Somatodendritic organization of pacemaker activity in midbrain dopamine neurons.

The slow and regular pacemaking activity of midbrain dopamine (DA) neurons requires proper spatial organization of the excitable elements between the soma and dendritic compartments, but the somatodendritic organization is not clear. Here, we show that the dynamic interaction between the soma and multiple proximal dendritic compartments (PDCs) generates the slow pacemaking activity in DA neurons. In multipolar DA neurons, spontaneous action potentials (sAPs) consistently originate from the axon-bearing dendrite. However, when the axon initial segment was disabled, sAPs emerge randomly from various primary PDCs, indicating that multiple PDCs drive pacemaking. Ca2+ measurements and local stimulation/perturbation experiments suggest that the soma serves as a stably-oscillating inertial compartment, while multiple PDCs exhibit stochastic fluctuations and high excitability. Despite the stochastic and excitable nature of PDCs, their activities are balanced by the large centrally-connected inertial soma, resulting in the slow synchronized pacemaking rhythm. Furthermore, our electrophysiological experiments indicate that the soma and PDCs, with distinct characteristics, play different roles in glutamate- induced burst-pause firing patterns. Excitable PDCs mediate excitatory burst responses to glutamate, while the large inertial soma determines inhibitory pause responses to glutamate. Therefore, we could conclude that this somatodendritic organization serves as a common foundation for both pacemaker activity and evoked firing patterns in midbrain DA neurons.

AST-001 Improves Social Deficits and Restores Dopamine Neuron Activity in a Mouse Model of Autism.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by impaired social communication and social interaction, restricted and repetitive behavior, and interests. The core symptoms of ASD are associated with deficits in mesocorticolimbic dopamine pathways that project from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC). AST-001 is an investigational product currently in a phase 3 clinical trial for treating the core symptoms of ASD, with L-serine as the API (active pharmaceutical ingredient). Because the causes of ASD are extremely heterogeneous, a single genetic ASD model cannot represent all autism models. In this paper, we used the VPA-exposed model, which is more general and widely used than a single genetic model, but this is also one of the animal models of autism. Herein, we conducted experiments to demonstrate the efficacy of AST-001 as L-Serine that alters the regulation of the firing rate in dopamine neurons by inhibiting small conductance Ca2+-activated K+ channels (SK channels). Through these actions, AST-001 improved sociability and social novelty by rescuing the intrinsic excitabilities of dopamine neurons in VPA-exposed ASD mouse models that showed ASD-related behavioral abnormalities. It is thought that this effect of improving social deficits in VPA-exposed ASD mouse models is due to AST-001 normalizing aberrant SK channel activities that slowed VTA dopamine neuron firing. Overall, these findings suggest that AST-001 may be a potential therapeutic agent for ASD patients, and that its mechanism of action may involve the regulation of dopamine neuron activity and the improvement of social interaction.

Proximal dendritic localization of NALCN channels underlies tonic and burst firing in nigral dopaminergic neurons.

In multipolar nigral dopamine (DA) neurons, the highly excitable proximal dendritic compartments (PDCs) and two Na+ -permeable leak channels, TRPC3 and NALCN, play a key role in pacemaking. However, the causal link between them is unknown. Here we report that the proximal dendritic localization of NALCN underlies pacemaking and burst firing in DA neurons. Our morphological analysis of nigral DA neurons reveals that TRPC3 is ubiquitously expressed in the whole somatodendritic compartment, but NALCN is localized within the PDCs. Blocking either TRPC3 or NALCN channels abolished pacemaking. However, only blocking NALCN, not TRPC3, degraded burst discharges. Furthermore, local glutamate uncaging readily induced burst discharges within the PDCs, compared with other parts of the neuron, and NALCN channel inhibition dissipated burst generation, indicating the importance of NALCN to the high excitability of PDCs. Therefore, we conclude that PDCs serve as a common base for tonic and burst firing in nigral DA neurons. KEY POINTS: Midbrain dopamine (DA) neurons are slow pacemakers that can generate tonic and burst firings, and the highly excitable proximal dendritic compartments (PDCs) and two Na+ -permeable leak channels, TRPC3 and NALCN, play a key role in pacemaking. We find that slow tonic firing 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. We find that TRPC3 is ubiquitously expressed in the entire somatodendritic compartment, but that NALCN exists only within the PDCs in nigral DA neurons. We show that NALCN channel localization confers high excitability on PDCs and is essential for burst generation in nigral DA neurons. These results suggest that PDCs serve as a common base for tonic and burst firing in nigral DA neurons.

TRPC3 and NALCN channels drive pacemaking in substantia nigra dopaminergic neurons.

Midbrain dopamine (DA) neurons are slow pacemakers that maintain extracellular DA levels. During the interspike intervals, subthreshold slow depolarization underlies autonomous pacemaking and determines its rate. However, the ion channels that determine slow depolarization are unknown. Here we show that TRPC3 and NALCN channels together form sustained inward currents responsible for the slow depolarization of nigral DA neurons. Specific TRPC3 channel blockade completely blocked DA neuron pacemaking, but the pacemaking activity in TRPC3 knock-out (KO) mice was perfectly normal, suggesting the presence of compensating ion channels. Blocking NALCN channels abolished pacemaking in both TRPC3 KO and wild-type mice. The NALCN current and mRNA and protein expression are increased in TRPC3 KO mice, indicating that NALCN compensates for TRPC3 currents. In normal conditions, TRPC3 and NALCN contribute equally to slow depolarization. Therefore, we conclude that TRPC3 and NALCN are two major leak channels that drive robust pacemaking in nigral DA neurons.

N-benzhydryl quinuclidine compounds are a potent and Src kinase-independent inhibitor of NALCN channels.

BACKGROUND AND PURPOSE: NALCN is a Na+ leak, GPCR-activated channel that regulates the resting membrane potential and neuronal excitability. Despite numerous possible roles for NALCN in both normal physiology and disease processes, lack of specific blockers hampers further investigation. EXPERIMENTAL APPROACH: The effect of N-benzhydryl quinuclidine compounds on NALCN channels was demonstrated using whole-cell patch-clamp recordings in HEK293T cells overexpressing NALCN and acutely isolated nigral dopaminergic neurons that express NALCN endogenously. Src kinase activity was measured using a Src kinase assay kit, and voltage and current-clamp recordings from nigral dopaminergic neurons were used to measure NALCN currents and membrane potentials. KEY RESULTS: N-benzhydryl quinuclidine compounds inhibited NALCN channels without affecting TRPC channels, another important route for Na+ leak. In HEK293T cells overexpressing NALCN, N-benzhydryl quinuclidine compounds potently suppressed muscarinic M3 receptor-activated NALCN currents. Structure-function relationship studies suggest that the quinuclidine ring with a benzhydryl group imparts the ability to inhibit NALCN currents regardless of Src family kinases. Moreover, N-benzhydryl quinuclidine compounds inhibited not only GPCR-activated NALCN currents but also background Na+ leak currents and hyperpolarized the membrane potential in native midbrain dopaminergic neurons that express NALCN endogenously. CONCLUSION AND IMPLICATIONS: These findings suggest that N-benzhydryl quinuclidine compounds have a pharmacological potential to directly inhibit NALCN channels and could be a useful tool to investigate functions of NALCN channels.

Small-molecule inhibitors of USP7 induce apoptosis through oxidative and endoplasmic reticulum stress in cancer cells.

USP7 is a deubiquitinating enzyme that involves the ubiquitin proteasome system (UPS) to maintain regulation of protein synthesis and degradation. The well-known substrate of USP7 is the Mdm2-p53 complex. In fact, several studies have reported that functional inhibition of USP7 induces cancer cell apoptosis through activation of p53. However, the contribution of oxidative or endoplasmic reticulum (ER) stress, which is commonly induced by inhibition of the UPS for USP7 inhibitor-mediated apoptosis in cancer cells, has not been investigated. In contrast to previous reports, we show that p53 is not critical during USP7 inhibitor-induced apoptosis in several cancer cells. Inhibition of deubiquitinating enzyme activities by USP7 inhibitors causes ER stress by accumulating polyubiquitinated proteins in cancer cells. Furthermore, we demonstrate that USP7 inhibitors increase intracellular reactive oxygen species and mainly cause cancer cell apoptosis. Taken together, our results reveal that oxidative and ER stress, rather than the Mdm2-p53 axis, mainly contributes to USP7 inhibitor-mediated apoptosis in cancer cells.

Coexistence of glutamatergic spine synapses and shaft synapses in substantia nigra dopamine neurons.

Dopamine neurons of the substantia nigra have long been believed to have multiple aspiny dendrites which receive many glutamatergic synaptic inputs from several regions of the brain. But, here, using high-resolution two-photon confocal microscopy in the mouse brain slices, we found a substantial number of common dendritic spines in the nigral dopamine neurons including thin, mushroom, and stubby types of spines. However, the number of dendritic spines of the dopamine neurons was approximately five times lower than that of CA1 pyramidal neurons. Immunostaining and morphological analysis revealed that glutamatergic shaft synapses were present two times more than spine synapses. Using local two-photon glutamate uncaging techniques, we confirmed that shaft synapses and spine synapses had both AMPA and NMDA receptors, but the AMPA/NMDA current ratios differed. The evoked postsynaptic potentials of spine synapses showed lower amplitudes but longer half-widths than those of shaft synapses. Therefore, we provide the first evidence that the midbrain dopamine neurons have two morphologically and functionally distinct types of glutamatergic synapses, spine synapses and shaft synapses, on the same dendrite. This peculiar organization could be a new basis for unraveling many physiological and pathological functions of the midbrain dopamine neurons.

Balance between the proximal dendritic compartment and the soma determines spontaneous firing rate in midbrain dopamine neurons.

Midbrain dopamine (DA) neurons are slow intrinsic pacemakers that require the elaborate composition of many ion channels in the somatodendritic compartments. Understanding the major determinants of the spontaneous firing rate (SFR) of midbrain DA neurons is important because they determine the basal DA levels in target areas, including the striatum. As spontaneous firing occurs synchronously at the soma and dendrites, the electrical coupling between the soma and dendritic compartments has been regarded as a key determinant for the SFR. However, it is not known whether this somatodendritic coupling is served by the whole dendritic compartments or only parts of them. In the rat substantia nigra pars compacta (SNc) DA neurons, we demonstrate that the balance between the proximal dendritic compartment and the soma determines the SFR. Isolated SNc DA neurons showed a wide range of soma size and a variable number of primary dendrites but preserved a quite consistent SFR. The SFR was not correlated with soma size or with the number of primary dendrites, but it was strongly correlated with the area ratios of the proximal dendritic compartments to the somatic compartment. Tetrodotoxin puff and local Ca(2+) perturbation experiments, computer simulation, and local glutamate uncaging experiments suggest the importance of the proximal dendritic compartments in pacemaker activity. These data indicate that the proximal dendritic compartments, not the whole dendritic compartments, play a key role in the somatodendritic balance that determines the SFR in DA neurons.

Homeostatic regulation mechanism of spontaneous firing determines glutamate responsiveness in the midbrain dopamine neurons.

Autonomous tonic firing of the midbrain dopamine neuron is essential for maintenance of ambient dopamine level in the brain, in which intracellular Ca2+ concentration ([Ca2+]c) plays a complex but pivotal role. However, little is known about Ca2+ signals by which dopamine neurons maintain an optimum spontaneous firing rate. In the midbrain dopamine neurons, we here show that spontaneous firing evoked [Ca2+]c changes in a phasic manner in the dendritic region but a tonic manner in the soma. Tonic levels of somatic [Ca2+]c strictly tallied with spontaneous firing rates. However, manipulatory raising or lowering of [Ca2+]c with caged compounds from the resting firing state proportionally suppressed or raised spontaneous firing rate, respectively, suggesting presence of the homeostatic regulation mechanism for spontaneous firing rate via tonic [Ca2+]c changes of the soma. More importantly, abolition of this homeostatic regulation mechanism significantly exaggerated the responses of tonic firings and high-frequency phasic discharges to glutamate. Therefore, we conclude that this Ca(2+)-dependent homeostatic regulation mechanism is responsible for not only maintaining optimum rate of spontaneous firing, but also proper responses to glutamate. Perturbation of this mechanism could cause dopamine neurons to be more vulnerable to glutamate and Ca2+ toxicities.

Regulation of dopaminergic neuron firing by heterogeneous dopamine autoreceptors in the substantia nigra pars compacta.

Dopamine (DA) receptors generate many cellular signals and play various roles in locomotion, motivation, hormone production, and drug abuse. According to the location and expression types of the receptors in the brain, DA signals act in either stimulatory or inhibitory manners. Although DA autoreceptors in the substantia nigra pars compacta are known to regulate firing activity, the exact expression patterns and roles of DA autoreceptor types on the firing activity are highly debated. Therefore, we performed individual correlation studies between firing activity and receptor expression patterns using acutely isolated rat substantia nigra pars compacta DA neurons. When we performed single-cell RT-PCR experiments, D(1), D(2)S, D(2)L, D(3), and D(5) receptor mRNA were heterogeneously expressed in the order of D(2)L > D(2)S > D(3) > D(5) > D(1). Stimulation of D(2) receptors with quinpirole suppressed spontaneous firing similarly among all neurons expressing mRNA solely for D(2)S, D(2)L, or D(3) receptors. However, quinpirole most strongly suppressed spontaneous firing in the neurons expressing mRNA for both D(2) and D(3) receptors. These data suggest that D(2) S, D(2)L, and D(3) receptors are able to equally suppress firing activity, but that D(2) and D(3) receptors synergistically suppress firing. This diversity in DA autoreceptors could explain the various actions of DA in the brain.