NeuroTrace 500/525 identifies human induced pluripotent stem cell-derived brain pericyte-like cells

In the CNS, pericytes are important for maintaining the blood–brain barrier (BBB) and for controlling blood flow. Recently, several methods were suggested for the differentiation of human pluripotent stem cells (hPSCs) into brain mural cells, specifically pericytes or vascular smooth muscle cells (vSMCs). Unfortunately, identifying the pericytes from among such hPSC-derived mural cells has been challenging. This is due both to the lack of pericyte-specific markers and to the loss of defining anatomical information inherent to culture conditions. We therefore asked whether NeuroTrace 500/525, a newly developed dye that shows cell-specific uptake into pericytes in the mouse brain, can help identify human induced pluripotent stem cell (hiPSC)-derived brain pericyte-like cells. First, we found that NeuroTrace 500/525 specifically stains primary cultured human brain pericytes, confirming its specificity in vitro. Second, we found that NeuroTrace 500/525 specifically labels hiPSC-derived pericyte-like cells, but not endothelial cells or vSMCs derived from the same hiPSCs. Last, we found that neuroectoderm-derived vSMCs, which have pericyte-like features, also take up NeuroTrace 500/525. These data indicate NeuroTrace 500/525 is useful for identifying pericyte-like cells among hiPSC-derived brain mural cells.

Progress on the roles of MEF2C in neuropsychiatric diseases

Myocyte Enhancer Factor 2 C (MEF2C), one of the transcription factors of the MADS-BOX family, is involved in embryonic brain development, neuronal formation and differentiation, as well as in the growth and pruning of axons and dendrites. MEF2C is also involved in the development of various neuropsychiatric disorders, such as autism spectrum disorders (ASD), epilepsy, schizophrenia and Alzheimer’s disease (AD). Here, we review the relationship between MEF2C and neuropsychiatric disorders, and provide further insights into the mechanism of these diseases.

Relief of neuropathic pain by cell-specific manipulation of nucleus accumbens dopamine D1- and D2-receptor-expressing neurons

Emerging evidence suggests that the mesolimbic dopaminergic network plays a role in the modulation of pain. As chronic pain conditions are associated with hypodopaminergic tone in the nucleus accumbens (NAc), we evaluated the effects of increasing signaling at dopamine D1/D2-expressing neurons in the NAc neurons in a model of neuropathic pain induced by partial ligation of sciatic nerve. Bilateral microinjection of either the selective D1-receptor (Gs-coupled) agonist Chloro-APB or the selective D2-receptor (Gi-coupled) agonist quinpirole into the NAc partially reversed nerve injury-induced thermal allodynia. Either optical stimulation of D1-receptor-expressing neurons or optical suppression of D2-receptor-expressing neurons in both the inner and outer substructures of the NAc also transiently, but significantly, restored nerve injury-induced allodynia. Under neuropathic pain-like condition, specific facilitation of terminals of D1-receptor-expressing NAc neurons projecting to the VTA revealed a feedforward-like antinociceptive circuit. Additionally, functional suppression of cholinergic interneurons that negatively and positively control the activity of D1- and D2-receptor-expressing neurons, respectively, also transiently elicited anti-allodynic effects in nerve injured animals. These findings suggest that comprehensive activation of D1-receptor-expressing neurons and integrated suppression of D2-receptor-expressing neurons in the NAc may lead to a significant relief of neuropathic pain.

Cell-type-specific DNA methylation analysis of the frontal cortices of mutant Polg1 transgenic mice with neuronal accumulation of deleted mitochondrial DNA

Bipolar disorder (BD) is a severe psychiatric disorder characterized by repeated conflicting manic and depressive states. In addition to genetic factors, complex gene–environment interactions, which alter the epigenetic status in the brain, contribute to the etiology and pathophysiology of BD. Here, we performed a promoter-wide DNA methylation analysis of neurons and nonneurons derived from the frontal cortices of mutant Polg1 transgenic (n = 6) and wild-type mice (n = 6). The mutant mice expressed a proofreading-deficient mitochondrial DNA (mtDNA) polymerase under the neuron-specific CamK2a promoter and showed BD-like behavioral abnormalities, such as activity changes and altered circadian rhythms. We identified a total of 469 differentially methylated regions (DMRs), consisting of 267 neuronal and 202 nonneuronal DMRs. Gene ontology analysis of DMR-associated genes showed that cell cycle-, cell division-, and inhibition of peptide activity-related genes were enriched in neurons, whereas synapse- and GABA-related genes were enriched in nonneurons. Among the DMR-associated genes, Trim2 and Lrpprc showed an inverse relationship between DNA methylation and gene expression status. In addition, we observed that mutant Polg1 transgenic mice shared several features of DNA methylation changes in postmortem brains of patients with BD, such as dominant hypomethylation changes in neurons, which include hypomethylation of the molecular motor gene and altered DNA methylation of synapse-related genes in nonneurons. Taken together, the DMRs identified in this study will contribute to understanding the pathophysiology of BD from an epigenetic perspective.

The emergence of molecular systems neuroscience

Systems neuroscience is focused on how ensemble properties in the brain, such as the activity of neuronal circuits, gives rise to internal brain states and behavior. Many of the studies in this field have traditionally involved electrophysiological recordings and computational approaches that attempt to decode how the brain transforms inputs into functional outputs. More recently, systems neuroscience has received an infusion of approaches and techniques that allow the manipulation (e.g., optogenetics, chemogenetics) and imaging (e.g., two-photon imaging, head mounted fluorescent microscopes) of neurons, neurocircuits, their inputs and outputs. Here, we will review novel approaches that allow the manipulation and imaging of specific molecular mechanisms in specific cells (not just neurons), cell ensembles and brain regions. These molecular approaches, with the specificity and temporal resolution appropriate for systems studies, promise to infuse the field with novel ideas, emphases and directions, and are motivating the emergence of a molecularly oriented systems neuroscience, a new discipline that studies how the spatial and temporal patterns of molecular systems modulate circuits and brain networks, and consequently shape the properties of brain states and behavior.

Cavβ surface charged residues contribute to the regulation of neuronal calcium channels

Voltage-gated calcium channels are essential regulators of brain function where they support depolarization-induced calcium entry into neurons. They consist of a pore-forming subunit (Cavα1) that requires co-assembly with ancillary subunits to ensure proper functioning of the channel. Among these ancillary subunits, the Cavβ plays an essential role in regulating surface expression and gating of the channels. This regulation requires the direct binding of Cavβ onto Cavα1 and is mediated by the alpha interacting domain (AID) within the Cavα1 subunit and the α binding pocket (ABP) within the Cavβ subunit. However, additional interactions between Cavα1 and Cavβ have been proposed. In this study, we analyzed the importance of Cavβ3 surface charged residues in the regulation of Cav2.1 channels. Using alanine-scanning mutagenesis combined with electrophysiological recordings we identified several amino acids within the Cavβ3 subunit that contribute to the gating of the channel. These findings add to the notion that additional contacts besides the main AID/ABP interaction may occur to fine-tune the expression and properties of the channel.

Stress-induced antinociception to noxious heat requires α1A-adrenaline receptors of spinal inhibitory neurons in mice

It is well known that acute exposure to physical stress produces a transient antinociceptive effect (called stress-induced analgesia [SIA]). One proposed mechanism for SIA involves noradrenaline (NA) in the central nervous system. NA has been reported to activate inhibitory neurons in the spinal dorsal horn (SDH), but its in vivo role in SIA remains unknown. In this study, we found that an antinociceptive effect on noxious heat after acute exposure to restraint stress was impaired in mice with a conditional knockout of α1A-adrenaline receptors (α1A-ARs) in inhibitory neurons (Vgat-Cre;Adra1aflox/flox mice). A similar reduction was also observed in mice treated with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, a selective neurotoxin for NAergic neurons in the locus coeruleus (LC). Furthermore, whole-cell patch-clamp recordings using spinal cord slices revealed that NA-induced increase in the frequency of spontaneous inhibitory postsynaptic currents in the substantia gelatinosa neurons was suppressed by silodosin, an α1A-AR antagonist, and by conditional knockout of α1A-ARs in inhibitory neurons. Moreover, under unstressed conditions, the antinociceptive effects of intrathecal NA and phenylephrine on noxious heat were lost in Vgat-Cre;Adra1aflox/flox mice. Our findings suggest that activation of α1A-ARs in SDH inhibitory neurons, presumably via LC-NAergic neurons, is necessary for SIA to noxious heat.

Effect of soluble amyloid precursor protein-alpha on adult hippocampal neurogenesis in a mouse model of Alzheimer’s disease

Soluble amyloid precursor protein-alpha (sAPPα) is a regulator of neuronal and memory mechanisms, while also having neurogenic and neuroprotective effects in the brain. As adult hippocampal neurogenesis is impaired in Alzheimer’s disease, we tested the hypothesis that sAPPα delivery would rescue adult hippocampal neurogenesis in an APP/PS1 mouse model of Alzheimer’s disease. An adeno-associated virus-9 (AAV9) encoding murine sAPPα was injected into the hippocampus of 8-month-old wild-type and APP/PS1 mice, and later two different thymidine analogues (XdU) were systemically injected to label adult-born cells at different time points after viral transduction. The proliferation of adult-born cells, cell survival after eight weeks, and cell differentiation into either neurons or astrocytes was studied. Proliferation was impaired in APP/PS1 mice but was restored to wild-type levels by viral expression of sAPPα. In contrast, sAPPα overexpression failed to rescue the survival of XdU+-labelled cells that was impaired in APP/PS1 mice, although it did cause a significant increase in the area density of astrocytes in the granule cell layer across both genotypes. Finally, viral expression of sAPPα reduced amyloid-beta plaque load in APP/PS1 mice in the dentate gyrus and somatosensory cortex. These data add further evidence that increased levels of sAPPα could be therapeutic for the cognitive decline in AD, in part through restoration of the proliferation of neural progenitor cells in adults.

Secretory carrier-associated membrane protein 2 (SCAMP2) regulates cell surface expression of T-type calcium channels

Low-voltage-activated T-type Ca2+ channels are key regulators of neuronal excitability both in the central and peripheral nervous systems. Therefore, their recruitment at the plasma membrane is critical in determining firing activity patterns of nerve cells. In this study, we report the importance of secretory carrier-associated membrane proteins (SCAMPs) in the trafficking regulation of T-type channels. We identified SCAMP2 as a novel Cav3.2-interacting protein. In addition, we show that co-expression of SCAMP2 in mammalian cells expressing recombinant Cav3.2 channels caused an almost complete drop of the whole cell T-type current, an effect partly reversed by single amino acid mutations within the conserved cytoplasmic E peptide of SCAMP2. SCAMP2-induced downregulation of T-type currents was also observed in cells expressing Cav3.1 and Cav3.3 channel isoforms. Finally, we show that SCAMP2-mediated knockdown of the T-type conductance is caused by the lack of Cav3.2 expression at the cell surface as evidenced by the concomitant loss of intramembrane charge movement without decrease of total Cav3.2 protein level. Taken together, our results indicate that SCAMP2 plays an important role in the trafficking of Cav3.2 channels at the plasma membrane.

The exit of axons and glial membrane from the developing Drosophila retina requires integrins

Coordinated development of neurons and glia is essential for the establishment of neuronal circuits during embryonic development. In the developing Drosophila visual system, photoreceptor (R cell) axons and wrapping glial (WG) membrane extend from the eye disc through the optic stalk into the optic lobe. Extensive studies have identified a number of genes that control the establishment of R-cell axonal projection pattern in the optic lobe. The molecular mechanisms directing the exit of R-cell axons and WG membrane from the eye disc, however, remain unknown. In this study, we show that integrins are required in R cells for the extension of R-cell axons and WG membrane from the eye disc into the optic stalk. Knockdown of integrins in R cells but not WG caused the stalling of both R-cell axons and WG membrane in the eye disc. Interfering with the function of Rhea (i.e. the Drosophila ortholog of vertebrate talin and a key player of integrin-mediated adhesion), caused an identical stalling phenotype. These results support a key role for integrins on R-cell axons in directing R-cell axons and WG membrane to exit the eye disc.