HRAS-Mutant Cardiomyocyte Model of Multifocal Atrial Tachycardia.

BACKGROUND: Germline HRAS gain-of-function pathogenic variants cause Costello syndrome (CS). During early childhood, 50% of patients develop multifocal atrial tachycardia, a treatment-resistant tachyarrhythmia of unknown pathogenesis. This study investigated how overactive HRAS activity triggers arrhythmogenesis in atrial-like cardiomyocytes (ACMs) derived from human-induced pluripotent stem cells bearing CS-associated HRAS variants. METHODS: HRAS Gly12 mutations were introduced into a human-induced pluripotent stem cells-ACM reporter line. Human-induced pluripotent stem cells were generated from patients with CS exhibiting tachyarrhythmia. Calcium transients and action potentials were assessed in induced pluripotent stem cell-derived ACMs. Automated patch clamping assessed funny currents. HCN inhibitors targeted pacemaker-like activity in mutant ACMs. Transcriptomic data were analyzed via differential gene expression and gene ontology. Immunoblotting evaluated protein expression associated with calcium handling and pacemaker-nodal expression. RESULTS: ACMs harboring HRAS variants displayed higher beating rates compared with healthy controls. The hyperpolarization activated cyclic nucleotide gated potassium channel inhibitor ivabradine and the Nav1.5 blocker flecainide significantly decreased beating rates in mutant ACMs, whereas voltage-gated calcium channel 1.2 blocker verapamil attenuated their irregularity. Electrophysiological assessment revealed an increased number of pacemaker-like cells with elevated funny current densities among mutant ACMs. Mutant ACMs demonstrated elevated gene expression (ie, ISL1, TBX3, TBX18) related to intracellular calcium homeostasis, heart rate, RAS signaling, and induction of pacemaker-nodal-like transcriptional programming. Immunoblotting confirmed increased protein levels for genes of interest and suppressed MAPK (mitogen-activated protein kinase) activity in mutant ACMs. CONCLUSIONS: CS-associated gain-of-function HRASG12 mutations in induced pluripotent stem cells-derived ACMs trigger transcriptional changes associated with enhanced automaticity and arrhythmic activity consistent with multifocal atrial tachycardia. This is the first human-induced pluripotent stem cell model establishing the mechanistic basis for multifocal atrial tachycardia in CS.

Astrocytic TDP-43 dysregulation impairs memory by modulating antiviral pathways and interferon-inducible chemokines.

Transactivating response region DNA binding protein 43 (TDP-43) pathology is prevalent in dementia, but the cell type-specific effects of TDP-43 pathology are not clear, and therapeutic strategies to alleviate TDP-43-linked cognitive decline are lacking. We found that patients with Alzheimer's disease or frontotemporal dementia have aberrant TDP-43 accumulation in hippocampal astrocytes. In mouse models, induction of widespread or hippocampus-targeted accumulation in astrocytic TDP-43 caused progressive memory loss and localized changes in antiviral gene expression. These changes were cell-autonomous and correlated with impaired astrocytic defense against infectious viruses. Among the changes, astrocytes had elevated levels of interferon-inducible chemokines, and neurons had elevated levels of the corresponding chemokine receptor CXCR3 in presynaptic terminals. CXCR3 stimulation altered presynaptic function and promoted neuronal hyperexcitability, akin to the effects of astrocytic TDP-43 dysregulation, and blockade of CXCR3 reduced this activity. Ablation of CXCR3 also prevented TDP-43-linked memory loss. Thus, astrocytic TDP-43 dysfunction contributes to cognitive impairment through aberrant chemokine-mediated astrocytic-neuronal interactions.

Bidirectional control of infant rat social behavior via dopaminergic innervation of the basolateral amygdala.

Social interaction deficits seen in psychiatric disorders emerge in early-life and are most closely linked to aberrant neural circuit function. Due to technical limitations, we have limited understanding of how typical versus pathological social behavior circuits develop. Using a suite of invasive procedures in awake, behaving infant rats, including optogenetics, microdialysis, and microinfusions, we dissected the circuits controlling the gradual increase in social behavior deficits following two complementary procedures-naturalistic harsh maternal care and repeated shock alone or with an anesthetized mother. Whether the mother was the source of the adversity (naturalistic Scarcity-Adversity) or merely present during the adversity (repeated shock with mom), both conditions elevated basolateral amygdala (BLA) dopamine, which was necessary and sufficient in initiating social behavior pathology. This did not occur when pups experienced adversity alone. These data highlight the unique impact of social adversity as causal in producing mesolimbic dopamine circuit dysfunction and aberrant social behavior.

Publisher Correction: Innate and plastic mechanisms for maternal behaviour in auditory cortex.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Innate and plastic mechanisms for maternal behaviour in auditory cortex.

Infant cries evoke powerful responses in parents1-4. Whether parental animals are intrinsically sensitive to neonatal vocalizations, or instead learn about vocal cues for parenting responses is unclear. In mice, pup-naive virgin females do not recognize the meaning of pup distress calls, but retrieve isolated pups to the nest after having been co-housed with a mother and litter5-9. Distress calls are variable, and require co-caring virgin mice to generalize across calls for reliable retrieval10,11. Here we show that the onset of maternal behaviour in mice results from interactions between intrinsic mechanisms and experience-dependent plasticity in the auditory cortex. In maternal females, calls with inter-syllable intervals (ISIs) from 75 to 375 milliseconds elicited pup retrieval, and cortical responses were generalized across these ISIs. By contrast, naive virgins were neuronally and behaviourally sensitized to the most common ('prototypical') ISIs. Inhibitory and excitatory neural responses were initially mismatched in the cortex of naive mice, with untuned inhibition and overly narrow excitation. During co-housing experiments, excitatory responses broadened to represent a wider range of ISIs, whereas inhibitory tuning sharpened to form a perceptual boundary. We presented synthetic calls during co-housing and observed that neurobehavioural responses adjusted to match these statistics, a process that required cortical activity and the hypothalamic oxytocin system. Neuroplastic mechanisms therefore build on an intrinsic sensitivity in the mouse auditory cortex, and enable rapid plasticity for reliable parenting behaviour.

Broadband Entrainment of Striatal Low-Threshold Spike Interneurons.

Striatal interneurons and spiny projection (SP) neurons are differentially tuned to spectral components of their input. Previous studies showed that spike responses of somatostatin/NPY-expressing low threshold spike (LTS) interneurons have broad frequency tuning, setting these cells apart from other striatal GABAergic interneurons and SP neurons. We investigated the mechanism of LTS interneuron spiking resonance and its relationship to non-spiking membrane impedance resonance, finding that abolition of impedance resonance did not alter spiking resonance. Because LTS interneurons are pacemakers whose rhythmic firing is perturbed by synaptic input, we tested the hypothesis that their spiking resonance arises from their phase resetting properties. Phase resetting curves (PRCs) were measured in LTS interneurons and SP neurons and used to make phase-oscillator models of both cell types. The models reproduced the broad tuning of LTS interneurons, and the differences from SP neurons. The spectral components of the PRC predicted each cell's sensitivity to corresponding input frequencies. LTS interneuron PRCs contain larger high-frequency components than SP neuron PRCs, providing enhanced responses to input frequencies above the cells' average firing rates. Thus, LTS cells can be entrained by input oscillations to which SP neurons are less responsive. These findings suggest that feedforward inhibition by LTS interneurons may regulate SP neurons' entrainment by oscillatory afferents.

Posterior amygdala regulates sexual and aggressive behaviors in male mice.

Sexual and aggressive behaviors are fundamental to animal survival and reproduction. The medial preoptic nucleus (MPN) and ventrolateral part of the ventromedial hypothalamus (VMHvl) are essential regions for male sexual and aggressive behaviors, respectively. While key inhibitory inputs to the VMHvl and MPN have been identified, the extrahypothalamic excitatory inputs essential for social behaviors remain elusive. Here we identify estrogen receptor alpha (Esr1)-expressing cells in the posterior amygdala (PA) as a main source of excitatory inputs to the hypothalamus and key mediators for mating and fighting in male mice. We find two largely distinct PA subpopulations that differ in connectivity, gene expression, in vivo responses and social behavior relevance. MPN-projecting PAEsr1+ cells are activated during mating and are necessary and sufficient for male sexual behaviors, while VMHvl-projecting PAEsr1+ cells are excited during intermale aggression and promote attacks. These findings place the PA as a key node in both male aggression and reproduction circuits.

A Hypothalamic Midbrain Pathway Essential for Driving Maternal Behaviors.

Maternal behaviors are essential for the survival of the young. Previous studies implicated the medial preoptic area (MPOA) as an important region for maternal behaviors, but details of the maternal circuit remain incompletely understood. Here we identify estrogen receptor alpha (Esr1)-expressing cells in the MPOA as key mediators of pup approach and retrieval. Reversible inactivation of MPOAEsr1+ cells impairs those behaviors, whereas optogenetic activation induces immediate pup retrieval. In vivo recordings demonstrate preferential activation of MPOAEsr1+ cells during maternal behaviors and changes in MPOA cell responses across reproductive states. Furthermore, channelrhodopsin-assisted circuit mapping reveals a strong inhibitory projection from MPOAEsr1+ cells to ventral tegmental area (VTA) non-dopaminergic cells. Pathway-specific manipulations reveal that this projection is essential for driving pup approach and retrieval and that VTA dopaminergic cells are reliably activated during those behaviors. Altogether, this study provides new insight into the neural circuit that generates maternal behaviors.

Cell-type-specific resonances shape the responses of striatal neurons to synaptic input.

Neurons respond to synaptic inputs in cell-type-specific ways. Each neuron type may thus respond uniquely to shared patterns of synaptic input. We applied statistically identical barrages of artificial synaptic inputs to four striatal cell types to assess differences in their responses to a realistic input pattern. Each interneuron type fired in phase with a specific input-frequency component. The fast-spiking interneuron fired in relation to the gamma-band (and higher) frequencies, the low-threshold spike interneuron to the beta-band frequencies, and the cholinergic neurons to the delta-band frequencies. Low-threshold spiking and cholinergic interneurons showed input impedance resonances at frequencies matching their spiking resonances. Fast-spiking interneurons showed resonance of input impedance but at lower than gamma frequencies. The spiny projection neuron's frequency preference did not have a fixed frequency but instead tracked its own firing rate. Spiny cells showed no input impedance resonance. Striatal interneurons are each tuned to a specific frequency band corresponding to the major frequency components of local field potentials. Their influence in the circuit may fluctuate along with the contribution of that frequency band to the input. In contrast, spiny neurons may tune to any of the frequency bands by a change in firing rate.

Genetic analysis of the neurosteroid deoxycorticosterone and its relation to alcohol phenotypes: identification of QTLs and downstream gene regulation.

BACKGROUND: Deoxycorticosterone (DOC) is an endogenous neurosteroid found in brain and serum, precursor of the GABAergic neuroactive steroid (3α,5α)-3,21-dihydroxypregnan-20-one (tetrahydrodeoxycorticosterone, THDOC) and the glucocorticoid corticosterone. These steroids are elevated following stress or ethanol administration, contribute to ethanol sensitivity, and their elevation is blunted in ethanol dependence. METHODOLOGY/PRINCIPAL FINDINGS: To systematically define the genetic basis, regulation, and behavioral significance of DOC levels in plasma and cerebral cortex we examined such levels across 47 young adult males from C57BL/6J (B6)×DBA/2J (D2) (BXD) mouse strains for quantitative trait loci (QTL) and bioinformatics analyses of behavior and gene regulation. Mice were injected with saline or 0.075 mg/kg dexamethasone sodium salt at 8:00 am and were sacrificed 6 hours later. DOC levels were measured by radioimmunoassay. Basal cerebral cortical DOC levels ranged between 1.4 and 12.2 ng/g (8.7-fold variation, p<0.0001) with a heritability of ∼0.37. Basal plasma DOC levels ranged between 2.8 and 12.1 ng/ml (4.3-fold variation, p<0.0001) with heritability of ∼0.32. QTLs for basal DOC levels were identified on chromosomes 4 (cerebral cortex) and 14 (plasma). Dexamethasone-induced changes in DOC levels showed a 4.4-fold variation in cerebral cortex and a 4.1-fold variation in plasma, but no QTLs were identified. DOC levels across BXD strains were further shown to be co-regulated with networks of genes linked to neuronal, immune, and endocrine function. DOC levels and its responses to dexamethasone were associated with several behavioral measures of ethanol sensitivity previously determined across the BXD strains by multiple laboratories. CONCLUSIONS/SIGNIFICANCE: Both basal and dexamethasone-suppressed DOC levels are positively correlated with ethanol sensitivity suggesting that the neurosteroid DOC may be a putative biomarker of alcohol phenotypes. DOC levels were also strongly correlated with networks of genes associated with neuronal function, innate immune pathways, and steroid metabolism, likely linked to behavioral phenotypes.

Differential effects of ethanol on serum GABAergic 3alpha,5alpha/3alpha,5beta neuroactive steroids in mice, rats, cynomolgus monkeys, and humans.

BACKGROUND: Acute ethanol administration increases plasma and brain levels of progesterone and deoxycorticosterone-derived neuroactive steroids (3alpha,5alpha)-3-hydroxypregnan-20-one (3alpha,5alpha-THP) and (3alpha,5alpha)-3,21-dihydroxypregnan-20-one (3alpha,5alpha-THDOC) in rats. However, little is known about ethanol effects on GABAergic neuroactive steroids in mice, nonhuman primates, or humans. We investigated the effects of ethanol on plasma levels of 3alpha,5alpha- and 3alpha,5beta-reduced GABAergic neuroactive steroids derived from progesterone, deoxycorticosterone, dehydroepiandrosterone, and testosterone using gas chromatography-mass spectrometry. METHODS: Serum levels of GABAergic neuroactive steroids and pregnenolone were measured in male rats, C57BL/6J and DBA/2J mice, cynomolgus monkeys, and humans following ethanol administration. Rats and mice were injected with ethanol (0.8 to 2.0 g/kg), cynomolgus monkeys received ethanol (1.5 g/kg) intragastrically, and healthy men consumed a beverage containing 0.8 g/kg ethanol. Steroids were measured after 60 minutes in all species and also after 120 minutes in monkeys and humans. RESULTS: Ethanol administration to rats increased levels of 3alpha,5alpha-THP, 3alpha,5alpha-THDOC, and pregnenolone at the doses of 1.5 g/kg (+228, +134, and +860%, respectively, p < 0.001) and 2.0 g/kg (+399, +174, and +1125%, respectively, p < 0.001), but not at the dose of 0.8 g/kg. Ethanol did not alter levels of the other neuroactive steroids. In contrast, C57BL/6J mice exhibited a 27% decrease in serum 3alpha,5alpha-THP levels (p < 0.01), while DBA/2J mice showed no significant effect of ethanol, although both mouse strains exhibited substantial increases in precursor steroids. Ethanol did not alter any of the neuroactive steroids in cynomolgus monkeys at doses comparable to those studied in rats. Finally, no effect of ethanol (0.8 g/kg) was observed in men. CONCLUSIONS: These studies show clear species differences among rats, mice, and cynomolgus monkeys in the effects of ethanol administration on circulating neuroactive steroids. Rats are unique in their pronounced elevation of GABAergic neuroactive steroids, while this effect was not observed in mice or cynomolgus monkeys at comparable ethanol doses.