DA neurons in the VTA play a key role in mood, reward, and emotion-related behaviors (Bromberg-Martin et al., 2010). These DA neurons project, through the mesocorticolimbic dopaminergic system, to brain regions including the nucleus accumbens (NAc), medial prefrontal cortex (mPFC), and basolateral amygdala (BLA), and modulate these target neuronal circuits through their regulated activity (firing of action potentials) (Lammel et al., 2014; Van den Heuvel and Pasterkamp, 2008). As such, understandably, the altered functional activity of the VTA DA neurons is found to be the key determinant in abnormal behaviors related to the diseased conditions such as depression- or anxiety-related states (Chaudhury et al., 2013). Thus, the study of the mechanism underlying the firing activity of the VTA DA neurons is crucial for understanding the function of the DA circuitry and the pathogenesis of related mental diseases (Chaudhury et al., 2013; Kabanova et al., 2015; Badrinarayan et al., 2012; Friedman et al., 2014; Krishnan et al., 2007).

The VTA DA neurons are slow intrinsic pacemakers, which are further modulated by synaptic inputs from multiple excitatory and inhibitory projections (Grace et al., 2007; Johnson and North, 1992). Firing activity controls the release of DA, thus the function of the VTA DA neurons. The altered activity of the VTA DA neurons in a pathological state includes altered firing frequency and switch of the firing patterns (Friedman et al., 2014; Zhong et al., 2018).

The research on the mechanism for the spontaneous firing of action potential (AP) is the key to understand functional regulation of the VTA DA neurons. A variety of ion channels (e.g. voltage-gated Na⁺ channels, high-threshold-activated Ca²⁺ channels, Kv2, large conductance calcium-activated potassium channels, A- and M-type potassium channels) are reported to be involved in the regulation of the spontaneous firing activity of midbrain DA neurons (including the substantia nigra pars compacta, SNc and VTA) (Bean, 2007; Guzman et al., 2009; Puopolo et al., 2007; Khaliq and Bean, 2010; Beckstead et al., 2004; Ford et al., 2007; Philippart et al., 2016; Dufour et al., 2014; Kimm et al., 2015; Li et al., 2017; Tarfa et al., 2017). However, it is not completely clear how this spontaneous firing is initiated. AP fires when the resting membrane potential (RMP) is depolarized to the activation threshold for the voltage-dependent Na⁺ channels, which results in an opening of Na⁺ channels and the upstroke of AP (Bean, 2007). Therefore, a controlled depolarization of the RMP to the activation threshold of AP is the first key step in a cascade leading to the firing of AP. In the VTA DA neurons, the identity of ion channels contributing to this subthreshold depolarization has not been unambiguously established.

The hyperpolarization-activated cyclic-nucleotide-modulated (HCN1-4) channel gene family, one non-selective cation channel that would be activated at a hyperpolarized membrane potential (Seutin et al., 2001), represents the molecular correlates of the pacemaker current in some cells with spontaneous firing of AP, such as If in cardiomyocytes and Ih in certain neurons including a subset of midbrain SNc DA neurons (Seutin et al., 2001). However, in the VTA DA neurons, results concerning the HCN channels to the spontaneous firing are controversial (Khaliq and Bean, 2010; Seutin et al., 2001; Neuhoff et al., 2002; Krashia et al., 2017).

Another non-selective cation channel, NALCN, unlike HCN, is a voltage-modulated channel (Chua et al., 2020), and is reported to be among the candidates for molecular correlates of the persistent background Na⁺ leak currents in the VTA DA neurons (Lu et al., 2009; Philippart and Khaliq, 2018). The NALCN-mediated background Na⁺ leak currents are found to be involved in the modulation of firing activity in a variety of neurons including DA neurons in the SNc (Philippart and Khaliq, 2018; Shi et al., 2016; Lutas et al., 2016; Ford et al., 2018; Um et al., 2021). However, it has not been reported if NALCN plays a role in the spontaneous firing of the VTA DA neurons, although the VTA DA neurons have larger Na⁺ leak currents than SNc DA neurons do (Khaliq and Bean, 2010).

TRP channels are a large class of non-selective cation channels, and are widely distributed in the peripheral and central nervous system. TRPC3, which has a high homology with TRPC6 (Kim et al., 2007), is found to be involved in the regulation of cardiac and SNc DA neuronal excitability, generating depolarizing currents, triggering action potentials, and participating in cellular rhythmic firing (Um et al., 2021; Zhou et al., 2008; Hof et al., 2019). The role of TRP channels, except TRPC4 (Klipec et al., 2016), in the regulation of VTA DA neuronal excitability has not been described.

In this study, we strove to find channel conductance which contribute to the subthreshold depolarization and thus the spontaneous firing of the VTA DA neurons. And furthermore, we investigated if these channels are the molecular mechanism for the altered function activity of the VTA DA neurons related to depression-like behaviors in mouse models of depression. We started by profiling the expression of non-selective cation channels (NSCCs) of the VTA DA neurons using the method of Patch-Seq from midbrain DA neurons projecting to different brain regions, and then focused on HCN, NALCN, TRPC6, and TRPV2 channels which are dominantly expressed in these DA neurons.

In this study, we made a systematic study of the molecular mechanism for the subthreshold depolarization that drives the spontaneous firing of the VTA DA neurons. We identified TRPC6 channels, alongside NALCN, as major contributors to this subthreshold depolarization and related spontaneous firing, and further importantly, we also demonstrated that TRPC6 contributed to the altered firing activity of the VTA DA neurons under states of chronic-stress-induced depression-like behaviors.

The nature of a relatively depolarized resting membrane potential in the VTA DA neurons imposes a need for a full understanding of the channel conductance underlying this depolarization. It is first clear from these current and previous studies that this conductance is mediated by a persistent Na⁺ influx. Unlike the DA neurons in the SNc, where Ca²⁺ influx is needed for the spontaneous firing (Kang and Kitai, 1993), the results in our and others’ studies indicate that Ca²⁺ influx is not necessary for the spontaneous firing of the VTA DA neurons. Replacement of Ca²⁺ ions in the extracellular fluid with Mg²⁺ accelerates the frequency of spontaneous firing (Figure 1B; Khaliq and Bean, 2010), possibly resulting from a reduced activity of the Ca²⁺-activated K⁺ currents and an increased activity of NALCN (Philippart and Khaliq, 2018; Lu et al., 2010). Consistent with this, removing extracellular Ca²⁺ did not hyperpolarize the RMP, which verifies more precisely for the role of Ca²⁺ influx on the subthreshold depolarization. On the other hand, consistent with the previous study (Khaliq and Bean, 2010), a TTX-insensitive Na⁺ influx clearly contributed to the subthreshold depolarization (Figure 1D).

A TTX-insensitive nature of this Na⁺ conductance suggests that cation conductance permeable to Na⁺ might be the molecular candidate. We first focused on the HCN channels because, (1) it has been suggested HCN channels are important regulators for the spontaneous firing of VTA DA neurons in adult male mice under states of depression-like behaviors (Friedman et al., 2014; Zhong et al., 2018); (2) Hcn are one of the more highly expressed NSCCs in the VTA DA neurons in our single-cell RNA-seq study; (3) HCN are permeable to Na⁺ (Benarroch, 2013). However, it is clear from our study that HCN does not contribute to the spontaneous firing of the VTA DA neurons in the adult male mice; none of the known HCN blockers we used had any significant effects on the firing activity. Presence of HCN has long been used as a signature of DA neurons (Mercuri et al., 1995), and also as a differentiating factor for the projection-specificity of the VTA DA neurons (Lammel et al., 2008). Nonetheless, the contribution of HCN to the spontaneous firing of the VTA DA neurons has been controversial; the HCN blockers ZD7288 and CsCl have been reported as both effective (Seutin et al., 2001; Neuhoff et al., 2002; Okamoto et al., 2006) and non-effective (Khaliq and Bean, 2010) on the spontaneous firing of the VTA DA neurons. A closer inspection of our and others’ results presents a possible explanation for these different results: in the younger rodents (less than 1-mo-old Chan et al., 2007 and less than 15 d old in our study), the HCN in the DA neurons play a more important role whereas in the adult mice (~8 wk Chan et al., 2007), like the male adult ones (6~8 wk) we used in this study, the HCN does not contribute to the spontaneous firing of the VTA DA neurons. This is likely due to a shift of activation gating of HCN channels in a hyperpolarization direction with age, thus the modulation of HCN on the firing activity diminishes with age, as it is described in the SNc DA neurons (Chan et al., 2007). Consistent with this, we found hyperpolarizing the RMP rendered the VTA DA neurons of the adult male mice with sensitivity to the HCN blockers. Thus, the VTA DA neurons in adult male mice do not use HCN to depolarize the membrane to generate action potentials.

NALCN is widely expressed in central neurons (Lee et al., 1999; Lu and Feng, 2012), and is a popular candidate considered for the ion mechanism of the subthreshold depolarization currents and Na⁺ leaky currents. NALCN has been shown to be an important component of background Na⁺ currents and to be important for neuronal excitability in the hippocampal neurons, posterior rhomboid nucleus (Retrotrapezoid nucleus/RTN) chemo-sensitive neurons (CO₂/H⁺-sensitive neurons), GABA neurons, and DA neurons in the substantia nigra, and spinal projection neurons (Philippart and Khaliq, 2018; Shi et al., 2016; Lutas et al., 2016; Ford et al., 2018; Um et al., 2021; Lu et al., 2007). Our results with single-cell RNA-seq, single-cell PCR, and immunofluorescence methods validated high-level expression of NALCN in the VTA DA neurons; both the pharmacological and the Nalcn-gene-knockout experiments demonstrated convincingly involvement of NALCN in subthreshold depolarization and spontaneous firing of the VTA DA neurons.

We did not detect alteration of NALCN protein expression in the depression model of CMUS in the VTA tissue. However, this does not completely exclude a possible contribution of NALCN to the altered functional activity of the VTA DA neurons which underlies the chronic-stress-induced depression-like behaviors. Future studies focusing on the functional property of NALCN in the cellular level of the VTA DA neurons under a state of depression are needed to clarify this issue. NALCN is after all a potential drug target in consideration of its important contribution to the resting membrane potential and functional activity of neurons.

The most interesting and important finding of this study is the role of TRPC6 in the regulation of the firing activity of the VTA DA neurons in both physiological and depression-state conditions; the experimental evidence from pharmacological, gene silencing, electrophysiological and behavior results unambiguously support this conclusion. It is interesting to note, although with an unclear neuronal circuit mechanism of action, the TRPC6 channel opener hyperforin was previously reported to have antidepressant effects in corticosterone-depressed mice, which were reversed by prior administration of the TRPC6 blocker larixyl acetate (Pochwat et al., 2018). Also, in a CMUS rat model, TRPC6 expression was found to be downregulated in the hippocampus, and administration of the TRPC6 opener hyperforin was effective in alleviating depression-like behavior (Liu et al., 2015). Our study provides strong and convincing evidence that TRPC6 is a key regulator of the VTA DA neurons which are known to be a key player in depression states (Friedman et al., 2014; Li et al., 2017; Tye et al., 2013; Chang and Grace, 2014; Cao et al., 2010).

The above-mentioned role for TRPC6 should come from its contribution to the subthreshold depolarization, through a persistent permeation to the influx of Na⁺. Although TRPC channels have been better recognized for their permeability to Ca²⁺ and their regulation of cellular Ca²⁺ homeostasis, the importance of TRPC channels for the permeability of monovalent cations, especially Na⁺, is now gradually being appreciated (Eder et al., 2005). In the substantia nigra GABA and DA neurons, TRPC3 has been reported to play an important role in maintaining depolarization membrane potential, peacemaking, and firing regularity (Um et al., 2021; Zhou et al., 2008). Otherwise, it has been reported that the slow tonic firing of SNc DA neurons, depends on the basal activity of both the NALCN and TRPC3 channels, but that burst firing does not require TRPC3 channels but relies only on NALCN channels (Hahn et al., 2023). TRPC6 and TRPC3 belong to the same TRPC family subclass and TRPC6 is more than 75% homologous to TRPC3 (Clapham et al., 2001). Interestingly, our results from single-cell RNA-seq and single-cell PCR demonstrated the rich expression of Trpc6 in the VTA DA neurons among all TRP channels but no expression of Trpc3 was found in these neurons. These results present an opportunity for selectively targeting a single TRPC channel to exert selective pharmacological results. Besides, TRPC4 expression was detected in a uniformly distributed subset of rat VTA DA neurons (Klipec et al., 2016), which differs with our results with single-cell RNA-seq and single-cell PCR, and TRPC4 KO rats showed decreased VTA DA neuron tonic firing and deficits in cocaine reward and social behaviors (Klipec et al., 2016).Trpc4 is detected to be expressed with a low level in the VTA DA neurons of male mice by single-cell RNA-seq (Figure 2) and in Trpc6 and Slc6a3 double-positive VTA cells by single-cell PCR (Figure 5—figure supplement 1). The different species used in this and our study may be one explanation, but the role of TRPC4 in the excitability of mice VTA TRPC6-negative DA neurons is worth to investigating further.

It has also been demonstrated in cell types other than neurons, such as rat portal vein smooth muscle cells, that the activation of the α-adrenergic receptor can open TRPC6, which leads to cell depolarization, activation of voltage-dependent Ca²⁺ channels, and ultimately the contraction of smooth muscle cell (Inoue and Mori, 2002); it is also shown that angiotensin II and endothelin I can activate TRPC3/C6 heterodimers via vascular G protein-coupled receptors (GPCR), thereby depolarizing cells (Nishida et al., 2010; Nishioka et al., 2011). Following this line, it would be very interesting to know if TRPC6 in the VTA DA neurons, where multiple GPCR reside receiving input and auto modulation by neuronal transmitters (Su et al., 2019; McCall et al., 2019), could also be a target of modulation with a similar mechanism. In consideration of the important role, we described here in this study, this type of modulation will present possible explanations for some unsolved mechanisms for physiological and pathophysiological functions of the VTA DA neurons.

The facts that down-regulation of TRPC6 proteins was correlated with reduced firing activity of the VTA DA neurons as well as the chronic-stress-induced depression-like behaviors of mice with both sexes, and that knocking down of TRPC6 in the VTA DA neurons confers the male mice with depression-like behaviors strongly suggest a crucial role for TRPC6 in the development of chronic-stress-induced depression-like behaviors under stressful conditions like CMUS. To reinforce this, down-regulation of TRPC6 was also found in another chronic-stress-induced depression model of CRS, a model with similar neuronal alteration of reduced firing activity in the VTA DA neurons to that described in the CMUS model (Qu et al., 2020). These facts with the fact that TRPC6 activator hyperforin is an effective antidepressant in multiple depression models (Pochwat et al., 2018; Liu et al., 2015), and that hyperforin is the principal component of St. John’s wort, a well-known antidepressant herb (Butterweck, 2003), present TRPC6 as a very attractive drug target for new lines of antidepressants.

All data generated or analysed during this study are included in the manuscript and supporting files; source data files have been provided for all Figures.

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Author details

1. Jing Wang

   1. Department of Pharmacology, Hebei Medical University, Shijiazhuang, China
   2. Department of Chinese Medicinal Chemistry, Hebei University of Chinese Medicine, Shijiazhuang, China
   3. The Key Laboratory of Neural and Vascular Biology, Ministry of Education, Hebei Medical University, Shijiazhuang, China
   4. The Key Laboratory of New Drug Pharmacology and Toxicology, Hebei Medical University, Shijiazhuang, China

   Contribution

   Data curation, Writing – original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6216-2919

2. Min Su

   1. Department of Pharmacology, Hebei Medical University, Shijiazhuang, China
   2. Yiling Pharmaceutical Company, Shijiazhuang, China

   Contribution

   Methodology

   Competing interests

   is affiliated with Yiling Pharmaceutical Company

3. Dongmei Zhang

   1. Department of Pharmacology, Hebei Medical University, Shijiazhuang, China
   2. Department of Clinical Pharmacy, Xingtai Ninth Hospital, Xingtai, China

   Contribution

   Data curation

   Competing interests

   No competing interests declared

4. Ludi Zhang

   Department of Pharmacology, Hebei Medical University, Shijiazhuang, China

   Contribution

   Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2061-4563

5. Chenxu Niu

   Department of Pharmacology, Hebei Medical University, Shijiazhuang, China

   Contribution

   Methodology

   Competing interests

   No competing interests declared

6. Chaoyi Li

   Department of Pharmacology, Hebei Medical University, Shijiazhuang, China

   Contribution

   Methodology

   Competing interests

   No competing interests declared

7. Shuangzhu You

   Department of Pharmacology, Hebei Medical University, Shijiazhuang, China

   Contribution

   Methodology

   Competing interests

   No competing interests declared

8. Yuqi Sang

   1. Department of Pharmacology, Hebei Medical University, Shijiazhuang, China
   2. College of Chemical Engineering, Shijiazhuang University, Shijiazhuang, China
   3. Shijiazhuang Key Laboratory of Targeted Drugs Research and Efficacy Evaluation, Shijiazhuang, China

   Contribution

   Methodology

   Competing interests

   No competing interests declared

9. Yongxue Zhang

   1. Department of Pharmacology, Hebei Medical University, Shijiazhuang, China
   2. Department of Pharmacy, Handan First Hospital, Handan, China

   Contribution

   Methodology

   Competing interests

   No competing interests declared

10. Xiaona Du

    Department of Pharmacology, Hebei Medical University, Shijiazhuang, China

    Contribution

    Project administration

    Competing interests

    No competing interests declared

11. Hailin Zhang

    1. Department of Pharmacology, Hebei Medical University, Shijiazhuang, China
    2. Department of Psychiatry, The First Hospital of Hebei Medical University, Mental Health Institute of Hebei Medical University, Shijiazhuang, China

    Contribution

    Funding acquisition, Writing – review and editing

    For correspondence

    zhanghl@hebmu.edu.cn

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-9305-9508

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