Cadmium versus Lanthanum Effects on Spontaneous Electrical Activity and Expression of Connexin Isoforms Cx26, Cx36, and Cx45 in the Human Fetal Cortex.

Electrical activity is important for brain development. In brain slices, human subplate neurons exhibit spontaneous electrical activity that is highly sensitive to lanthanum. Based on the results of pharmacological experiments in human fetal tissue, we hypothesized that hemichannel-forming connexin (Cx) isoforms 26, 36, and 45 would be expressed on neurons in the subplate (SP) zone. RNA sequencing of dissected human cortical mantles at ages of 17-23 gestational weeks revealed that Cx45 has the highest expression, followed by Cx36 and Cx26. The levels of Cx and pannexin expression between male and female fetal cortices were not significantly different. Immunohistochemical analysis detected Cx45- and Cx26-expressing neurons in the upper segment of the SP zone. Cx45 was present on the cell bodies of human SP neurons, while Cx26 was found on both cell bodies and dendrites. Cx45, Cx36, and Cx26 were strongly expressed in the cortical plate, where newborn migrating neurons line up to form cortical layers. New information about the expression of 3 "neuronal" Cx isoforms in each cortical layer/zone (e.g., SP, cortical plate) and pharmacological data with cadmium and lanthanum may improve our understanding of the cellular mechanisms underlying neuronal development in human fetuses and potential vulnerabilities.

Mechanisms of Spontaneous Electrical Activity in the Developing Cerebral Cortex-Mouse Subplate Zone.

Subplate (SP) neurons exhibit spontaneous plateau depolarizations mediated by connexin hemichannels. Postnatal (P1-P6) mice show identical voltage pattern and drug-sensitivity as observed in slices from human fetal cortex; indicating that the mouse is a useful model for studying the cellular physiology of the developing neocortex. In mouse SP neurons, spontaneous plateau depolarizations were insensitive to blockers of: synaptic transmission (glutamatergic, GABAergic, or glycinergic), pannexins (probenecid), or calcium channels (mibefradil, verapamil, diltiazem); while highly sensitive to blockers of gap junctions (octanol), hemichannels (La3+, lindane, Gd3+), or glial metabolism (DLFC). Application of La3+ (100 µM) does not exert its effect on electrical activity by blocking calcium channels. Intracellular application of Gd3+ determined that Gd3+-sensitive pores (putative connexin hemichannels) reside on the membrane of SP neurons. Immunostaining of cortical sections (P1-P6) detected connexins 26, and 45 in neurons, but not connexins 32 and 36. Vimentin-positive glial cells were detected in the SP zone suggesting a potential physiological interaction between SP neurons and radial glia. SP spontaneous activity was reduced by blocking glial metabolism with DFLC or by blocking purinergic receptors by PPADS. Connexin hemichannels and ATP release from vimentin-positive glial cells may underlie spontaneous plateau depolarizations in the developing mammalian cortex.

Domain-selective BET inhibition attenuates transcriptional and behavioral responses to cocaine.

Epigenetic pharmacotherapies have emerged as a promising treatment option for substance use disorder (SUD) due to their ability to reverse maladaptive transcriptional and behavioral responses to drugs of abuse. In particular, inhibitors of bromodomain and extra terminal domain (BET) reader proteins have been shown to reduce cocaine- and opioid-seeking behaviors in rodents. However, only pan-BET inhibitors, small molecules that bind to both bromodomains (BD1 and BD2) with all BET proteins, have been investigated in animal models of SUD. Given the potential side effects associated with pan-BET inhibitors, safer and more selective strategies are needed to advance BET therapeutics as a potential treatment for SUD. Here, we show that RVX-208, a clinically tested, BD2-selective BET inhibitor, dose-dependently reduced cocaine conditioned place preference in male and female mice, similar to the pan-BET inhibitor JQ1. In other behavioral experiments, RVX-208 treatment did not alter distance traveled, anxiety-like behavior, or novel object recognition memory. At the transcriptional level, RVX-208 attenuated the expression of multiple cocaine-induced genes in the nucleus accumbens in a sex-dependent manner. RVX-208 produced a distinct transcriptional response in stimulated primary neurons compared to JQ1 but had little effect on gene expression in non-stimulated neurons. Together, these data indicate that targeting domain-specific BET mechanisms may be an effective and safer strategy to reduce cocaine-induced neurobehavioral adaptations.

BET bromodomains as novel epigenetic targets for brain health and disease.

Epigenetic pharmacotherapy for CNS-related diseases is a burgeoning area of research. In particular, members of the bromodomain and extra-terminal domain (BET) family of proteins have emerged as intriguing therapeutic targets due to their putative involvement in an array of brain diseases. With their ability to bind to acetylated histones and act as a scaffold for chromatin modifying complexes, BET proteins were originally thought of as passive epigenetic 'reader' proteins. However, new research depicts a more complex reality where BET proteins act as key nodes in lineage-specific and signal-dependent transcriptional mechanisms to influence disease-relevant functions. Amid a recent wave of drug development efforts from basic scientists and pharmaceutical companies, BET inhibitors are currently being studied in several CNS-related disease models, but safety and tolerability remain a concern. Here we review the progress in understanding the neurobiological mechanisms of BET proteins and the therapeutic potential of targeting BET proteins for brain health and disease.

Contribution of extrasynaptic N-methyl-D-aspartate and adenosine A1 receptors in the generation of dendritic glutamate-mediated plateau potentials.

Thin basal dendrites can strongly influence neuronal output via generation of dendritic spikes. It was recently postulated that glial processes actively support dendritic spikes by either ceasing glutamate uptake or by actively releasing glutamate and adenosine triphosphate (ATP). We used calcium imaging to study the role of NR2C/D-containing N-methyl-d-aspartate (NMDA) receptors and adenosine A1 receptors in the generation of dendritic NMDA spikes and plateau potentials in basal dendrites of layer 5 pyramidal neurons in the mouse prefrontal cortex. We found that NR2C/D glutamate receptor subunits contribute to the amplitude of synaptically evoked NMDA spikes. Dendritic calcium signals associated with glutamate-evoked dendritic plateau potentials were significantly shortened upon application of the NR2C/D receptor antagonist PPDA, suggesting that NR2C/D receptors prolong the duration of calcium influx during dendritic spiking. In contrast to NR2C/D receptors, adenosine A1 receptors act to abbreviate dendritic and somatic signals via the activation of dendritic K(+) current. This current is characterized as a slow-activating outward-rectifying voltage- and adenosine-gated current, insensitive to 4-aminopyridine but sensitive to TEA. Our data support the hypothesis that the release of glutamate and ATP from neurons or glia contribute to initiation, maintenance and termination of local dendritic glutamate-mediated regenerative potentials.

Branch specific and spike-order specific action potential invasion in basal, oblique, and apical dendrites of cortical pyramidal neurons.

In neocortical pyramidal neurons, action potentials (APs) propagate from the axon into the dendritic tree to influence distal synapses. Traditionally, AP backpropagation was studied in the thick apical trunk. Here, we used the principles of optical imaging developed by Cohen to investigate AP invasion into thin dendritic branches (basal, oblique, and tuft) of prefrontal cortical L5 pyramidal neurons. Multisite optical recordings from neighboring dendrites revealed a clear dichotomy between two seemingly equal dendritic branches belonging to the same cell ("sister branches"). We documented the variable efficacy of AP invasion in basal and oblique branches by revealing their AP voltage waveforms. Using fast multisite calcium imaging, we found that trains of APs are filtered differently between two apical tuft branches. Although one dendritic branch passes all spikes in an AP train, another branch belonging to the same neuron, same cortical layer, and same path distance from the cell body, experiences only one spike. Our data indicate that the vast differences in dendritic voltage and calcium transients, detected in dendrites of pyramidal neurons, arise from a nonuniform distribution of A-type [Formula: see text] conductance, an aggregate number of branch points in the path of the AP propagation and minute differences in dendritic diameter.

Spiny neurons of amygdala, striatum, and cortex use dendritic plateau potentials to detect network UP states.

SPINY NEURONS OF AMYGDALA, STRIATUM, AND CEREBRAL CORTEX SHARE FOUR INTERESTING FEATURES: (1) they are the most abundant cell type within their respective brain area, (2) covered by thousands of thorny protrusions (dendritic spines), (3) possess high levels of dendritic NMDA conductances, and (4) experience sustained somatic depolarizations in vivo and in vitro (UP states). In all spiny neurons of the forebrain, adequate glutamatergic inputs generate dendritic plateau potentials ("dendritic UP states") characterized by (i) fast rise, (ii) plateau phase lasting several hundred milliseconds, and (iii) abrupt decline at the end of the plateau phase. The dendritic plateau potential propagates toward the cell body decrementally to induce a long-lasting (longer than 100 ms, most often 200-800 ms) steady depolarization (∼20 mV amplitude), which resembles a neuronal UP state. Based on voltage-sensitive dye imaging, the plateau depolarization in the soma is precisely time-locked to the regenerative plateau potential taking place in the dendrite. The somatic plateau rises after the onset of the dendritic voltage transient and collapses with the breakdown of the dendritic plateau depolarization. We hypothesize that neuronal UP states in vivo reflect the occurrence of dendritic plateau potentials (dendritic UP states). We propose that the somatic voltage waveform during a neuronal UP state is determined by dendritic plateau potentials. A mammalian spiny neuron uses dendritic plateau potentials to detect and transform coherent network activity into a ubiquitous neuronal UP state. The biophysical properties of dendritic plateau potentials allow neurons to quickly attune to the ongoing network activity, as well as secure the stable amplitudes of successive UP states.

Neurogenic potential of hESC-derived human radial glia is amplified by human fetal cells.

The efficient production of human neocortical neurons from human embryonic stem cells (hESC) is the primary requirement for studying early stages of human cortical development. We used hESC to obtain radial glial cells (hESC-RG) and then compared them with RG cells isolated from human fetal forebrain. Fate of hESC-RG cells critically depends on intrinsic and extrinsic factors. The expression of Pax6 (intrinsic factor) has a similar neurogenic effect on hESC-RG differentiation as reported for human fetal RG cells. Factors from the microenvironment also play a significant role in determining hESC-RG cell fate. In contrast to control cultures, wherein hESC-RG generate mainly astroglia and far fewer neurons, in co-cultures with human fetal forebrain cells, the reverse was found to be true. This neurogenic effect was partly due to soluble factors from human fetal brain cultures. The detected shift towards neurogenesis has significance for developing future efficient neuro-differentiation protocols. Importantly, we established that hESC-RG cells are similar in many respects to human fetal RG cells, including their proliferative capacity, neurogenic potential, and ability to generate various cortical neuronal sub-types. Unlike fetal RG cells, the hESC-RG cells are readily available and can be standardized, features that have considerable practical advantages in research and clinics.