Changed firing activity of nigra dopaminergic neurons in Parkinson's disease

Parkinson's disease is the second most common neurodegenerative disease which is characterized by selective degeneration of dopaminergic neurons in the substantia nigra pars compacta. The intrinsic neuronal firing activity is critical for the functional organization of brain and the specific deficits of neuronal firing activity may be associated with different brain disorders. It is known that the surviving nigra dopaminergic neurons exhibit altered firing activity, such as decreased spontaneous firing frequency, reduced number of firing neurons and increased burst firing in Parkinson's disease. Several ionic mechanisms are involved in changed firing activity of dopaminergic neurons under parkinsonian state. In this review, we summarize the changes of spontaneous firing activity as well as the possible mechanisms of nigra dopaminergic neurons in Parkinson's disease. This review may let us clearly understand the involvement of neuronal firing activity of nigra dopaminergic neurons in Parkinson's disease.


Introduction
Parkinson's disease is the second most common neurodegenerative disease with the selective loss of dopaminergic neurons in the substantia nigra pars compacta. Although the exact cause of Parkinson's disease is unknown, several factors of genes and environmental toxins appear to be associated with the occurrence of Parkinson's disease. The processes of mitochondrial dysfunction, oxidative stress, excitotoxin and neuroinflammatory are considered to contribute to the death of nigra dopaminergic neurons. Recently, Stoker and Barker (2020) reviewed the current and planned clinical trials for the treatment of Parkinson's disease. For example, deep brain stimulation (DBS) of the subthalamic nucleus in patients has been demonstrated as a new and effective treatment for parkinsonian motor symptoms of bradykinesia, resting tremor and muscular rigidity (Okun, 2012;Faggiani and Benazzouz, 2017;Reich et al., 2022).
It has been demonstrated that the intrinsic electrophysiological properties of neurons are critical for the functional organization in central nervous system (Llinás, 1988(Llinás, , 2014. The specific changes of neuronal firing activity may be associated with different brain functions as well as some brain disorders. Early electrophysiological studies revealed that nigra dopaminergic neurons show a long duration and low frequency spontaneous discharge (Grace and Bunney 1983;Fà et al., 2003). The firing patterns of nigra dopaminergic neurons exhibit a continuum running from burst firing to random pattern to pacemaker-like regular firing (Wilson et al., 1977;Bunney, 1984a, 1984b;Freeman et al., 1985;Tepper et al., 1995;Hyland et al., 2002). Several ion channels are involved in the spontaneous firing activity of nigra dopaminergic neurons, such as hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels (Neuhoff et al., 2002), L-type calcium channels (Guzman et al., 2009;Branch et al., 2014), T-type calcium channels (Yung et al., 1991;Wolfart and Roeper, 2002), calcium-activated potassium channels (Wolfart et al., 2001), ATP-sensitive potassium (K-ATP) channels (Schiemann et al., 2012), and small conductance calcium-activated potassium (SK) channels (Ping and Shepard, 1999). The spontaneous firing and ion channel activity may change under different physiological conditions. For example, at the onset of movement, the firing rate of nigra dopaminergic neurons reduces significantly (Dodson et al., 2016). Blockade of Cav1.3 calcium channels reverses the pacemaking form from adulthood dopaminergic neurons to that of juvenile dopaminergic neurons (Chan et al., 2007).
The spontaneous firing activity of nigra dopaminergic neurons displays different changes under parkinsonian state, which may be associated with the manifestation of parkinsonian motor symptoms. Manipulation of defective firing activity of nigra dopaminergic neurons may be effective in the treatment of Parkinson's diseases. In Parkinson's disease, the nigra dopaminergic neurons mainly exhibit decreased firing rate and bursting firing. Increasing neuronal excitability of dopaminergic neurons using optogenetic techniques could rescue parkinsonian locomotion defects (Qi et al., 2017). Furthermore, mild excitatory stimulating nigra dopaminergic neurons may improve neuronal survival in Parkinson's disease (Michel et al., 2013). Indeed, several neuropeptides such as ghrelin, orexin, and cholecystokinin (CCK) increase the neuronal excitability, and meanwhile exert neuroprotective effects on nigra dopaminergic neurons in Parkinson's disease. In this review, we summarize the changes of spontaneous firing rate and patterns of dopaminergic neurons in the substantia nigra pars compacta of Parkinson's disease. We also review the possible ionic mechanisms involved in the changes of firing properties of nigra dopaminergic neurons (Table 1).  Due to progress of disease Liu et al. (2022) α-syn: α-synuclein.

Reduction of firing rate and reduced number of active nigra dopaminergic neurons under parkinsonian state
It is well known that the spontaneous firing activity of dopaminergic neurons in the substantia nigra pars compacta is disrupted in Parkinson's disease. The reduction of neuronal firing rate has been reported in the substantia nigra pars compacta of both genetic (Good et al., 2011;Janezic et al., 2013;Chou et al., 2014;Guatteo et al., 2017) and neurotoxin-based (Liss et al., 2005;Chiba et al., 2015;Abe et al., 2016;Chang et al., 2020;Heo et al., 2020;Hill et al., 2021) parkinsonian animal models. In vitro patch clamp recordings revealed that application of 1-methyl-4-phenylpyridinium iodide (MPP + ) inhibits the frequency of both spontaneous and evoked action potential of nigra dopaminergic neurons (Chang et al., 2020). Furthermore, 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP) induces changes of firing rate in a concentration-dependent manner. For example, low to middle doses of MPTP induce a transient but not potent increase in firing rate, while higher doses of MPTP produce a sustained reduction in firing rate (Chiba et al., 2015;Abe et al., 2016). Using both in vivo and in vitro electrophysiological recordings, Heo et al. (2020) found that the firing frequency of nigra dopaminergic neurons decreases significantly in either MPTP or 6-hydroxydopamine (6-OHDA) parkinsonian animal models. It is well known that accumulation of α-synuclein (α-syn) oligomers leads to dysfunction of dopaminergic neurons. Recently, Hill et al. (2021) showed that application of α-syn aggregates into single dopaminergic neurons induces a time-dependent decrease of neuronal firing rate.
The firing activity of nigra dopaminergic neurons also changes in several genetic parkinsonian models. In bacterial artificial chromosome transgenic mice (SNCA-OVX), a familial and sporadic parkinsonian model, the nigra dopaminergic neurons exhibit an age-dependent reduction in spontaneous firing rate (Janezic et al., 2013). The nigra dopaminergic neurons of eight-month-old (G2019S) leucine-rich repeat kinase 2 (LRRK2) transgenic mice display a reduced spontaneous firing rate as well as a reduction of dopamine release in the striatum (Chou et al., 2014). In addition, the movement-related firing pauses of nigra dopaminergic neurons are lost in SNCA-OVX transgenic parkinsonian mouse model which may suggest some disrupted activity dynamics in parkinsonism (Dodson et al., 2016).
In addition to reduction of spontaneous firing rate, previous studies revealed the reduced number of nigra dopaminergic neurons with spontaneous firing activity in Parkinson's disease. Tozzi et al. (2021) reported that α-syn-preformed fibrils reduce the number of active dopaminergic neurons in early stage of 6 weeks. In a genetic mitochondrial model of Parkinson's disease, the membrane potential of nigra dopaminergic neurons changes to hyperpolarization which leads to absence of the characteristic pacemaker firing (Good et al., 2011). In 6-OHDA partially lesioned rats, the number of nigra dopaminergic neurons with spontaneous firing also reduces with the decreased regularity of spikes (Harden and Grace, 1995;Bilbao et al., 2006;Gui et al., 2011). Furthermore, treatment with levodopa significantly reverses the irregularity of neuronal firing activity (Bilbao et al., 2006) as well as the reduced number of spontaneously active dopaminergic neurons (Harden and Grace, 1995) in partial 6-OHDA lesioned parkinsonian rats.
We assumed that, at least in partial nigra dopaminergic neurons, the decreased neuronal firing is a remarkable and probably reversible pathological change in the process of parkinsonian neuronal death. Indeed, the decreased firing activity is observed long before the death of nigra dopaminergic neurons (Good et al., 2011). In Parkinson's disease, dysfunction of mitochondria may lead to disturbance of membrane potential, subsequently decreases firing activity of nigra dopaminergic neurons (Kriete 2012), and consequently reduces dopamine release in the striatum and causes locomotion deficit (González-Cabrera et al., 2017). In early stage of Parkinson's disease, application of mild stimulation triggers the silent nigra neurons to fire in a normal pacemaker activity (Good et al., 2011), prevents the death of dopaminergic neurons and therefore alleviates locomotion deficits (Salthun-Lassalle et al., 2004;Qi et al., 2017).
In summary, decrease of the spontaneous firing rate of surviving nigra dopaminergic neurons may be a characteristic change of neuronal firing activity in animal models of both genetic and neurotoxin-based Parkinson's disease. The decreased firing rate of nigra dopaminergic neurons is associated with age and concentration of neurotoxin. Consistent with decrease of spontaneous firing rate, reduced number of active nigra dopaminergic neurons may also suggest the decreased excitability of neurons in Parkinson's disease. The decreased neuronal excitability is probably a reversible change in the early stage of Parkinson's disease, which could be rescued by mild stimulation.

Reduction of firing activity of nigra dopaminergic neurons with aging and sex
What we have described above are the reduction of firing activity of nigra dopaminergic neurons under different parkinsonian animal models. It is well known that Parkinson's disease is a progressive neurodegenerative disease. Although early electrophysiological recordings did not show any significant difference in the firing rate of nigra dopaminergic neurons between young (3 months) and aged (24-28 months) male rats (Freeman et al., 1989), recent studies did demonstrate a reduced spontaneous firing activity of nigra dopaminergic neurons with increasing age. For example, in vitro extracellular recordings revealed that firing rate of dopaminergic neurons in old (25-30 months) male mice is significantly slower than that of 2-7 month-young counterparts (Branch et al., 2014). The age-dependent downregulation of L-type calcium channel currents may be involved in the lower firing rate of old mice (Branch et al., 2014). Consistently, recent in vivo electrophysiological study revealed that blockade of L-type calcium channels by isradipine reduces the spontaneous firing rate of nigra dopaminergic neurons (Shin et al., 2022). Interestingly, the L-type calcium channels selectively modulate the neuronal firing rate in lateral nigrostriatal dopaminergic neurons, in which the neuronal degeneration appears earlier and more serious in Parkinson's disease (German et al., 1989;Gibb and Lees, 1991;Betarbet et al., 2000). Therefore, one may hypothesize that decline of firing activity of nigra dopaminergic neurons in aged animals may provide some early characteristics in Parkinson's disease state. In addition, other in vivo recordings also showed that the percentage of lower firing rate dopaminergic neurons is much more in old rats than in young adults (Mendoza-Ramírez et al., 1995). Furthermore, intrastriatal graft of nerve growth factor cultured chromaffin cells could maintain the basal firing activity and increase the survival of nigra dopaminergic neurons in aged rats, and in turn induce motor improvement (Mendoza-Ramírez et al., 1995).
It is known that sex is another largest risk factor for Parkinson's disease with males having nearly twice higher incidence than females. Recently, a decline of firing rate and an increase in firing variability of nigra dopaminergic neurons have been reported in male C57 mice, but not females, by the age of 18 months. The increased expression of PARK2 in males is probably associated with the higher incidence of Parkinson's disease in male (Howell et al., 2020).
In summary, the nigra dopaminergic neurons in aged and/or male animals exhibit a reduction of spontaneous firing rate, which is consistent with the age and gender related Parkinson's disease.

Increased burst firing of nigra dopaminergic neurons under parkinsonian state
In addition to the changes of spontaneous firing rate, firing patterns of nigra dopaminergic neurons also change in both parkinsonian animals and patients. Bursting firing activity has been revealed to be a key pathological characteristic. In Parkinson's disease patients, nigra dopaminergic neurons exhibit twofold higher burst firing activity (Zaghloul et al., 2009;Schiemann et al., 2012). Chronic 6-OHDA exposure to nigra dopaminergic neurons could change the regular rhythmic pacemaking firing to an irregular burst firing (Wang et al., 2015). Recently, Liu et al. (2022) reported that the burst firing activity of dopaminergic neurons increases significantly in 6-and 9-month-old A53T α-syn mice. Studies in both Parkinson's disease patients (Schiemann et al., 2012) and A53T mice (Liu et al., 2022) demonstrated that upregulation of K-ATP channel subunit SUR1 is associated with the increased burst firing of nigra dopaminergic neurons. In addition, the increased activity of SK channels is also associated with 6-OHDA-induced switch of firing activity from regular to irregular bursting (Wang et al., 2015). In parkinQ311X mice, a model of human parkin-induced toxicity, the burst firing activity of dopaminergic neurons increases accompanying with mitochondrial vacuolization in the substantia nigra pars compacta (Regoni et al., 2021).
In summary, nigra dopaminergic neurons show a strong burst firing activity in both Parkinson's disease patients and animal models. Increased activity of K-ATP channels and SK channels may be associated with the burst firing activity under parkinsonian state.

Increase of firing activity of nigra dopaminergic neurons under parkinsonian state and the possible mechanisms of the diversified changes of firing activity
In addition to decreased firing activity of nigra dopaminergic neurons under parkinsonian state, many studies indicated that the increased firing rate and bursting firing may be associated with increased death of nigro-striatal neurons (Doble, 1995;Brown et al., 2009;Good et al., 2011;Subramaniam et al., 2014;Tozzi et al., 2021;Liu et al., 2022). Overactive dopaminergic neurons may tend to die more rapidly (Good et al., 2011). The increased burst firing activity increases the concentration of dopamine in the striatum and may ameliorate locomotion deficits to some degree (Daniel et al., 2021). However, the consequence of increased burst firing and firing rate may deteriorate the degeneration of nigra dopaminergic neurons. It is known that an increase of intracellular calcium concentration is inevitable during the burst firing activity (Kriete 2012). In the condition of Parkinson's disease, the increased intracellular calcium could promote the endoplasmic reticulum stress and oxidative stress and therefore the death of nigra dopaminergic neurons.
The diverse changes of firing rate and patterns of nigra dopaminergic neurons may depend on both the heterogeneity of dopaminergic neurons and disease progression. It is well known that there are different subtypes of dopaminergic neurons in the substantia nigra pars compacta (Carmichael et al., 2021;Mannal et al., 2021). Indeed, early study showed that the average firing rate of nigra dopaminergic neurons in 6-OHDA parkinsonian rats is not significantly different from that of intact rats (Harden and Grace, 1995). Furthermore, the firing frequency exhibits no change, decrease or increase in three types of nigra dopaminergic neurons in MitoPark mice (Good et al., 2011). On the other hand, the spontaneous firing activity of nigra dopaminergic neurons is disturbed with the progress of Parkinson's disease. For example, Good et al. (2011) suggested that the three types of neurons with different changes of firing rate may be associated with a continuous impairment of dopaminergic neurons. Consistently, Tozzi et al. (2021) reported that intrastriatal application of α-syn-preformed fibrils increases the firing rate of dopaminergic neurons in the stage of 12 weeks although the number of active dopaminergic neurons reduces in the early stage of 6 weeks. Similarly, recent study revealed that the firing rate of nigra dopaminergic neurons increases in 9-month A53T mice, while no change in firing rate was found in 3-and 6-month A53T mice (Liu et al., 2022). In addition, it was reported that the firing rate of dopaminergic neurons in MitoPark mice increases with age (Branch et al., 2016).
Early studies indicated that the death of nigra dopaminergic neurons exhibits regional inconsistency. The ventrolateral region of the substantia nigra pars compacta presents earlier and more serious neuronal death in Parkinson's disease patients, and the Lewy body first appears in the ventrolateral region (German et al., 1989;Gibb and Lees, 1991).
Consistent regional inconsistency was also observed in parkinsonian animal models, with the neurons in the lateral portion and ventral tier of the substantia nigra pars compacta most vulnerable to neurotoxin (Betarbet et al., 2000). The regional difference of genetic expression (Duke et al., 2007) and neuromelatonin or neurotrophin expression between lateral and medial substantia nigra pars compacta is probably involved in the differential vulnerability (Gibb and Lees, 1991;Nishio et al., 1998;Barroso-Chinea et al., 2005). Therefore, the regional inconsistency of neuronal death may also be associated with the changed firing activities under parkinsonian state.
A very recent study demonstrated at least three distinct thalamic circuits (Zhang et al., 2022). It is known that in addition to motor deficit, Parkinson's disease patients also exhibit motor learning impairments and depression. The three distinct thalamic circuits are associated with parkinsonian locomotion, motor learning and mood symptoms, respectively. In parkinsonian mice, activation of different parafascicular thalamic subpopulations projecting to caudate putamen, subthalamic nucleus and nucleus accumbens rescues locomotion, motor learning, and depression, respectively (Zhang et al., 2022). DBS of the subthalamic nucleus is a useful treatment for Parkinson's disease. DBS located in the subthalamic nucleus modulates the spontaneous firing rate and pattern of neurons in the basal ganglia (Wichmann et al., 2011). It is reported that high frequency DBS increases the concentration of dopamine and the firing rate of striatal medium spiny neurons which in turn improves motor symptoms (Xiao et al., 2020). Consistently, DBS of the subthalamic nucleus in Parkinson's disease may exert benefits in motor symptoms and side-effects of cognitive decline through influencing different thalamocortical circuits (Okun, 2012;Faggiani and Benazzouz, 2017;Reich et al., 2022). In addition to motor control, the nigra dopaminergic neurons are also involved in reward-based learning (Joshua et al., 2009), which may suggest that the complex functional connectivity of nigra dopaminergic neurons probably has some association with the different changes of neuronal firing activities under parkinsonian state.
In summary, nigra dopaminergic neurons may appear different changes of firing activity, such as no change, reduced firing rate and increased firing rate, at different stages of Parkinson's disease. The firing rate of dopaminergic neurons may increase in advanced Parkinson's disease. The diverse changes of firing activity may depend on the heterogeneity of dopaminergic neurons and disease progression. In addition, the regional inconsistency and distinct functional circuits may also be associated with the different changes of firing activity of dopaminergic neurons under parkinsonian state.

Possible mechanisms involved in decrease and increase of firing activity of nigra dopaminergic neurons under parkinsonian state
Several types of ion channels have been implicated in the pathological firing activity of nigra dopaminergic neurons in Parkinson's disease (Liss et al., 2001(Liss et al., , 2005Good et al., 2011;Zhang et al., 2020). Recently, Daniel et al. (2021) reviewed that ion channels in the substantia nigra regulate spontaneous firing activity of dopaminergic neurons and in turn may function as potential therapeutic targets for Parkinson's disease.
The expression of ion channel subunits, such as Cav1.2, Cav1.3, HCN1, Nav1.2, and Nav1.3, is upregulated in older MitoPark mice which may be associated with the disrupted firing regularity (Branch et al., 2016). Firstly, dysfunction of mitochondrial metabolism in MPTP parkinsonian model activates K-ATP channels and in turn reduces the spontaneous firing activity of dopaminergic neurons (Liss et al., 2005), while blockade of K-ATP channels could reverse α-syn-induced decline of firing rate further suggesting the involvement of K-ATP channels (Hill et al., 2021). Secondly, the reduced activity of HCN channels may be associated with hyperpolarization and absence of pacemaker firing of dopaminergic neurons in MitoPark mitochondrial model of Parkinson's disease (Good et al., 2011). In vitro patch clamp recordings demonstrated that inhibition of HCN channels is associated with MPP + -induced decrease of firing rate of nigra dopaminergic neurons (Chang et al., 2020). Although nigra dopaminergic neurons in spontaneous α-syn overexpressing rats do not show a clear alteration in both spontaneous and evoked firing activity, the HCN channel currents (Ih) and the spontaneous excitatory postsynaptic currents (sEPSCs) decrease clearly (Guatteo et al., 2017). In (G2019S) LRRK2 mice, overphosphorylation of some ion channels may also be associated with the decreased firing rate of nigra dopaminergic neurons (Chou et al., 2014). In addition, reactive astrocytes could synthesize gamma-aminobutyric acid (GABA) via monoamine oxidase-B (MAO-B) (Jo et al., 2014). The astrocytic GABA is involved in the suppression of dopaminergic neurons in parkinsonian animals. Application of GABA A receptor antagonist or MAO-B inhibitor recovers the decreased firing rate of dopaminergic neurons (Abe et al., 2016;Heo et al., 2020). Further experiments revealed that GABA released from reactive astrocytes in the substantia nigra pars compacta inhibits the firing activity of nigra dopaminergic neurons. In addition, application of MAO-B inhibitor also recovers the number of nigra dopaminergic neurons in parkinsonian animals (Heo et al., 2020). Clinically, inhibition of MAO-B by Safinamide has been approved to improve motor symptoms in Parkinson's disease patients (Borgohain et al., 2014).
In contrast, other studies revealed the possible ionic mechanisms involved in the increased firing activity under parkinsonian state. For example, in a mouse model overexpressing mutant α-syn (A53T-SNCA), an impairment of A-type Kv4.3 potassium channels may be associated with the increased firing rate of dopaminergic neurons (Subramaniam et al., 2014).
It is well known that somatodendritic release of dopamine could activate D2 autoreceptors and in turn inhibit the firing activity of nigra dopaminergic neurons (Lacey et al., 1987;Beckstead et al., 2004;Wang et al., 2020;Hikima et al., 2021). Changes of dopamine D2 receptor-mediated autoinhibition in Parkinson's disease may play an important role in the firing rate and patterns of dopaminergic neurons. When the large number of dopaminergic neurons die to produce clinical Parkinson's disease symptoms, the reduced somatodendritic release of dopamine leads to increased firing rate and bursting firing of residual neurons, same as that of blockade of D2 autoreceptors by antagonists.
Recent study demonstrated that α-syn decreases functional availability of D2 receptors in both α-syn triplication mutation (AST)-derived dopaminergic neurons and mouse dopaminergic neurons overexpressing α-syn (Lin et al., 2021). Consistently, activation of D2 receptors restores the changed firing activity of AST-derived dopaminergic neurons to normal levels.
Although no direct evidence indicates the possible modulation of firing activity of dopaminergic neurons, several preclinical or clinical trials did report that drugs targeting on different types of ion channels are useful in the treatment of Parkinson's disease (Daniel et al., 2021). For example, the broad spectrum of voltage-dependent potassium channel blocker, 4-AP, has completed clinical phase-2 experiment successfully (Luca and Singer, 2013). Furthermore, the T-type calcium channel blocker has already been used clinically to improve motor symptoms of Parkinson's disease patients (Murata, 2004;Kothare and Kaleyias, 2008;Li et al., 2020). However, drugs activating HCN channels are still in preclinical evidence for the treatment of Parkinson's disease (Daniel et al., 2021).
In summary, the increased activity of K-ATP channels and decreased activity of HCN channels are closely associated with reduction of firing activity of nigra dopaminergic neurons under parkinsonian state. The expression of several ion channels within the substantia nigra pars compacta may change in parkinsonian animal model. In addition, the reactive astrocyte-induced GABA release is involved in decreased firing activity of nigra dopaminergic neurons. However, impairment of A-type Kv4.3 potassium channels is one of the possible reasons of increased firing activity. The reduced dopamine release decreases functional availability of D2 autoreceptors which in turn leads to increased firing rate.

Conclusion
The spontaneous neuronal firing activity of dopaminergic neurons is critical for dopamine release and motor control. Under parkinsonian state, the nigra dopaminergic neurons exhibit different changes in spontaneous firing rate and patterns. The abnormal firing activities including decreased firing rate, reduced number of firing neurons, increased burst firing and increased firing rate have been reported under parkinsonian state (Table 1). The different alteration of firing activity may depend on disease progression, neuronal heterogeneity and distinct functional circuits under parkinsonian state. Altered expressions or functions of several ion channels such as K-ATP channels, HCN channels, K-ATP channels, SK channels, A-type Kv4.3 potassium channels, as well as D2 autoreceptors and reactive astrocyte-induced GABA release are possible mechanisms of changed firing activities in Parkinson's disease. The changed firing activity of dopaminergic neurons must be elucidated in detail before one could better understand the roles of dopaminergic neurons in Parkinson's disease.

Authors' contribution
Xin-Yi Chen contributed to the writing of the original draft and review of the manuscript. Cui Liu contributed to the revision of the manuscript, and Yan Xue contributed to the table of the manuscript. Lei Chen contributed to the conception, design and revision of the manuscript. All authors listed have approved the submitted version.

Declaration of competing interest
The authors have no conflict of interest to declare.

Data availability
No data was used for the research described in the article.