamatergic neurons may accelerate nigrostriatal and PPN neurodegeneration and may be exacerbated by simultaneous loss of PPN cholinergic neurons. PPN hyperactivity may also arise due to loss of nigral DA and raphe serotonin afferents in PD, as PPN neurons contain the inhibitory DA D2R and 5HT1AR (Leonard and Llinás, 1994). Conversely, PPN hypoactivity (Jang et al., 2012) caused by increased SNr and GP inputs may lead to more severe PD symptoms (Nandi et al., 2002). Perhaps, PD heterogeneity could be partially explained by the order of cellular degeneration and by differential PPN activity. Future research should investigate the timing of neuronal loss, affected projections, and behaviors resulting from cell loss in these two lesion paradigms.

Although under-investigated, a recent study in preclinical models indicates that galanin may rescue PPN cholinergic neurons in PD. Following SNc lactacystin lesion, galanin is upregulated in rostral PPN cholinergic neurons (Elson et al., 2018). Galanin inhibits cholinergic transmission and may be neuroprotective, as it guards basal forebrain cholinergic neurons from amyloid beta toxicity in Alzheimer's disease (Ding et al., 2006; Pirondi et al., 2010; Elliot-Hunt et al., 2011). In PD, galanin could reduce the likelihood of apoptosis in response to Lewy bodies or neuroinflammation (however, see also, Crawley et al., 2002).

#### PPN Involvement in Movement

The role of the PPN in generating locomotion remains controversial; however, early studies suggest PPN involvement in gait, an assertion that has been partially supported and further refined based on recent studies. Early research shows that the PPN and cuneiform nucleus make up the mesencephalic locomotor region (MLR), a functional neurocircuit modulating movement, rhythm, and speed upon electrical stimulation (Garcia-Rill et al., 1987; Garcia-Rill and Skinner, 1988; see Figure 1A for PPN projections related to PD motor symptoms). MLR projections to the lateral paragigantocellular nucleus (LPGi) modulate activity of motorneurons and ventral spinal laminae "central pattern generators" that regulate patterns of locomotor activity (Grillner and Wallen, 1985). In support of the PPN's role in gait, fMRI studies demonstrate that PPN activity increases during fast imagined walking in healthy individuals (Bakker et al., 2008; Karachi et al., 2010); however, whether the PPN exerts its effects through ascending or descending pathways in actual fast walking is unknown. Although both PPN glutamatergic and cholinergic neurons may exert effects through LGPi efferents, recent data from preclinical models show that these neurons play different roles in locomotion. Based on these studies, glutamatergic neurons control flexor muscles to stop locomotion and are involved in slow-paced movement and exploratory behavior (Caggiano et al., 2018; Josset et al., 2018), whereas cholinergic neurons control extensor muscles and prolong the stance phase of locomotion and can also increase speed (Roseberry et al., 2016).

Ascending PPN efferents are also well-positioned to influence movement, although most existing data on this subject stem from research in preclinical models. Most importantly, the PPN projects to thalamic nuclei, which guide movements via thalamospinal projections. Additionally, although less direct, PPN glutamatergic and cholinergic inputs to SNc and striatum may affect both direct and indirect pathways. Relatedly, PPN cholinergic terminals in SNc drive locomotion (Xiao et al., 2016), whereas the motor outcomes of PPN afferents to striatum remain enigmatic, but are likely modulatory in nature (Dautan et al., 2016; Assous and Tepper, 2019). Finally, PPN cholinergic and GABAergic inputs to SNr exert influence over the nigrostriatal pathway. There, PPN ACh inhibits MSN terminals via M4R, inhibiting movement (Moehle et al., 2017), and PPN GABAergic projections improve motor learning through thalamic disinhibition (Li and Spitzer, 2019). Though PPN neurons likely contribute to normal gait, the role of neurons in the basal forebrain and cuneiform nucleus should also be carefully considered (Xiang et al., 2013; Sarter et al., 2014).

## PPN Involvement in PD Gait and Motor Deficits

Nearly 20%–60% of PD patients experience gait dysfunction including freezing of gait (FOG) that is not consistently improved by DA replacement therapy (Giladi et al., 2001). FOG and falls are more common in PD patients exhibiting decreased ACh metabolism and reuptake in the thalamus (Bohnen et al., 2009; Bohnen and Albin, 2011; Bohnen et al., 2019), where the PPN is the main source of ACh. Cell death and synaptic inhibition from the GPi and SNr likely contribute to this decrease in cholinergic tone. In animal models, chemogenetic activation of PPN ACh neurons rescues motor deficits, indicating that cholinergic neurons remain which may be suitable therapeutic targets in PD (Pienaar et al., 2015). Similarly, cholinesterase inhibitors decrease falls in some PD patients (Chung et al., 2010).

Given its role in locomotion, the PPN has been considered as a site for deep brain stimulation (DBS) to improve otherwise intractable postural instability, FOG, and falling in PD and atypical Parkinsonism. Results of this approach have been varied. Multiple studies demonstrate that PPN-DBS can ameliorate FOG or general gait parameters in PD patients (Pereira et al., 2008; Ferraye et al., 2010; Khan et al., 2011; Thevathasan et al., 2011; Khan et al., 2012a; Khan et al., 2012b; Mazzone et al., 2014; Welter et al., 2015; Mestre et al., 2016). Although unilateral (Moro et al., 2010) and bilateral (Ferraye et al., 2010) approaches decrease falls, double-blind studies indicate the superiority of bilateral PPN stimulation for improving PD-related gait symptoms (Thevathasan et al., 2010; Thevathasan et al., 2012). Conversely, postural instability is not consistently improved with PPN-DBS (Moro et al., 2010; Thevathasan et al., 2010). Despite symptomatic improvements in some studies, open questions remain on the clinical relevance of PPN-DBS, as it cannot eliminate PD gait symptoms, and exact mechanisms underlying PPN-DBS effects are unknown. Furthermore, side effects ranging from oscillopsia, paresthesia, and even worsening of FOG have occurred in some patients (Hamani et al., 2007).

Multiple factors explain inconsistent PPN-DBS clinical outcomes. First, electrode placements in the PPN of PD patients vary. MRI placements correlated with patient outcomes suggest bilateral lead placement in caudal PPN is most efficacious for gait improvement (Goetz et al., 2018; Khan et al., 2012b). Relatedly, preclinical data show that rostral PPN lead placement has deleterious effects on movement and that caudal PPN controls stepping behavior (Gut and Winn, 2015; Garcia-Rill, 1986; Garcia-Rill and Skinner, 1988; Garcia-Rill, 1991). Second, stimulation parameters for PPN-DBS differ from one study to another. Many paradigms employ frequencies within beta and gamma ranges, but some have also employed stimulation at very low frequencies. Finally, small sample sizes, differences in disease progression, and the heterogeneity of PD signs and symptoms likely contribute to differential results regarding PPN-DBS.

#### PPN Contribution to L-DOPA Response

DA replacement therapy with L-DOPA is standard treatment for PD. However, chronic use results in debilitating abnormal involuntary movements termed L-DOPA-induced dyskinesia (LID) in up to 90% of PD patients (Ahlskog and Muenter, 2001). Severe LID can lead to hospitalization and reduced quality of life (Péchevis et al., 2005; Lyoo and Lee, 2011). Although the PPN is not well investigated for its role in L-DOPA's motor efficacy and in LID, existing evidence from patients undergoing PPN-DBS and preclinical evidence on the PPN's involvement in motor stereotypy suggest that the PPN may contribute.

For instance, PD patients decrease their L-DOPA dose following long-term treatment with PPN-DBS (Mazzone et al., 2014), suggesting that PPN-DBS has pro-motor effects and may potentiate L-DOPA's motor efficacy. Prior combination of PPN-DBS with L-DOPA shows additional motor benefit beyond L-DOPA alone (Jenkinson et al., 2008; Stefani et al., 2007). Additionally, combining caudal PPN-DBS with L-DOPA in preclinical models results in attenuation of LID (Gut and Winn, 2015). Similarly, immediate early genes associated with development and expression of LID are upregulated in the PPN of dyskinetic rodents (Bastide et al., 2014), implicating the PPN in LID expression.

The PPN has long been known to contribute to motor stereotypy in preclinical models; contextualizing this literature may provide useful insights into the role of the PPN in LID, as research on the PPN's contribution to LID is sparse. PPN lesion produces orolingual stereotypy in response to d-amphetamine and apomorphine (Inglis et al., 1994), implying that PPN neurons suppress stereotyped movements in the normal brain. Furthermore, existing evidence shows that PPN GABAergic and cholinergic neurons have differing effects on motor stereotypy. PPN infusion of GABAergic antagonists reduces motor stereotypy produced by apomorphine and SNr microinfusion of muscimol (Childs and Gale, 1983), intimating that PPN GABA projections inhibit SNr neurons to promote motor stereotypy, or that SNr GABA projections to PPN promote motor stereotypy. Conversely, PPN infusion of mAChR antagonists increases motor stereotypy and exacerbates LID (Mathur et al., 1997; Chambers et al., 2019). Taken together, these results suggest that decreasing GABAergic tone or increasing cholinergic tone in the PPN may decrease LID, but this must be examined further. If true, this provides further support for hypoactivity of the PPN in PD. Future research should address specific contributions of PPN neuronal subtypes, and investigate the role of PPN efferents to the SNr and striatum.

# PPN Involvement in Sleep

The PPN is part of the reticular activating system (RAS), a group of nuclei regulating consciousness and attention. The PPN receives lateral hypothalamus orexin projections and SNr GABA projections that regulate muscle tone during sleep and wakefulness (Takakusaki et al., 2011, see Figure 1B). As mentioned previously, SNr and GPi GABA transmission to PPN is enhanced in PD. Importantly, when GABA B receptors are bound, REM sleep is inhibited (Sakai and Koyama, 1996; Ulloor et al., 2004; Datta, 2007). This alone may account for a significant portion of REM sleep problems in PD.

PPN cholinergic neurons promote EEG desynchronization and changes in state of consciousness via thalamic projections which regulate cortical activity (Hallanger et al., 1987; Steriade et al., 1990; Kobayashi et al., 2002; Mena-Segovia et al., 2008; Kotagal et al., 2012). PPN cholinergic neurons are active during REM sleep and wakefulness, manifesting alpha activity at rest and gamma oscillations related to behavior during waking (Steriade et al., 1991; Steriade et al., 1996; Kezunovic et al., 2011). During REM sleep, cholinergic neurons promote muscle atonia through projections to the subcoeruleus dorsalis (Baghdoyan et al., 1984; Sanford et al., 1994), and nucleus reticularis gigantocellularis neurons which project to motorneurons (Takakusaki et al., 2011). Cholinergic neurons promote REM and pontogeniculooccipital waves characteristic of REM sleep via dorsolateral geniculate nucleus and frontal eye field efferents (Sakai et al., 1976; Callaway et al., 1987; Shouse and Siegel, 1992; Rye, 1997). During slow wave sleep cholinergic neurons also promote nested gamma oscillations associated with memory replay and neuronal plasticity in cortex and hippocampus (Lee and Wilson, 2002).

#### PPN Involvement in PD-Related Sleep Dysfunction

Sleep disturbances resulting in excessive daytime sleepiness occur in 98% of PD patients (for review see Comella, 2007). While prevalence of PD-related sleep disorders alone is concerning, sleep dysfunction, particularly REM sleep behavior

TABLE 1 | Potential targets for PPN-mediated improvement of PD motor and non-motor symptoms.

disorder (RBD), is linked to increased risk of cognitive impairment in PD patients (Marion et al., 2008; Gagnon et al., 2009). RBD is present in an estimated 50% of PD patients and is characterized by lack of muscle atonia during REM sleep which leads to patients engaging in complex motor behaviors during sleep. Intriguingly, diagnosis with idiopathic RBD often precedes PD and other synucleinopathies and may represent a prodromal phase of these illnesses (Boeve et al., 2001).

PPN ACh and substance-P-expressing neurons may contribute to the pathogenesis of RBD. Despite similar age of onset and disease duration, PD patients with RBD show decreased cholinergic transmission in cortex and thalamus relative to PD patients without RBD (Kotagal et al., 2012; however see also Bedard et al., 2019). Similarly, AChE inhibitors decrease symptoms of RBD, meaning that RBD may arise due to loss of cholinergic tone (Ringman and Simmons, 2000). Substance P is co-expressed in 27% of caudal PPN ACh neurons and is co-released onto the pontoreticular formation, where it has additive effects beyond those of ACh on initiation and maintenance of sleep (Kohlmeier et al., 2002). Substance P neurons degenerate in PD, which likely contributes to REM sleep atonia in PD patients. Congruent with PPN involvement in sleep dysfunction, PPN-DBS increases sleep efficiency, REM and stage 2 sleep, and decreases awakenings in PD patients (Romigi et al., 2008). Although understudied, these findings demonstrate that PPN-DBS could be used in the future to improve sleep for PD patients.

#### Therapeutic Strategies/Future Directions

Interpreting PPN-DBS outcomes is impeded by small sample sizes, differences in electrode placement, stimulation paradigms, and disease progression. Additionally, PPN-DBS paradigms employ open-loop DBS which does not consider the situation and/or symptoms the patient is experiencing, nor brain activity in other areas which influence PD symptoms. Employing closedloop PPN-DBS may be more effective (Rosin et al., 2011; Hebb et al., 2014; Sun and Morrell, 2014), as different stimulation frequencies have varying effects on PD symptoms. Using machine learning strategies, clinicians could correlate specific patterns of EEG readout with problems with FOG or other symptoms, and then use this information to promote more efficacious PPN-DBS. Future research should also address effects of exercise on the PPN, as recent data show that similar to hippocampal and basal forebrain cholinergic neurons (Molteni et al., 2008; Hall and Savage, 2016), exercise may induce PPN neuroplasticity beneficial for motor learning (Li and Spitzer, 2018). Finally, gene-therapy strategies focusing on neuroprotection through galanin or modulating L-type calcium channels should be considered, as well as gene therapy strategies that target specific PPN projections for symptomatic relief (see Table 1).

# CONCLUSION

Overall, through innervation of ascending and descending motor structures, RAS, and cortex, the PPN is well-positioned to modify PD motor and sleep symptoms. However, to develop effective PPN-centered treatment strategies, there is a need for cellspecific manipulations in the PPN. Currently, this is challenging, as the contribution of specific PPN pathways to gait and RBD symptoms, and pathways that govern PPN's modulation of L-DOPA's effects are not well understood. Additionally, receptor types specific to each PPN neuron and how they change in health and disease are not well-defined. Therefore, future research should elucidate the contribution of specific PPN afferent and efferent connections to behavior and identify receptors exclusive to each type of PPN neuron. Additionally, as PD is a heterogeneous disorder, it is especially important that the therapeutic strategies employed are customtailored to the patient based on his or her symptoms.

# AUTHOR CONTRIBUTIONS

Contributed to writing of the manuscript: NC, CB Created figures: KL.

## FUNDING

American Parkinson's Disease Association Research.

# REFERENCES

tone by motor pathway: A virally mediated trans-synaptic tracing study in surgically sympathectomized mice. Brain 136, 1–4. doi: 10.1093/brain/ awt123

Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Chambers, Lanza and Bishop. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Neuropsychiatric Disorders in Parkinson's Disease: What Do We Know About the Role of Dopaminergic and Non-dopaminergic Systems?

#### Kathy Dujardin<sup>1</sup> \* and Véronique Sgambato<sup>2</sup> \*

1 Inserm U1171 Degenerative and Vascular Cognitive Disorders, Lille University Medical Center, Lille, France, <sup>2</sup> CNRS, Institut des Sciences Cognitives Marc Jeannerod, UMR 5229, Lyon University, Bron, France

Besides the hallmark motor symptoms (rest tremor, hypokinesia, rigidity, and postural instability), patients with Parkinson's disease (PD) have non-motor symptoms, namely neuropsychiatric disorders. They are frequent and may influence the other symptoms of the disease. They have also a negative impact on the quality of life of patients and their caregivers. In this article, we will describe the clinical manifestations of the main PD-related behavioral disorders (depression, anxiety disorders, apathy, psychosis, and impulse control disorders). We will also provide an overview of the clinical and preclinical literature regarding the underlying mechanisms with a focus on the role of the dopaminergic and non-dopaminergic systems.

#### Edited by:

Andrew C. McCreary, Uniqure B.V., Netherlands

#### Reviewed by:

Susan Fox, University Health Network (UHN), Canada Philippe Huot, McGill University, Canada

#### \*Correspondence:

Kathy Dujardin<EMAIL_ADDRESS>Véronique Sgambato<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neuropharmacology, a section of the journal Frontiers in Neuroscience

Received: 09 September 2019 Accepted: 10 January 2020 Published: 29 January 2020

#### Citation:

Dujardin K and Sgambato V (2020) Neuropsychiatric Disorders in Parkinson's Disease: What Do We Know About the Role of Dopaminergic and Non-dopaminergic Systems? Front. Neurosci. 14:25. doi: 10.3389/fnins.2020.00025 Keywords: depression, anxiety, apathy, psychosis, impulse control disorders, Parkinson's disease, basal ganglia, animal models

## INTRODUCTION

Parkinson's disease (PD) is a neurodegenerative disease resulting from progressive death of the dopaminergic neurons in the brainstem, particularly the substantia nigra pars compacta (SNc). Besides the hallmark motor symptoms (hypokinesia, rest tremor, rigidity, and postural instability), PD patients have non-motor symptoms including neuropsychiatric disorders (Chaudhuri et al., 2006). These behavioral disorders can be separated into two categories according to their pathogenesis. Depression, anxiety, apathy, and partly psychosis are consequences of the neurodegenerative process while some psychotic symptoms and impulse control disorders (ICDs) occur as adverse effects of some antiparkinsonian drugs. In this article, we will describe the clinical manifestations of these behavioral disorders and provide some elements of epidemiology. We will also provide an overview of the current literature regarding the underlying mechanisms with a focus on the role of the dopaminergic and non-dopaminergic systems at both the clinical and preclinical levels.

## SEARCH STRATEGY

A structured search in PubMed for articles written in English up to September 2019 was performed. The following keywords and MeSH terms were used: Parkinson's disease, depression, depressive, anxiety, anxious, apathy, apathetic, psychosis, hallucination, hallucinatory, delusion, ICDs,

**536**

impulsivity, impulsive, compulsion, compulsive, gambling, hypersexuality, animal model, 6-OHDA, MPTP, alpha-synuclein, pathophysiology, pathophysiological mechanisms, neurotransmission, neuromodulation, catecholamine, dopamine, dopaminergic system, dopaminergic pathway, non-dopaminergic, non-dopaminergic pathway, serotonin, norepinephrine, noradrenaline, acetylcholine, glutamate, GABA.

## DEPRESSION

The prevalence of clinically relevant depressive symptoms in patients with PD ranges from 30 to 35% according to most studies (Reijnders et al., 2008; Aarsland et al., 2011). A study on a very large sample of PD patients showed that this prevalence was higher in female than males, at advanced stages of the disease and in patients with dementia (Riedel et al., 2010).

It is noteworthy that 10 to 27% of early, untreated PD patients have significant depressive symptoms (Ravina et al., 2007b; Dujardin et al., 2014; Weintraub et al., 2015b). Moreover, depression may be a prodromal symptom of PD (Schapira et al., 2017) even though it is not a risk factor for the disease (Ascherio and Schwarzschild, 2016). In PD, there is an increased risk of depression among women, in the advanced stages of the disease and in patients with cognitive impairment (Aarsland et al., 2011).

Core symptoms of depression in PD are sadness, depressed mood, loss of pleasure, feelings of worthless, and guilt. Diagnosis is difficult because some symptoms of depression like slowness, loss of weight, sleep disturbances, or poor emotional expression are also common symptoms of PD and frequently occur in non-depressed PD patients (Okun and Watts, 2002; Brown et al., 2011). According to the last version of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5), patients with PD may present minor, major or persistent depressive disorders. Depression is often comorbid with anxiety in PD (Brown et al., 2011).

The pathophysiology of depression in PD remains largely unknown and multiple factors (genetic, inflammatory, cellular regulation, signaling pathways, . . .) are probably incriminated. The loss of dopamine (DA) in the mesocortical and mesolimbic pathways, leading to dysfunction of the orbito-frontal area, is likely involved (Vriend et al., 2014a), as well as the loss of striatal DA, particularly in the caudate (Vriend et al., 2014b). Molecular neuroimaging studies have shown that noradrenaline (NA) (Remy et al., 2005) and serotonin (5- HT) (Boileau et al., 2008; Politis et al., 2010; Ballanger et al., 2012; Maillet et al., 2016) also play a role. The severity of depression in early untreated PD patients correlates with the reduction of the serotonergic innervation in the anterior cingulate cortex (Maillet et al., 2016). Cholinergic deficits may also be involved in PD patients with depression and cognitive deficits (Bohnen et al., 2007). Looking for blood-based biomarkers in PD, one study reported that lower plasma levels of 5-HT and its metabolite 5HIAA correlate with more severe depression in PD patients (Tong et al., 2015). However, data are few and sometimes inconsistent (Aarsland et al., 2011). Further studies are needed to determine how DA and non-DA systems interact in the regulation of mood in PD.

Most of PD patients with depression are treated with selective serotonin reuptake inhibitors (SSRIs) (Starkstein and Brockman, 2017). The efficacy of dopamine agonists to counteract depression is still controversial as pramipexole induced positive results (Barone et al., 2010) but not rotigotine (Chung et al., 2016). One clinical trial has been completed on the safety and efficacy of pimavanserin, a selective 5-HT2A antagonist/inverse agonist, to treat depression in PD but there are no results available (ClinicalTrials.gov Identifier: NCT03482882). Finally, there was a clinical trial on the use of atomoxetine, a potent NA reuptake inhibitor (ClinicalTrials.gov Identifier: NCT00304161) which has been completed and gave negative results as already shown by the same group previously (Weintraub et al., 2010a).

These clinical data are to be compared with experimental data obtained in rodents (rat and mouse) and non-human primate (NHP) models of PD (see **Table 1**). These models afford substantial resources to investigate the etiology and pathophysiology of PD as well as to develop therapeutic approaches. Animal models of PD include acute toxin models, such as 6-hydroxydopamine (6-OHDA) or 1-methyl-4phenyl-1,2,3,6-tertrahydropyridine (MPTP), as well as genetic models, such as α-synuclein, Parkin and monoamine-related alterations. Depressive-like behavior can be observed in animals using several behavioral paradigms, such as the tail suspension test (Cryan et al., 2005) and the forced swim test (Porsolt et al., 1977). These tests have been used in animal models of PD and most of the studies showed that partial and bilateral DA lesion of the nigrostriatal pathway induced depression-like behaviors, which were predominantly counteracted by dopaminergic medication, although some studies found positive effects of SSRIs. Results are far less strong for genetic studies, which showed either no difference or an increase of depression.

By looking more specifically at each of these models, MPTPtreated mice (with a moderate non-selective DA lesion) exhibited an increased depressive-like behavior (Mori et al., 2005; Vuckovic et al., 2008), which was counteracted by L-DOPA or a D2 agonist (Mori et al., 2005). However, these MPTP mice studies did not specifically target the nigrostriatal or mesostriatal dopaminergic pathways and 5-HT levels were also downregulated by MPTP.

The unilateral lesion of the substantia nigra pars compacta (SNc) with the toxin 6-OHDA also induced a depressive-like behavior and a hyperactivity in the lateral habenula. Interestingly, injection of muscimol (a GABA<sup>A</sup> agonist) in this area had antidepressant-like effects by decreasing the firing rate of the lateral habenula neurons and increasing serotonin release within the medial prefrontal cortex (Wang T. et al., 2017). Degeneration of the nigrostriatal pathway may impair GABA transmission in the lateral habenula, which play a role in regulating PD mood. Furthermore, the modulation of different 5-HT receptors within the lateral habenula also impacted depressive-like behavior in hemiparkinsonian rats (Han et al., 2015, 2016) and lesions of the lateral habenula cause a decrease in depressive-like behavior and an increase of 5-HT levels in the raphe nuclei (Luo et al., 2015).

#### TABLE 1 | Summary of the preclinical studies investigating the pathophysiology of PD-related neuropsychiatric-like disorders.

(Continued)

#### TABLE 1 | Continued

In association to the substantia nigra and raphe, the lateral habenula then probably contributes to the pathophysiological mechanisms of depression. After a moderate bilateral DA lesion of the SNc (noradrenergic neurons were protected from 6-OHDA with desipramine), rats had no locomotion impairment but exhibited more depressive-like behaviors which were reversed by L-DOPA, ropinirole (a D2/D3 agonist), SKF-38393 (a selective D1 agonist), sumanirole (a selective D2 agonist) and PD-128907 (a preferred D3 agonist) but not citalopram (a SSRI) (Carnicella et al., 2014; Drui et al., 2014). This suggests that the injury of the DA nigrostriatal pathway is involved in the pathogenesis of depressive-like behavior in this PD animal model. In agreement with these data, both L-DOPA and pramipexole had antidepressant effects in the forced swim test and both normalized decreased neurogenesis in the hippocampus in mice with bilateral 6-OHDA intra-nigral lesions (Chiu et al., 2015). Administration of ketamine (an antagonist of NMDA glutamatergic receptors) or imipramine (noradrenalin and serotonin reuptake inhibitor) can also improve depressive-like behavior in rats (Vecchia et al., 2018). Another study has shown that after bilateral intranigral injection of either MPTP or 6-OHDA (leading both to a 50% DA cell loss in the SNc), rats exhibited depressive-like behaviors which were associated to hippocampal reductions of DA, 5-HT and NA, suggesting an involvement of these monoamines in depression after nigral lesions (Santiago et al., 2010). Interestingly, after a partial (around 50%) 6-OHDA lesion of DA neurons from the olfactory bulb, rats exhibited an olfactory impairment, but also depressive-like behaviors (Ilkiw et al., 2019). Moreover, this lesion exacerbated the depressive-like behaviors, classically induced by the bilateral and partial lesion of SNc (Ilkiw et al., 2019), evidencing that the degeneration of the DA pathways within the olfactory bubble also impacts depression in PD.

Regarding the potential involvement of the ventral tegmental area (VTA), conflicting results have been obtained since rats with a bilateral but partial (around 50%) lesion of the medial part of VTA displayed no motor deficits, nor depressive-like behavior (Drui et al., 2014) but anhedonia, a major symptom of depression (Dardou et al., 2014). Another study showed that combined lesions of SNc and VTA led to no motor deficits but a depressivelike behavior, whose severity increased with nigral or the VTA lesions. It was reversed by both citalopram and L-DOPA (Winter et al., 2007). This suggests that SSRIs might be efficient at least partly by modulating the DA system (Ainsworth et al., 1998; Gambarana et al., 1999).

Unilateral 6-OHDA injections into the medial forebrain bundle (MFB) can also be used to model PD in rodents even though it leads to an extensive and not selective DA lesion. Several studies have reported depressive-like behaviors in such lesioned rats without any motor deficit (Winter et al., 2007; Zhang et al., 2011). L-DOPA failed to reverse these behaviors (Eskow Jaunarajs et al., 2010) while acute administration of sarizotan, an agonist at 5-HT receptors and partial agonist at D2 DA receptors had an antidepressant effect (Zhang et al., 2011). Again, these results fit well with the known involvement of the serotonin system in the modulation of depression. Activation of prelimbic 5-HT1A receptors produced antidepressant effects while their blockade favored depressive-like behavior in the MFB-lesioned rats (Hui et al., 2014). It has also been shown that the modulation of 5-HT<sup>6</sup> or 5-HT<sup>7</sup> receptors within either the prelimbic cortex or the hippocampus had an impact on depressive-like behaviors (Liu et al., 2015; Zhang et al., 2015, 2016). After bilateral injection of 6-OHDA in the MFB, rats exhibited depressive-like behavior, which was dramatically exacerbated when lesions of serotonergic and noradrenergic neurons were combined with

the DA one (Faggiani et al., 2015). These data emphasize again the involvement of not only DA but also other monoamine pathways in the pathophysiology of depressive-like behavior in animal models of PD.

Intra-striatal 6-OHDA injections can also be used to model PD, especially at early stages. After bilateral lesion (around 70%) of the dorsolateral striatum (with or without using desipramine to protect noradrenergic neurons), mice displayed slight motor deficits and longer immobility times in forced swim test and tail suspension test (Bonito-Oliva et al., 2014). This depressivelike behavior could be reversed by pramipexole (D2/D3 agonist) as well as by a noradrenaline inhibitor but not by L-DOPA. In another study, rats injected with bilateral 6-OHDA into the dorsal striatum (no use of desipramine), exhibited a mild reduction (36%) of striatal DA associated with increased depressive-like behaviors (Branchi et al., 2008). Other studies have shown that the partial and restricted lesion (50%) of the ventrolateral area of the dorsal striatum triggered no motor deficits but increased depressive-like behavior with monoamines alterations (Tadaiesky et al., 2008; da Silva et al., 2016). Interestingly, while anhedonia-like behaviors were observed shortly after a partial bilateral 6-OHDA lesion of the dorsolateral striatum, rats exhibited depressive-like behavior later, when anhedonia was no more present, indicating a temporal dissociation between the dorsolateral striatum and the prefrontal cortex (Matheus et al., 2016). The dorsolateral part of the striatum has motor and nonmotor functions and undergoes extensive DA depletion. This leads to dysfunction of other regions such as the prefrontal cortex which has been associated with the occurrence of anhedonia and depression at premotor stages of PD.

A few studies using genetic models of PD have reported depressive-like behaviors. Mice deficient for the DA D3 receptor do not show significant deficits in locomotion when tested in the open field but exhibit depressive-like behaviors (Moraga-Amaro et al., 2014). In the same line, heterozygote mice for Engrailed 1, a developmental gene controlling the survival of DA neurons, display a progressive loss of the mesencephalic DA neurons, motor deficits as well as depressive-like behaviors (Le Pen et al., 2008). Mice deficient for the vesicular monoamine transporter-2 (VMAT-2) display enhanced depression-like behaviors, which worsen with advancing age (Taylor et al., 2009). The MitoPark mouse is a genetic model of PD, replicating several essential features of PD, including adult onset of DA neuron loss, slow progressive neurodegeneration, formation of intra-neuronal inclusions albeit without α-synuclein, responsiveness to L-DOPA treatment and non-motor deficits. MitoPark mice show a depressive-like phenotype (Cong et al., 2016). This depressivelike phenotype is observed besides several other motor and behavioral deficits and brain structural changes. Several groups have overexpressed α-synuclein in animals as a progressive model of PD (Ulusoy et al., 2010). After bilateral injection of the wild type human α-synuclein in the substantia nigra, which produces a partial DA cell loss (43% in the SNc, 30% in the VTA, 31% in the striatum), rats display depressive-like behaviors, independently of mild locomotor deficits (Caudal et al., 2015).

Up to now, no study has measured or observed a depressivelike behavior in a NHP model of PD. Such studies would be interesting since ethological investigations have shown that some monkeys are more prone to depression than others (Camus et al., 2014).

# ANXIETY

The prevalence of anxiety disorders in PD ranges from 25 to 43% (Dissanayaka et al., 2014). It is higher than in other diseases causing similar disability. A recent systematic review considering data from a total of 2399 patients found that the point prevalence of anxiety disorders in PD is 31% (Broen et al., 2016). When referring to the DSM-IV-R diagnostic criteria, generalized anxiety is the most frequent, followed by agoraphobia, social phobia and panic disorder (see **Figure 1**). A large proportion of patients have multiple anxiety disorders (Leentjens et al., 2011). Moreover, a recurrent observation of the studies is the large proportion of patients (13.3% according to the systematic review) with significant anxiety symptoms that do not fit usual criteria for anxiety disorders (Dissanayaka et al., 2014; Broen et al., 2016). It is considered as "not otherwise specified" (NOS) anxiety disorder (Pontone et al., 2009). It refers to PD-specific anxiety, like phobia of falling, of driving, social phobia related to potentially embarrassing symptoms (drooling, dysarthria, . . .), anxiety related to withdrawal of DA medication or to wearing-off of medication in patients with fluctuations, panic-like disorder related to OFF periods, among others. Adverse effects of DA medication can also generate significant anxiety as in patients with ICD, DA dysregulation syndrome, or hallucinations. By consequence, anxiety disorders are probably largely underestimated in PD as triggers (predisposing and precipitating factors) are multiple.

The main manifestations of anxiety in PD are inability to relax, feeling tense, excessive concern, restlessness. Somatic symptoms may also be observed as palpitations, shortness of breath, sweating, digestive upset, etc. Usually, all the parkinsonian symptoms worsen with anxiety. Moreover, anxiety frequently implies emotion regulation deficits, irritability, excessive tiredness and difficulties falling asleep.

The association between anxiety and motor fluctuations, the significant exacerbation of anxiety provoked by DA withdrawal suggest that neurodegeneration of the DA pathways is involved in anxiety in PD. This is also supported by a study showing that in de novo patients, lower DA uptake at the caudate was associated with more severe trait anxiety (Picillo et al., 2017). However, the pathophysiology of PD-related anxiety disorders is complex and remains to be elucidated. In humans, several neuroimaging studies tried to decipher the mechanisms underlying anxiety in PD. Few specifically focused on anxiety.  Three were volumetric studies (Tinaz et al., 2011; Vriend et al., 2016; Wee et al., 2016). Overall, the functional studies showed the involvement of the striatum and of the DA, 5-HT and NA pathways in the occurrence of anxiety manifestations in PD. The anatomical studies showed reductions in the volume of several brain areas, namely the amygdala, the anterior cingulate cortex and the orbito-frontal cortex. However, most of these studies were correlation studies including PD patients whatever their status in terms of anxiety disorders. Moreover, the anxiety measures used were not always optimal. There is thus a real need of further investigations.

It is absolutely necessary to continue studies to decipher the mechanisms of anxiety in PD. To date, no interventional clinical trial specifically targeting PD-related anxiety has been published. Two trials have just ended but their results are not published. One concerns rotigotine, a D2–D3 agonist (ClinicalTrials.gov Identifier: NCT02365870). The other concerns buspirone, a 5-HTA1 agonist (ClinicalTrials.gov Identifier: NCT02803749).

Anxiety-like behaviors can be observed in animals using several behavioral paradigms such as the elevated plus maze, the Light/Dark box and the open field. These tests have been used in animal models of PD and most of the literature report an increased anxiety-like behavior in 6-OHDA- or MPTP-lesioned animals (**Table 1**). In contrast, genetic models of the disease either show no difference or a decrease of anxiety-like behavior.

Specifically, after a MPTP intoxication that causes an almost complete depletion of striatal DA, mice exhibited motor deficits and an increased anxiety-like behavior, concomitant with a reduction of 5-HT levels in the basolateral nucleus of the amygdala (BLA) (Gorton et al., 2010). Several studies also reported an increase in anxiety-like behavior after bilateral intranigral injection of MPTP in rats (Wang et al., 2009; Sy et al., 2010).

After 6-OHDA bilateral lesion of nigral neurons within the SNc (inducing a maximal DA loss of about 70% in the dorsal striatum, noradrenergic neurons being protected with desipramine), rats exhibited no motor deficits but an increased anxiety-like behavior (Carnicella et al., 2014; Drui et al., 2014). A significant correlation was found between the latency of response and the striatal DA loss, suggesting that the increased anxiety-like response was related to the degree of striatal DA depletion (Drui et al., 2014). However, no similar correlations were found in the elevated plus-maze. DA agonists (SKF-38193, Sumanirole, and PD-128907) all reduced anxiety-like behavior in these 6-OHDA bilaterally lesioned rats (Carnicella et al., 2014). Another study showed that after a partial (less than 45%) and bilateral 6-OHDA lesion of the SNc (desipramine use), rats displayed an increased anxiety-like behavior as well as motor deficits (Campos et al., 2013). Bilateral partial (48%) lesion of the SNc induces significant deficits in the elevated plus maze, which were not reversed by either acute or chronic treatment with L-DOPA (Loiodice et al., 2019). This lesion was not associated to motor impairment.

The partial and bilateral lesion of the medial VTA failed to induce anxiety (Drui et al., 2014). On the contrary, after unilateral 6-OHDA MFB lesion, rats exhibited a mild increase of anxiety-like behavior, which could not be improved by chronic L-DOPA (Eskow Jaunarajs et al., 2010). However, other studies using the same type of lesion have shown anxiety-like behaviors, which could be improved by chronic L-DOPA or diazeapam (O'Connor et al., 2016), but not by sarizotan (Zhang et al., 2011) or diazepam (O'Connor et al., 2016). Several studies have shown that acting on 5-HT1A, 5-HT<sup>6</sup> or 5-HT<sup>7</sup> receptor subtypes within the amygdala, the hippocampus or the prelimbic cortex can modulate anxiety-like behavior (Sun et al., 2015, 2018; Du et al., 2018; Zhang et al., 2018; Liu et al., 2019), underlying the major role of the serotonergic system in anxiety-like behavior in PD animal models. However, another study has shown, in 6-OHDA bilaterally MFB lesioned rats, that the additional depletion of serotonin or of noradrenalin had no further effects on anxietylike behavior (Faggiani et al., 2015). However, L-DOPA enhanced the firing rate of amygdala and significantly decreased anxiety in these animals (Faggiani et al., 2018). This suggests that the increased activity of serotonin neurons may enhance the anxiolytic action of L-DOPA.

In mice, bilateral 6-OHDA lesion (75%) of the dorsolateral striatum also led to an increased anxiety-like behavior with slight motor deficits (Bonito-Oliva et al., 2014). This anxiety-like behavior was corrected by pramipexole (but not by L-DOPA) and reboxetine (a selective noradrenaline reuptake inhibitor), and was independent of noradrenaline depletion as the use of desipramine to protect noradrenergic neurons from the toxicity of 6-OHDA did not modify this anxiety-like behavior (Bonito-Oliva et al., 2014). Again, this suggests that the depletion of DA caused by 6-OHDA is sufficient to induce affective-like symptoms. Other studies showed increased anxiety in rats with a bilateral and partial (less than 50%) lesion of the striatum inducing slight motor deficits as well (Tadaiesky et al., 2008; Chen et al., 2011a; da Silva et al., 2016). The administration of an antagonist of metabotropic receptors was efficient to counteract both behavioral and neuronal changes (Tadaiesky et al., 2008; Chen et al., 2011a,b; da Silva et al., 2016).

A few studies have reported anxiety-like symptoms in genetic mouse models of PD. For instance, Zhu and colleagues reported increased anxiety-like behavior in Parkin deficient mice (Zhu et al., 2007). VMAT-2 deficient mice display an enhanced anxiety-like behavior, worsening with aging (Taylor et al., 2009). Compared to α-synuclein deficient mice and wild-type controls, mice overexpressing the human mutated form A53T of α-synuclein exhibited, besides early and late stage cognitive and sensorimotor deficits, a reduced anxiety-like

behavior (George et al., 2008). These results indicate a possible role for α-synuclein in anxiety-like behavior.

Regarding studies performed on monkey models of PD, only one study mentioned the case of a transgenic macaque monkey expressing the mutated form of α-synuclein and exhibiting an enhanced anxiety-like behavior (Niu et al., 2015).

# APATHY

Clinically, apathy refers to a set of behavioral, emotional and cognitive manifestations, such as reduced interest and participation in the main activities of daily life, loss of initiative, lack of perseverance, indifference, and flattening of affect. As noted by Marin, apathy can exist per se as a syndrome. It is not just a symptom of depression or dementia (Marin, 1991). The definition of Marin was anchored on the motivational component of apathy: "diminished motivation not attributable to diminished level of consciousness, cognitive impairment, or emotional distress" (Marin, 1996). Due to the difficulty of addressing the concept of motivation, a more operational definition of apathy is currently preferred. According to the recent revision of the clinical diagnosis criteria (see **Table 2**), apathy corresponds to a quantitative reduction of goal-directed activity either in behavioral, cognitive, emotional, or social dimensions in comparison to the patient's previous level of functioning in these areas (Robert et al., 2018).

According to a meta-analysis on a large set of data, the mean prevalence of apathy in PD is 39.8% (n = 5,388; 95% CI 34.6–45.0%) (den Brok et al., 2015). Patients with apathy are on average older than the non-apathetic patients, have more impaired cognition in general and executive function in particular, have more severe motor symptoms and an increased risk of co-morbid depression. Apathy may be a predictive factor for dementia and cognitive decline over time (Dujardin et al., 2009; Pedersen et al., 2009).

Regarding the mechanisms underlying apathy, most neuropathological and neuroimaging studies in apathetic patients show abnormalities in a network of brain regions involved in motivated behavior, especially the dorsal part of the anterior cingulate cortex, the medial orbitofrontal cortex and the ventral striatum, under the influence of the mesolimbic DA system originating in the VTA (Heron et al., 2018). However, only few studies have examined the pathophysiology of apathy in PD patients. Reijnders et al. (2010) used voxelbased morphometry to identify the anatomical correlates of apathy in a group of 60 PD patients (16% were apathetic). High apathy scores were correlated with lower cognitive efficiency, more depressive symptoms and low gray matter density in the posterior cingulate, precuneus, insula and inferior parietal gyrus. Carriere et al. (2014) compared with shape analysis the volume of the striatum in apathetic vs. non-apathetic PD patients and showed a remodeling of the left caudate and left accumbens in apathetic patients. Most other studies in PD have used functional imaging. Remy et al. (2005) used [11C]-RTI-32 Positron emission tomography (PET), an in vivo marker of both DA and NA membrane transporters, with low affinity for 5-HT transporters. They found that apathy scores were negatively correlated with binding potential values in the ventral striatum bilaterally, suggesting that catecholamine denervation of this

Criterion A: A quantitative reduction of goal-directed activity either in behavioral, cognitive, emotional, or social dimensions in comparison to the patient's previous level of functioning in these areas. These changes may be reported by the patient themselves or by observation of others.

Criterion B: The presence of at least 2 of the 3 following dimensions for a period of at least 4 weeks and present most of the time.

B1. BEHAVIOR AND COGNITION

#### B2. EMOTION

Loss of, or diminished, emotion as evidenced by at least one of the following:

B3. SOCIAL INTERACTION

Loss of, or diminished engagement in social interaction as evidenced by at least one of the following:

Criterion C: These symptoms (A–B) cause clinically significant impairment in personal, social, occupational, or other important areas of functioning.

Criterion D: The symptoms (A–B) are not exclusively explained or due to physical disabilities (e.g., blindness and loss of hearing), to motor disabilities, to a diminished level of consciousness, to the direct physiological effects of a substance (e.g., drug of abuse, medication), or to major changes in the patient's environment.

TABLE 2 | Criteria for clinical diagnosis of apathy.

Loss of, or diminished, goal-directed behavior or cognitive activity as evidenced by at least one of the following:

area has a role in apathy. We used [11C]DASB and [11C]PE2I PET, radioligands, binding to the serotonergic and dopaminergic transporters, respectively (Maillet et al., 2016) and found that the severity of apathy in 12 de novo PD patients was mainly related to specific serotonergic lesions within the right-sided anterior caudate nucleus and the orbitofrontal cortex. Robert et al. (2012) used <sup>18</sup>FDG PET to measure glucose metabolism in 45 patients with PD and reported a correlation between the severity of apathy symptoms and cerebral metabolism in the inferior frontal gyrus, middle frontal gyrus, cuneus and anterior insula, at the right side. As apathy is a possible complication of the treatment of PD with deep brain stimulation of the subthalamic nucleus (STN-DBS), several studies have used the model of STN stimulation to investigate the pathophysiology of apathy in PD. Jeune et al. (2009) observed a worsening of apathetic symptoms after surgery and showed that this change was correlated with greater glucose metabolism in the right frontal middle gyrus (Brodmann area 10) and right inferior frontal gyrus (Brodmann areas 46 and 47) and lower glucose metabolism in the right posterior cingulate gyrus (Brodmann area 31) and left medial frontal lobe (Brodmann area 9). According to Jeune et al. (2009), stimulation of the STN could have a negative effect on the limbic territory of the STN, thus destabilizing the limbic circuit. The same team showed that reduced preoperative glucose metabolism within the right ventral striatum was associated with the occurrence of post-surgery apathy (Robert et al., 2014).

Thobois et al. (2010) used PET with an antagonist of DA D2 receptors to investigate the role of DA denervation in post-surgery apathy. They compared 12 patients who had developed apathy after initiation of DBS and 13 control PD patients who had not developed apathy after stimulation. Binding potential values were higher in apathetic patients compared with non-apathetic patients, in the orbito-frontal, dorsolateral and posterior cingulate cortex, bilaterally, as well as in the left striatum and right amygdala. After a challenge with methylphenidate, a drug that inhibits the reuptake of DA and NA and increases DA concentration at dopaminergic terminals, they observed that apathetic patients had lower reserve of endogenous DA. Although these results are consistent with a hypodopaminergic etiology of apathy in PD, other neurotransmitter systems are probably involved. As already mentioned, we found a prominent role of serotonergic degeneration in the occurrence of apathy at early stage of the disease (Maillet et al., 2016). This was confirmed by a complementary study by Prange et al. (2019) showing microstructural changes specifically associated with apathy in the limbic system in de novo patients with apathy compared with non-apathetic patients and healthy controls. These changes overlapped with the functional alteration of the serotoninergic terminals but not with the dopaminergic abnormalities (Prange et al., 2019). Given the links between apathy and cognitive decline in PD patients, a role of cholinergic denervation is also possible. The reduction of apathy symptoms by administration of rivastigmine, an inhibitor of acetylcholinesterase, reinforced such an assumption (Devos et al., 2013). Further confirmation of the role of non-dopaminergic systems in the PD apathy is still needed.

The first studies searching for a pharmacological treatment of apathy targeted the dopaminergic pathway. Czernecki et al. (2008) were the first to describe an improvement of post-surgery apathy after administration of ropinirole in a small group of patients. However, the level of evidence was very low. Later, Thobois et al. (2013) tested the effectiveness of piribedil to reduce post-surgery apathy in PD. They observed a significant reduction of the symptoms after 12 weeks of pharmacotherapy (Thobois et al., 2013). The effectiveness of rotigotine, another D2/D3 dopamine agonist, on apathy was also tested but there was no effect on the symptom severity (Hauser et al., 2016). Methylphenidate, a drug enhancing mesolimbic dopaminergic stimulation, was shown to improve apathy in an 82-yearold patient. Its effectiveness was not explored further in PD (Chatterjee and Fahn, 2002). As stated above, the efficacy of rivastigmine, an acetylcholinesterase inhibitor, on apathy in non-demented, non-depressed patients was also tested (Devos et al., 2013). Compared with placebo, there was a significant improvement of apathy in the rivastigmine group, particularly on the cognitive and initiation components of apathy.

Apathy is difficult to assess in animals. Some behavioral conditions considered as "activities of daily life" can be used in rodents such as the burrowing test or the nest building test. Other tests can also be used such as the runway task for food, operant sucrose self-administration and the saccharin preference test. There are very few studies on apathy-like behavior in PD animal models (**Table 1**). Three studies from the same group have evidenced motivational deficits after the selective, partial (less than 70%) and bilateral 6-OHDA lesion of the SNc in rats (Carnicella et al., 2014; Drui et al., 2014; Favier et al., 2014). But these animals also exhibited depressive-like and anxiouslike behaviors. This apathy-like behavior, which was detected with the operant sucrose self-administration, was only improved by drugs preferentially acting on D3 type of DA receptors such as ropinirole, pramipexole, and PD-128907. Lesion of the medial part of VTA did not induce such a behavior, indicating that damage of the nigrostriatal pathway is a key risk factor to develop apathy-like behavior in the 6-OHDA rat model of PD. Another study did report behavioral signs of apathy in aged mice deficient for the vesicular monoamine transporter 2 (Baumann et al., 2016).

We have shown that apathy-like behavior (a hypoactive state with loss of motivation for food) can be induced by non-DA pharmacological dysfunction of the primate ventral striatum (Sgambato-Faure et al., 2016; Voon et al., 2017; Sgambato and Tremblay, 2018). This behavior involved a circuit including the orbital and medial prefrontal cortex, anterior insula, and lateral parts of medial output basal ganglia regions. This lack of motivation was induced by bicuculline (a GABA<sup>A</sup> antagonist) in moderately DA-depleted monkeys, suggesting that dopamine might only modulate the expression of apathy rather than cause it. However, another group has shown in MPTP-treated monkeys that the dopaminergic pathways play a key role in apathy-like behavior (Brown et al., 2012). Specifically, they tested the impact of MPTP on the monkeys' willingness to attempt goal-directed behaviors (Brown et al., 2012). Using PET imaging and postmortem analysis, they showed that apathy-like scores correlated

with the degree of lesion of the DA mesostriatal pathway, and that dysfunction of the mesostriatal pathway predicted apathylike behavior better than DA nigrostriatal dysfunction. More recently, the same group has extended these findings by showing that DA dysfunction in cortical regions also contributed to the development of apathetic behavior in NHP (Tian et al., 2015). Apathy scores correlated with DA injury (detected by PET tracers) in the dorsolateral prefrontal cortex, ventromedial prefrontal cortex, and insular cortex. Furthermore, only VTA cell counts could predict DAT changes in the insular cortex, suggesting a particular role for this pathway in the manifestation of apathy in this monkey model of PD. Contrary to the results in rodents, these data indicate a key role for the VTA involving both subcortical and cortical regions in the pathophysiology of apathy. Further studies investigating the role of both DA and non-DA pathways in apathy-like behavior in PD animal models are thus warranted.

# PSYCHOSIS

In PD, psychosis encompasses a set of symptoms including illusions, hallucinations, delusions, and related symptoms. These symptoms are typically visual, more rarely in other modalities. They usually form a continuum progressing with the course of the disease (Ffytche and Aarsland, 2017). Passage (feeling like something is passing at the outer visual field) and presence (feeling that someone is close by) hallucinations as well as illusions (mis- or distorted perception of an actual stimulus) are the most common symptoms at early stage of the disease. Passage and presence hallucinations may concern 50% of the patients (Wood et al., 2015). As the disease progresses, visual hallucinations in PD may be complex and formed, usually vision of animals, people or objects. The scene is often stereotyped. They tend to occur in a low sensory environment. The hallucinations usually ignore the patient and disappear when approaching. Initially, insight is preserved but later, it is lost and contribute to the development of delusions.

There are few prevalence studies and frequencies vary widely. Point prevalence of complex hallucinations ranges from 22 to 38% (Fenelon and Alves, 2010). The prevalence of minor psychotic symptoms is much more variable. Fénelon et al. (2000) reported that 26% of a sample of 216 consecutive patients taking medication had minor hallucinations. In early diagnosed untreated patients, we observed that 6.32% had minor hallucinations (Dujardin et al., 2014) while another study reported a much higher prevalence of 42% (Pagonabarraga et al., 2016). The risk of developing visual hallucinations increases with age, severity of motor and cognitive symptoms, disease duration and medication (Fenelon and Alves, 2010). Moreover, visual hallucinations have been shown to be the main risk factor for nursing home placement (Aarsland et al., 2000). It increases the risk of cognitive impairment and dementia (Guo et al., 2019) and mortality (Goetz and Stebbins, 1995).

The mechanisms behind visual hallucinations (VH) remain largely unknown. Changes in visual information processing are incriminated. For example, compared with patients without visual hallucinations and healthy controls, PD patients with visual hallucinations had a thinning of the retinal nerve fiber layer, as measured by optical coherence tomography (Lee et al., 2014). Some studies have shown that PD patients with visual hallucinations have lower performance in visuospatial tasks, but findings are not consistent probably due to a lack of sensitivity of the tests and variability of the populations (for a review, see Ffytche and Aarsland, 2017). However, changes in the networks involved in perceptual and cognitive processing of visual information have been shown by anatomical and functional neuroimaging studies in patients with visual hallucinations compared with those without visual hallucinations. Most anatomical studies reported an atrophy in areas corresponding to dorsal and ventral visual streams (for a review, see Ffytche and Aarsland, 2017). Functional MRI (fMRI) studies reported reduced activity of the visual areas compensated by an increased activity in the associative cortical areas (Diederich et al., 2009; Shine et al., 2012; Yao et al., 2014, 2015, 2016; Shine et al., 2015). However, a better understanding of the pathophysiology of chronic visual hallucinations in PD lacks a direct exploration of the brain changes at the time of hallucination (i.e., capture studies). Because of the challenging nature of state explorations of hallucinations, only two studies were in PD. One single-case study reported the results of an fMRI scan performed in a 66-year old PD patient at time of hallucinations (Goetz et al., 2014). When comparing epochs with and without VH, there was an increased activation in a large attentional network and decreased activation in the right primary visual cortex. Recently, we measured brain functional changes when VH occurred in seven PD patients. We observed increased connectivity in visual networks concomitant to VH (Dujardin et al., 2019).

According to clinical experience, DA agonists may cause the onset of hallucinations, suggesting a role of the dopaminergic pathways in the occurrence of psychosis. However, the role of medication is very controversial since visual hallucinations are observed even in drug-naïve patients. As suggested by a work group on psychosis in PD, anti-parkinson medication is more to consider as a modifier than a trigger of hallucinations (Ravina et al., 2007a; Dujardin et al., 2014; Weintraub et al., 2015a). Regarding the other neuromodulation systems, the benefit of acetylcholinesterase inhibitors on symptoms suggests an involvement of the cortical cholinergic pathway (Burn et al., 2006; Oh et al., 2015). Moreover, an atrophy of the substantia innominate has been observed in PD patients with hallucinations (Shin et al., 2012). A contribution of 5-HT has also been suggested since a 5-HT depletion in the ventral occipitotemporal regions and bilateral frontal cortex was found in PD patients with visual hallucinations (Ballanger et al., 2010). However, further investigations are really needed to better understand the respective role of DA and non-DA pathways in the occurrence of psychosis in PD.

Pharmacological treatments currently available to reduce psychosis in PD patients have an action on these DA and 5-HT pathways. Benefits on psychotic symptoms have been reported in several placebo-controlled trials with clozapine and pimavanserin while quietapine and olanzapine provided no significant benefit (Wilby et al., 2017).

The number of studies in which psychosis-like symptoms have been observed in animal models of PD is still limited (**Table 1**). Behavioral assays such as head switches, hallucinatorylike responses, amphetamine-induced locomotor activity and disrupted prepulse inhibition are frequently used to assess antipsychotic activity. These tests have been used in animal models of PD.

Animals with bilateral 6-OHDA lesions of the substantia nigra display motor impairments as well as increased head twitches, locomotor activity, and disrupted prepulse inhibition (McFarland et al., 2011; Hubbard et al., 2013). Pimavanserin, a selective 5-HT2A antagonist/inverse agonist, reverse psychosislike behaviors, suggesting that 5HT2A antagonism/inverse agonism may treat psychosis in PD.

A psychosis-like behavior rating scale has been developed and used in MPTP-treated marmosets (Fox et al., 2006; Visanji et al., 2006). This scale allows to assess four types of psychosis-like behaviors: agitation, hallucinatory-like responses to non-apparent stimuli, obsessive grooming, and stereotypies. It has been shown that L-DOPA, apomorphine, pramipexole, pergolide, ropinirole reversed motor symptoms and induced psychosis-like behaviors in MPTP-intoxicated marmosets, and that amantadine exacerbated them (Fox et al., 2006; Visanji et al., 2006). Finally, while haloperidol reduced those behaviors but increases motor symptoms, clozapine and quietapine reduced them without exacerbating parkinsonian disability. We have shown that L-DOPA triggered such psychosis-like behaviors in moderately-lesioned macaque monkeys and that these psychosislike behaviors were reduced following lesion of serotonergic fibers by MDMA (Beaudoin-Gobert et al., 2015). Altogether, these data suggest both DA, 5-HT and glutamatergic mechanisms are involved in the pathophysiology of psychosis-like behavior.

# IMPULSE CONTROL DISORDERS

Impulse control disorders refer to a class of psychiatric disorders characterized by impulsivity, i.e., an urge or failure to resist to temptation. In the DSM-5, it is included in a new section labeled "disruptive, impulse control, and conduct disorders." ICDs result in behaviors performed repetitively, excessively, and compulsively to an extent that interferes with major areas of daily life (Weintraub and Claassen, 2017). The individual pursues certain reward-based activities without taking account of the potentially deleterious consequences of these repetitive activities. The semiology is diverse.  Other impulsive/compulsive behaviors are linked, namely DA dysregulation syndrome (Giovannoni et al., 2000), an addiction to DA medication, particularly high-potency and shortacting drugs (e.g., subcutaneous apomorphine or dispersible formulation of levodopa). Related phenomena have also been identified as punding (repetitive, purposeless, and stereotyped behaviors) (Spencer et al., 2011), hobbyism (excessive exercise or creative activities) and hoarding (acquisition and keeping of a large number of items with little or no objective value, e.g., used gloves) (O'Sullivan et al., 2010). Very often, those patients also develop hypomania which interferes at lot with their familial and social life.

Regarding prevalence, the main available data come from the DOMINION study, which included 3090 PD patients from movement disorders clinics in North America and assessed the frequency of the four main ICDs (Weintraub et al., 2010b). It revealed that 13.6% of patients had one or more ICDs (compulsive buying in 5.7%, gambling in 5%, binge eating disorders in 4.3% and compulsive sexual behavior in 3.5%) and 3.9% had two or more ICDs. Similar prevalence data have been reported in studies conducted in different countries around the world (for a review, see Weintraub and Claassen, 2017). The Italian multi-center prospective ICARIUS study reported a higher point prevalence with 28.6% of the 1069 patients having at least one ICD (Antonini et al., 2017). The 2-year incidence was of 20.6% (Barone et al., 2019). The prevalence of the other impulsive/compulsive disorders is not well-documented.

Dopamine replacement therapy, namely DA agonist use, is the main risk factor for ICDs. In the DOMINION study, ICDs were more frequent in patients receiving DA agonists (17.1%) than in patients only treated by levodopa (6.9%). An analysis on the baseline data of the Parkinson's Progression Markers Initiative (PPMI) cohort, including 168 untreated PD patients and 143 healthy control subjects, revealed that the frequencies of ICDs and related symptoms did not differ in both groups (Weintraub et al., 2013). After 24-month followup, there was no increase of ICDs prevalence in the PD patient group, overall. However, in those who were on DA replacement therapy for at least 1 year, the incidence of ICDs was significantly higher than in patients still untreated or who initiate DA replacement therapy for less than 1 year (de la Riva et al., 2014). Hence, these results suggest that PD itself does not increase the risk to develop ICDs but reinforce the hypothesis that DA medication plays a major role in the occurrence of such disorders. The risk is greater with DA agonists having a preferential selectivity for D3 and D2 receptors, suggesting that the mesocortical and mesolimbic dopaminergic pathways are most likely involved. These pathways play a key role in reward-based learning and decision-making (Ballard et al., 2011; Haber, 2014; Hauser et al., 2017). Dysfunction of this DA system was confirmed by several neuroimaging studies (Cilia et al., 2008; Steeves et al., 2009; Voon et al., 2010, 2014). However, ICDs seem to be multi-determined and dysfunction of the mesocorticolimbic system due to DA replacement therapy is not the only underlying mechanism. Other clinical features (early onset disease, comorbid depression, and anxiety), premorbid (personality traits, genetic polymorphisms, past history of addiction), environmental (ease to access to the internet, pornography, casino, etc.), demographical (higher frequency of hypersexuality in men than women while the inverse is observed for compulsive buying) and cultural factors, cognitive bias (altered executive functions and decision-making) also influence the occurrence of ICDs (Weintraub et al., 2015b). Other neuromodulation systems are probably also involved, as suggested by studies in animal models of PD but further investigations are needed in patients.

To date, there is no available treatment for ICDs (Gatto and Aldinio, 2019). One clinical trial should recruit PD patients soon. It concerns pimavanserin, a selective 5-HT2A inverse agonist (ClinicalTrials.gov Identifier: NCT03947216). Another trial has started recruiting PD patients. It will test the efficacy of clonidine, an α2 noradrenergic agonist (ClinicalTrials.gov Identifier: NCT03552068).

The number of studies in which impulsive/compulsive-like behavior has been modeled in animal models of PD is still quite limited (**Table 1**). Several models try to recapitulate the DA deficit of early or more advanced PD and the impulsive or compulsive traits provoked by DA loss in association with dopatherapy, in order to better understand pathophysiological mechanisms. Impulsive or compulsive behaviors can be observed in animals using several behavioral tasks, such as food-related instrumental learning tasks (5-choice reaction time task, delay-discounting task, rat gambling task, rodent betting task, etc.). Some of these tests have been used in animal models of PD.