Ca
2+
-influx through L-type Ca
2+
-channels (LTCCs) is associated with activity-related stressful oscillations of Ca
2+
levels within dopaminergic (DA) neurons in the substantia nigra (SN), which ...may contribute to their selective degeneration in Parkinson's disease (PD). LTCC blockers were neuroprotective in mouse neurotoxin models of PD, and isradipine is currently undergoing testing in a phase III clinical trial in early PD. We report no evidence for neuroprotection by
in vivo
pretreatment with therapeutically relevant isradipine plasma levels, or Ca
v
1.3 LTCC deficiency in 6-OHDA-treated male mice. To explain this finding, we investigated the pharmacological properties of human LTCCs during SN DA-like and arterial smooth muscle (aSM)-like activity patterns using whole-cell patch-clamp recordings in HEK293 cells (Ca
v
1.2 α1-subunit, long and short Ca
v
1.3 α1-subunit splice variants; β3/α2δ1). During SN DA-like pacemaking, only Ca
v
1.3 variants conducted Ca
2+
current (I
Ca
) at subthreshold potentials between action potentials. SN DA-like burst activity increased integrated I
Ca
during (Ca
v
1.2 plus Ca
v
1.3) and after (Ca
v
1.3) the burst. Isradipine inhibition was splice variant and isoform dependent, with a 5- to 11-fold lower sensitivity to Ca
v
1.3 variants during SN DA-like pacemaking compared with Ca
v
1.2 during aSM-like activity. Supratherapeutic isradipine concentrations reduced the pacemaker precision of adult mouse SN DA neurons but did not affect their somatic Ca
2+
oscillations. Our data predict that Ca
v
1.2 and Ca
v
1.3 splice variants contribute differentially to Ca
2+
load in SN DA neurons, with prominent Ca
v
1.3-mediated I
Ca
between action potentials and after bursts. The failure of therapeutically relevant isradipine levels to protect SN DA neurons can be explained by weaker state-dependent inhibition of SN DA LTCCs compared with aSM Ca
v
1.2.
SIGNIFICANCE STATEMENT
The high vulnerability of dopamine (DA) neurons in the substantia nigra (SN) to neurodegenerative stressors causes Parkinson's disease (PD). Ca
2+
influx through voltage-gated L-type Ca
2+
channels (LTCCs), in particular Ca
v
1.3, appears to contribute to this vulnerability, and the LTCC inhibitor isradipine is currently being tested as a neuroprotective agent for PD in a phase III clinical trial. However, in our study isradipine plasma concentrations approved for therapy were not neuroprotective in a PD mouse model. We provide an explanation for this observation by demonstrating that during SN DA-like neuronal activity LTCCs are less sensitive to isradipine than Ca
v
1.2 LTCCs in resistance blood vessels (mediating dose-limiting vasodilating effects) and even at supratherapeutic concentrations isradipine fails to reduce somatic Ca
2+
oscillations of SN DA neurons.
Human cerebral organoids, derived from induced pluripotent stem cells, offer a unique in vitro research window to the development of the cerebral cortex. However, a key player in the developing ...brain, the microglia, do not natively emerge in cerebral organoids. Here we show that erythromyeloid progenitors (EMPs), differentiated from induced pluripotent stem cells, migrate to cerebral organoids, and mature into microglia-like cells and interact with synaptic material. Patch-clamp electrophysiological recordings show that the microglia-like population supported the emergence of more mature and diversified neuronal phenotypes displaying repetitive firing of action potentials, low-threshold spikes and synaptic activity, while multielectrode array recordings revealed spontaneous bursting activity and increased power of gamma-band oscillations upon pharmacological challenge with NMDA. To conclude, microglia-like cells within the organoids promote neuronal and network maturation and recapitulate some aspects of microglia-neuron co-development in vivo, indicating that cerebral organoids could be a useful biorealistic human in vitro platform for studying microglia-neuron interactions.
Dopaminergic (DA) midbrain neurons within the substantia nigra (SN) display an autonomous pacemaker activity that is crucial for dopamine release and voluntary movement control. Their progressive ...degeneration is a hallmark of Parkinson's disease. Their metabolically demanding activity-mode affects Ca
homeostasis, elevates metabolic stress, and renders SN DA neurons particularly vulnerable to degenerative stressors. Accordingly, their activity is regulated by complex mechanisms, notably by dopamine itself, via inhibitory D2-autoreceptors and the neuroprotective neuronal Ca
sensor NCS-1. Analyzing regulation of SN DA neuron activity-pattern is complicated by their high vulnerability. We studied this activity and its control by dopamine, NCS-1, and glucose with extracellular multi-electrode array (MEA) recordings from midbrain slices of juvenile and adult mice. Our tailored MEA- and spike sorting-protocols allowed high throughput and long recording times. According to individual dopamine-responses, we identified two distinct SN cell-types, in similar frequency: dopamine-inhibited and dopamine-excited neurons. Dopamine-excited neurons were either silent in the absence of dopamine, or they displayed pacemaker-activities, similar to that of dopamine-inhibited neurons. Inhibition of pacemaker-activity by dopamine is typical for SN DA neurons, and it can undergo prominent desensitization. We show for adult mice, that the number of SN DA neurons with desensitized dopamine-inhibition was increased (~60-100%) by a knockout of NCS-1, or by prevention of NCS-1 binding to D2-autoreceptors, while time-course and degrees of desensitization were not altered. The number of neurons with desensitized D2-responses was also higher (~65%) at high glucose-levels (25 mM), compared to lower glucose (2.5 mM), while again desensitization-kinetics were unaltered. However, spontaneous firing-rates were significantly higher at high glucose-levels (~20%). Moreover, transient glucose-deprivation (1 mM) induced a fast and fully-reversible pacemaker frequency reduction. To directly address and quantify glucose-sensing properties of SN DA neurons, we continuously monitored their electrical activity, while altering extracellular glucose concentrations stepwise from 0.5 mM up to 25 mM. SN DA neurons were excited by glucose, with EC
values ranging from 0.35 to 2.3 mM. In conclusion, we identified a novel, common subtype of dopamine-excited SN neurons, and a complex, joint regulation of dopamine-inhibited neurons by dopamine and glucose, within the range of physiological brain glucose-levels.
Understanding the mechanisms underlying neural progenitor differentiation and neuronal fate specification is critical for the use of embryonic stem cells (ESCs) for regenerative medicine. Cortical ...interneurons are of particular interest for cell transplantation; however, only a limited subset of these neurons can be generated from ESCs. Here we uncover a pivotal role for Activin in regulating the differentiation and identity of telencephalic neural precursors derived from mouse and human ESCs. We show that Activin directly inhibits the mitogenic sonic hedgehog pathway in a Gli3-dependent manner while enhancing retinoic acid signalling, the pro-neurogenic pathway. In addition, we demonstrate that Activin provides telencephalic neural precursors with positional cues that specifically promote the acquisition of a calretinin interneuron fate by controlling the expression of genes that regulate cortical interneuron identity. This work demonstrates a novel means for regulating neuronal differentiation and specification of subtype identity.
The dorsal raphe nucleus (DRN) and ventrolateral periaqueductal grey (vlPAG) regions contain populations of dopamine neurons, often considered to be a dorsal caudal extension of the A10 group mostly ...found in the ventral tegmental area (VTA). Recent studies suggest they are involved in promoting wakefulness and mediate some of the antinociceptive and rewarding properties of opiates. However, little is known about their electrophysiological properties. To address this, we used Pitx3‐GFP and tyrosine hydroxylase (TH)‐GFP mice to carry out targeted whole‐cell recordings from this population in acute brain slices. We found that DRN/vlPAG dopamine neurons have characteristics similar to most VTA dopamine neurons, but distinct from dorsal raphe serotonin neurons. They fire broad action potentials at a relatively slow, regular rate, exhibit a hyperpolarization‐activated inward current and delayed repolarization, and show spike‐frequency adaptation in response to prolonged depolarization. In addition, they receive fast excitatory and inhibitory synaptic inputs. Moreover, we found co‐expression of vasoactive intestinal polypeptide in small, periaqueductal dopamine neurons, but generally not in larger, more ventral dopamine neurons.
Dopamine neurons in the DRN/vlPAG are electrophysiologically similar to VTA dopamine neurons, but distinct from serotonin neurons. Vasoactive‐intestinal polypeptide is co‐expressed in a subset of these dopamine neurons, typically those positioned more dorsally, close to the aqueduct. These neurons receive fast excitatory and inhibitory synaptic inputs in brainslices.
Human pluripotent stem cell (hPSC)-derived neuron cultures have emerged as models of electrical activity in the human brain. Microelectrode arrays (MEAs) measure changes in the extracellular electric ...potential of cell cultures or tissues and enable the recording of neuronal network activity. MEAs have been applied to both human subjects and hPSC-derived brain models. Here, we review the literature on the functional characterization of hPSC-derived two- and three-dimensional brain models with MEAs and examine their network function in physiological and pathological contexts. We also summarize MEA results from the human brain and compare them to the literature on MEA recordings of hPSC-derived brain models. MEA recordings have shown network activity in two-dimensional hPSC-derived brain models that is comparable to the human brain and revealed pathology-associated changes in disease models. Three-dimensional hPSC-derived models such as brain organoids possess a more relevant microenvironment, tissue architecture and potential for modeling the network activity with more complexity than two-dimensional models. hPSC-derived brain models recapitulate many aspects of network function in the human brain and provide valid disease models, but certain advancements in differentiation methods, bioengineering and available MEA technology are needed for these approaches to reach their full potential.
Dopamine (DA) neurons of the ventrolateral periaqueductal gray (vlPAG) and dorsal raphe nucleus (DRN) fire spontaneous action potentials (APs) at slow, regular patterns
in vitro
but a detailed ...account of their intrinsic membrane properties responsible for spontaneous firing is currently lacking. To resolve this, we performed a voltage-clamp electrophysiological study in brain slices to describe their major ionic currents and then constructed a computer model and used simulations to understand the mechanisms behind autorhythmicity
in silico
. We found that vlPAG/DRN DA neurons exhibit a number of voltage-dependent currents activating in the subthreshold range including, a hyperpolarization-activated cation current (I
H
), a transient, A-type, potassium current (I
A
), a background, ‘persistent’ (I
NaP
) sodium current and a transient, low voltage activated (LVA) calcium current (I
CaLVA
). Brain slice pharmacology, in good agreement with computer simulations, showed that spontaneous firing occurred independently of I
H
, I
A
or calcium currents. In contrast, when blocking sodium currents, spontaneous firing ceased and a stable, non-oscillating membrane potential below AP threshold was attained. Using the DA neuron model we further show that calcium currents exhibit little activation (compared to sodium) during the interspike interval (ISI) repolarization while, any individual potassium current alone, whose blockade positively modulated AP firing frequency, is not required for spontaneous firing. Instead, blockade of a number of potassium currents simultaneously is necessary to eliminate autorhythmicity. Repolarization during ISI is mediated initially
via
the deactivation of the delayed rectifier potassium current, while a sodium background ‘persistent’ current is essentially indispensable for autorhythmicity by driving repolarization towards AP threshold.
Cellular perturbations underlying Alzheimer’s disease (AD) are primarily studied in human postmortem samples and model organisms. Here, we generated a single-nucleus atlas from a rare cohort of ...cortical biopsies from living individuals with varying degrees of AD pathology. We next performed a systematic cross-disease and cross-species integrative analysis to identify a set of cell states that are specific to early AD pathology. These changes—which we refer to as the early cortical amyloid response—were prominent in neurons, wherein we identified a transitional hyperactive state preceding the loss of excitatory neurons, which we confirmed by acute slice physiology on independent biopsy specimens. Microglia overexpressing neuroinflammatory-related processes also expanded as AD pathology increased. Finally, both oligodendrocytes and pyramidal neurons upregulated genes associated with β-amyloid production and processing during this early hyperactive phase. Our integrative analysis provides an organizing framework for targeting circuit dysfunction, neuroinflammation, and amyloid production early in AD pathogenesis.
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•Single-nucleus profiling of human cortex biopsies uncovers amyloid-associated states•Upper-layer pyramidal neurons show hyperactivity prior to degeneration•Microglial states correlate with pathological and clinical progression•Signatures of amyloid production identified in both neurons and oligodendrocytes
Generating single-nucleus atlas from cortical biopsies of living individuals at early stage of Alzheimer’s disease, cell states of neurons, microglia, and oligodendrocytes associated with AD pathology are identified.
The preferential degeneration of Substantia nigra dopamine midbrain neurons (SN DA) causes the motor-symptoms of Parkinson's disease (PD). Voltage-gated L-type calcium channels (LTCCs), especially ...the Cav1.3-subtype, generate an activity-related oscillatory Ca(2+) burden in SN DA neurons, contributing to their degeneration and PD. While LTCC-blockers are already in clinical trials as PD-therapy, age-dependent functional roles of Cav1.3 LTCCs in SN DA neurons remain unclear. Thus, we analysed juvenile and adult Cav1.3-deficient mice with electrophysiological and molecular techniques. To unmask compensatory effects, we compared Cav1.3 KO mice with pharmacological LTCC-inhibition. LTCC-function was not necessary for SN DA pacemaker-activity at either age, but rather contributed to their pacemaker-precision. Moreover, juvenile Cav1.3 KO but not WT mice displayed adult wildtype-like, sensitised inhibitory dopamine-D2-autoreceptor (D2-AR) responses that depended upon both, interaction of the neuronal calcium sensor NCS-1 with D2-ARs, and on voltage-gated T-type calcium channel (TTCC) activity. This functional KO-phenotype was accompanied by cell-specific up-regulation of NCS-1 and Cav3.1-TTCC mRNA. Furthermore, in wildtype we identified an age-dependent switch of TTCC-function from contributing to SN DA pacemaker-precision in juveniles to pacemaker-frequency in adults. This novel interplay of Cav1.3 L-type and Cav3.1 T-type channels, and their modulation of SN DA activity-pattern and D2-AR-sensitisation, provide new insights into flexible age- and calcium-dependent activity-control of SN DA neurons and its pharmacological modulation.
Ca2+-influx through L-type Ca2+-channels (LTCCs) is associated with activity-related stressful oscillations of Ca2+ levels within dopaminergic (DA) neurons in the substantia nigra (SN), which may ...contribute to their selective degeneration in Parkinson's disease (PD). LTCC blockers were neuroprotective in mouse neurotoxin models of PD, and isradipine is currently undergoing testing in a phase III clinical trial in early PD. We report no evidence for neuroprotection by in vivo pretreatment with therapeutically relevant isradipine plasma levels, or Cav1.3 LTCC deficiency in 6-OHDA-treated male mice. To explain this finding, we investigated the pharmacological properties of human LTCCs during SN DA-like and arterial smooth muscle (aSM)-like activity patterns using whole-cell patch-clamp recordings in HEK293 cells (Cav1.2 α1-subunit, long and short Cav1.3 α1-subunit splice variants; β3/α2δ1). During SN DA-like pacemaking, only Cav1.3 variants conducted Ca2+ current (ICa) at subthreshold potentials between action potentials. SN DA-like burst activity increased integrated ICa during (Cav1.2 plus Cav1.3) and after (Cav1.3) the burst. Isradipine inhibition was splice variant and isoform dependent, with a 5- to 11-fold lower sensitivity to Cav1.3 variants during SN DA-like pacemaking compared with Cav1.2 during aSM-like activity. Supratherapeutic isradipine concentrations reduced the pacemaker precision of adult mouse SN DA neurons but did not affect their somatic Ca2+ oscillations. Our data predict that Cav1.2 and Cav1.3 splice variants contribute differentially to Ca2+ load in SN DA neurons, with prominent Cav1.3-mediated ICa between action potentials and after bursts. The failure of therapeutically relevant isradipine levels to protect SN DA neurons can be explained by weaker state-dependent inhibition of SN DA LTCCs compared with aSM Cav1.2.
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