Sleep Fragmentation, Electroencephalographic Slowing, and Circadian Disarray in a Mouse Model for Intensive Care Unit Delirium.

BACKGROUND: We aimed to further validate our previously published animal model for delirium by testing the hypothesis that in aged mice, Anesthesia, Surgery and simulated ICU conditions (ASI) induce sleep fragmentation, electroencephalographic (EEG) slowing, and circadian disarray consistent with intensive care unit (ICU) patients with delirium. METHODS: A total of 41 mice were used. Mice were implanted with EEG electrodes and randomized to ASI or control groups. ASI mice received laparotomy, anesthesia, and simulated ICU conditions. Controls did not receive ASI. Sleep was recorded at the end of ICU conditions, and hippocampal tissue was collected on EEG recording. Arousals, EEG dynamics, and circadian gene expression were compared with t tests. Two-way repeated measures analysis of variance (RM ANOVA) was used to assess sleep according to light. RESULTS: ASI mice experienced frequent arousals (36.6 ± 3.2 vs 26.5 ± 3.4; P = .044; 95% confidence interval [CI], 0.29-19.79; difference in mean ± SEM, 10.04 ± 4.62) and EEG slowing (frontal theta ratio, 0.223 ± 0.010 vs 0.272 ± 0.019; P = .026; 95% CI, -0.091 to -0.007; difference in mean ± SEM, -0.05 ± 0.02) relative to controls. In ASI mice with low theta ratio, EEG slowing was associated with a higher percentage of quiet wakefulness (38.2 ± 3.6 vs 13.4 ± 3.8; P = .0002; 95% CI, -35.87 to -13.84; difference in mean ± SEM, -24.86 ± 5.19). ASI mice slept longer during the dark phases of the circadian cycle (nonrapid eye movement [NREM], dark phase 1 [D1]: 138.9 ± 8.1 minutes vs 79.6 ± 9.6 minutes, P = .0003, 95% CI, -95.87 to -22.69, predicted mean difference ± SE: -59.28 ± 13.89; NREM, dark phase 2 (D2): 159.3 ± 7.3 minutes vs 112.6 ± 15.5 minutes, P = .006, 95% CI, -83.25 to -10.07, mean difference ± SE, -46.66 ± 13.89; rapid eye movement (REM), D1: 20.5 ± 2.1 minutes vs 5.8 ± 0.8 minutes, P = .001, 95% CI, -24.60 to -4.71, mean difference ± SE, -14. 65 ± 3.77; REM, D2: 21.0 ± 2.2 minutes vs 10.3 ± 1.4 minutes, P = .029, 95% CI, -20.64 to -0.76, mean difference ± SE, -10.70 ± 3.77). The expression of essential circadian genes was also lower in ASI mice (basic helix-loop-helix ARNT like [BMAL1] : -1.3 fold change; circadian locomotor output cycles protein kaput [CLOCK] : -1.2). CONCLUSIONS: ASI mice experienced EEG and circadian changes mimicking those of delirious ICU patients. These findings support further exploration of this mouse approach to characterize the neurobiology of delirium.

Intrinsic Adrenal TWIK-Related Acid-Sensitive TASK Channel Dysfunction Produces Spontaneous Calcium Oscillations Sufficient to Drive AngII (Angiotensin II)-Unresponsive Hyperaldosteronism.

BACKGROUND: Ion channel mutations in calcium regulating genes strongly associate with AngII (angiotensin II)-independent aldosterone production. Here, we used an established mouse model of in vivo aldosterone autonomy, Cyp11b2-driven deletion of TWIK-related acid-sensitive potassium channels (TASK-1 and TASK-3, termed zona glomerulosa [zG]-TASK-loss-of-function), and selective pharmacological TASK channel inhibition to determine whether channel dysfunction in native, electrically excitable zG cell rosette-assemblies: (1) produces spontaneous calcium oscillatory activity and (2) is sufficient to drive substantial aldosterone autonomy. METHODS: We imaged calcium activity in adrenal slices expressing a zG-specific calcium reporter (GCaMP3), an in vitro experimental approach that preserves the native rosette assembly and removes potentially confounding extra-adrenal contributions. In parallel experiments, we measured acute aldosterone production from adrenal slice cultures. RESULTS: Absent from untreated WT slices, we find that either adrenal-specific genetic deletion or acute pharmacological TASK channel inhibition produces spontaneous oscillatory bursting behavior and steroidogenic activity (2.4-fold) that are robust, sustained, and equivalent to activities evoked by 3 nM AngII in WT slices. Moreover, spontaneous activity in zG-TASK-loss-of-function slices and inhibitor-evoked activity in WT slices are unresponsive to AngII regulation over a wide range of concentrations (50 pM to 3 µM). CONCLUSIONS: We provide proof of principle that spontaneous activity of zG cells within classic rosette assemblies evoked solely by a change in an intrinsic, dominant resting-state conductance can be a significant source of AngII-independent aldosterone production from native tissue.

AMPK-mediated potentiation of GABAergic signalling drives hypoglycaemia-provoked spike-wave seizures.

Metabolism regulates neuronal activity and modulates the occurrence of epileptic seizures. Here, using two rodent models of absence epilepsy, we show that hypoglycaemia increases the occurrence of spike-wave seizures. We then show that selectively disrupting glycolysis in the thalamus, a structure implicated in absence epilepsy, is sufficient to increase spike-wave seizures. We propose that activation of thalamic AMP-activated protein kinase, a sensor of cellular energetic stress and potentiator of metabotropic GABAB-receptor function, is a significant driver of hypoglycaemia-induced spike-wave seizures. We show that AMP-activated protein kinase augments postsynaptic GABAB-receptor-mediated currents in thalamocortical neurons and strengthens epileptiform network activity evoked in thalamic brain slices. Selective thalamic AMP-activated protein kinase activation also increases spike-wave seizures. Finally, systemic administration of metformin, an AMP-activated protein kinase agonist and common diabetes treatment, profoundly increased spike-wave seizures. These results advance the decades-old observation that glucose metabolism regulates thalamocortical circuit excitability by demonstrating that AMP-activated protein kinase and GABAB-receptor cooperativity is sufficient to provoke spike-wave seizures.

Respiratory alkalosis provokes spike-wave discharges in seizure-prone rats.

Hyperventilation reliably provokes seizures in patients diagnosed with absence epilepsy. Despite this predictable patient response, the mechanisms that enable hyperventilation to powerfully activate absence seizure-generating circuits remain entirely unknown. By utilizing gas exchange manipulations and optogenetics in the WAG/Rij rat, an established rodent model of absence epilepsy, we demonstrate that absence seizures are highly sensitive to arterial carbon dioxide, suggesting that seizure-generating circuits are sensitive to pH. Moreover, hyperventilation consistently activated neurons within the intralaminar nuclei of the thalamus, a structure implicated in seizure generation. We show that intralaminar thalamus also contains pH-sensitive neurons. Collectively, these observations suggest that hyperventilation activates pH-sensitive neurons of the intralaminar nuclei to provoke absence seizures.

Mice Harboring a Non-Functional CILK1/ICK Allele Fail to Model the Epileptic Phenotype in Patients Carrying Variant CILK1/ICK.

CILK1 (ciliogenesis associated kinase 1)/ICK (intestinal cell kinase) is a highly conserved protein kinase that regulates primary cilia structure and function. CILK1 mutations cause a wide spectrum of human diseases collectively called ciliopathies. While several CILK1 heterozygous variants have been recently linked to juvenile myoclonic epilepsy (JME), it remains unclear whether these mutations cause seizures. Herein, we investigated whether mice harboring either a heterozygous null Cilk1 (Cilk1+/-) mutation or a heterozygous loss-of-function Cilk1 mutation (Cilk1R272Q/+) have epilepsy. We first evaluated the spontaneous seizure phenotype of Cilk1+/- and Cilk1R272Q/+ mice relative to wildtype littermates. We observed no electrographic differences among the three mouse genotypes during prolonged recordings. We also evaluated electrographic and behavioral responses of mice recovering from isoflurane anesthesia, an approach recently used to measure seizure-like activity. Again, we observed no electrographic or behavioral differences in control versus Cilk1+/- and Cilk1R272Q/+ mice upon isoflurane recovery. These results indicate that mice bearing a non-functional copy of Cilk1 fail to produce electrographic patterns resembling those of JME patients with a variant CILK1 copy. Our findings argue against CILK1 haploinsufficiency being the mechanism that links CILK1 variants to JME.

Nonlinearities between inhibition and T-type calcium channel activity bidirectionally regulate thalamic oscillations.

Absence seizures result from 3 to 5 Hz generalized thalamocortical oscillations that depend on highly regulated inhibitory neurotransmission in the thalamus. Efficient reuptake of the inhibitory neurotransmitter GABA is essential, and reuptake failure worsens human seizures. Here, we show that blocking GABA transporters (GATs) in acute rat brain slices containing key parts of the thalamocortical seizure network modulates epileptiform activity. As expected, we found that blocking either GAT1 or GAT3 prolonged oscillations. However, blocking both GATs unexpectedly suppressed oscillations. Integrating experimental observations into single-neuron and network-level computational models shows how a non-linear dependence of T-type calcium channel gating on GABAB receptor activity regulates network oscillations. Receptor activity that is either too brief or too protracted fails to sufficiently open T-type channels necessary for sustaining oscillations. Only within a narrow range does prolonging GABAB receptor activity promote channel opening and intensify oscillations. These results have implications for therapeutics that modulate inhibition kinetics.

Angiotensin II induces coordinated calcium bursts in aldosterone-producing adrenal rosettes.

Aldosterone-producing zona glomerulosa (zG) cells of the adrenal gland arrange in distinct multi-cellular rosettes that provide a structural framework for adrenal cortex morphogenesis and plasticity. Whether this cyto-architecture also plays functional roles in signaling remains unexplored. To determine if structure informs function, we generated mice with zG-specific expression of GCaMP3 and imaged zG cells within their native rosette structure. Here we demonstrate that within the rosette, angiotensin II evokes periodic Cav3-dependent calcium events that form bursts that are stereotypic in form. Our data reveal a critical role for angiotensin II in regulating burst occurrence, and a multifunctional role for the rosette structure in activity-prolongation and coordination. Combined our data define the calcium burst as the fundamental unit of zG layer activity evoked by angiotensin II and highlight a novel role for the rosette as a facilitator of cell communication.

VIPergic neurons of the infralimbic and prelimbic cortices control palatable food intake through separate cognitive pathways.

The prefrontal cortex controls food reward seeking and ingestion, playing important roles in directing attention, regulating motivation towards reward pursuit, and the assignment of reward salience and value. The cell types that mediate these behavioral functions, however, are not well described. We report here that optogenetic activation of vasoactive peptide expressing (VIP) interneurons in both the infralimbic (IL) and prelimbic (PL) divisions of the medial prefrontal cortex in mice is sufficient to reduce acute, binge-like intake of high calorie palatable food in the absence of any effect on low calorie rodent chow intake in the sated animal. In addition, we discovered that the behavioral mechanisms associated with these changes in feeding differed between animals that underwent either IL or PL VIPergic stimulation. While IL VIP neurons showed the ability to reduce palatable food intake, this effect was dependent upon the novelty and relative value of the food source. In addition, IL VIP neuron activation significantly reduced novel object- and novel social investigative behavior. Activation of PL VIP neurons, however, produced a reduction in high calorie palatable food intake that was independent of food novelty. Neither IL nor PL VIP excitation changed motivation to obtain food reward. Our data show how neurochemically-defined populations of cortical interneurons can regulate specific aspects of food reward-driven behavior, resulting in a selective reduction in intake of highly valued food.

Disruption of Rapid Eye Movement Sleep Homeostasis in Adolescent Rats after Neonatal Anesthesia.

BACKGROUND: Previous studies suggest that rapid eye movement sleep rebound and disruption of rapid eye movement sleep architecture occur during the first 24 h after general anesthesia with volatile anesthetics in adult rats. However, it is unknown whether rapid eye movement sleep alterations persist beyond the anesthetic recovery phase in neonatal rats. This study tested the hypothesis that rapid eye movement sleep disturbances would be present in adolescent rats treated with anesthesia on postnatal day 7. METHODS: Forty-four neonatal rats were randomly allocated to treatment with anesthesia consisting of midazolam, nitrous oxide, and isoflurane or control conditions for 2 h or 6 h. Electroencephalographic and electromyographic electrodes were implanted and recordings obtained between postnatal days 26 and 34. The primary outcome was time spent in rapid eye movement sleep. Data were analyzed using two-tailed unpaired t tests and two-way repeated measures analysis of variance. RESULTS: Rats treated with midazolam, nitrous oxide, and isoflurane exhibited a significant increase in rapid eye movement sleep three weeks later when compared with control rats, regardless of whether they were treated for 2 h (174.0 ± 7.2 min in anesthetized, 108.6 ± 5.3 in controls, P < 0.0001) or 6 h (151.6 ± 9.9 min in anesthetized, 108.8 ± 7.1 in controls, P = 0.002). CONCLUSIONS: Treatment with midazolam, nitrous oxide, and isoflurane on postnatal day 7 increases rapid eye movement sleep three weeks later in rats.

Cracklin' Fish Brains.

Whole-Brain Neuronal Activity Displays Crackling Noise Dynamics Ponce-Alvarez A, Jouary A, Privat M, Deco G, Sumbre G. Neuron. 2018;100(6):1446-1459.e6. Previous studies suggest that the brain operates at a critical point in which phases of order and disorder coexist, producing emergent patterned dynamics at all scales and optimizing several brain functions. Here, we combined light-sheet microscopy with GCaMP zebrafish larvae to study whole-brain dynamics in vivo at near single-cell resolution. We show that spontaneous activity propagates in the brain's 3-dimensional space, generating scale-invariant neuronal avalanches with time courses and recurrence times that exhibit statistical self-similarity at different magnitude, temporal, and frequency scales. This suggests that the nervous system operates close to a nonequilibrium phase transition, where a large repertoire of spatial, temporal, and interactive modes can be supported. Finally, we show that gap junctions contribute to the maintenance of criticality and that, during interactions with the environment (sensory inputs and self-generated behaviors), the system is transiently displaced to a more ordered regime, conceivably to limit the potential sensory representations and motor outcomes.

The effect of rho kinase inhibition on morphological and electrophysiological maturity in iPSC-derived neurons.

Induced pluripotent stem cell (iPSC)-derived neurons permit the study of neurogenesis and neurological disease in a human setting. However, the electrophysiological properties of iPSC-derived neurons are consistent with those observed in immature cortical neurons, including a high membrane resistance depolarized resting membrane potential and immature firing properties, limiting their use in modeling neuronal activity in adult cells. Based on the proven association between inhibiting rho kinase (ROCK) and increased neurite complexity, we seek to determine if short-term ROCK inhibition during the first 1-2 weeks of differentiation would increase morphological complexity and electrophysiological maturity after several weeks of differentiation. While inhibiting ROCK resulted in increased neurite formation after 24 h, this effect did not persist at 3 and 6 weeks of age. Additionally, there was no effect of ROCK inhibition on electrophysiological properties at 2-3, 6, or 12 weeks of age, despite an increase in evoked and spontaneous firing and a more hyperpolarized resting membrane potential over time. These results indicate that while there is a clear effect of time on electrophysiological maturity, ROCK inhibition did not accelerate maturity.

Out of thin air: Hyperventilation-triggered seizures.

Voluntary hyperventilation triggers seizures in the vast majority of people with absence epilepsy. The mechanisms that underlie this phenomenon remain unknown. Herein, we review observations - many made long ago - that provide insight into the relationship between breathing and absence seizures.

Tenuous Inhibitory GABAergic Signaling in the Reticular Thalamus.

Maintenance of a low intracellular Cl- concentration ([Cl-]i) is critical for enabling inhibitory neuronal responses to GABAA receptor-mediated signaling. Cl- transporters, including KCC2, and extracellular impermeant anions ([A]o) of the extracellular matrix are both proposed to be important regulators of [Cl-]i Neurons of the reticular thalamic (RT) nucleus express reduced levels of KCC2, indicating that GABAergic signaling may produce excitation in RT neurons. However, by performing perforated patch recordings and calcium imaging experiments in rats (male and female), we find that [Cl-]i remains relatively low in RT neurons. Although we identify a small contribution of [A]o to a low [Cl-]i in RT neurons, our results also demonstrate that reduced levels of KCC2 remain sufficient to maintain low levels of Cl- Reduced KCC2 levels, however, restrict the capacity of RT neurons to rapidly extrude Cl- following periods of elevated GABAergic signaling. In a computational model of a local RT network featuring slow Cl- extrusion kinetics, similar to those we found experimentally, model RT neurons are predisposed to an activity-dependent switch from GABA-mediated inhibition to excitation. By decreasing the activity threshold required to produce excitatory GABAergic signaling, weaker stimuli are able to propagate activity within the model RT nucleus. Our results indicate the importance of even diminished levels of KCC2 in maintaining inhibitory signaling within the RT nucleus and suggest how this important activity choke point may be easily overcome in disorders such as epilepsy.SIGNIFICANCE STATEMENT Precise regulation of intracellular Cl- levels ([Cl-]i) preserves appropriate, often inhibitory, GABAergic signaling within the brain. However, there is disagreement over the relative contribution of various mechanisms that maintain low [Cl-]i We found that the Cl- transporter KCC2 is an important Cl- extruder in the reticular thalamic (RT) nucleus, despite this nucleus having remarkably low KCC2 immunoreactivity relative to other regions of the adult brain. We also identified a smaller contribution of fixed, impermeant anions ([A]o) to lowering [Cl-]i in RT neurons. Inhibitory signaling among RT neurons is important for preventing excessive activation of RT neurons, which can be responsible for generating seizures. Our work suggests that KCC2 critically restricts the spread of activity within the RT nucleus.

Histone Deacetylase Inhibitor Entinostat (MS-275) Restores Anesthesia-induced Alteration of Inhibitory Synaptic Transmission in the Developing Rat Hippocampus.

Recent evidence strongly supports the idea that common general anesthetics (GAs) such as isoflurane (Iso) and nitrous oxide (N2O; laughing gas), as well as sedative drugs such as midazolam are neurotoxic for the developing mammalian brain having deleterious effects on neural circuits involved in cognition, learning and memory. However, to date, very little is known about epigenetic mechanisms involved in GA-induced plasticity of synaptic transmission in the hippocampus, the main memory-processing region in the brain. Here, we used patch-clamp recordings of miniature inhibitory post-synaptic currents (mIPSCs) from hippocampal neurons in slice cultures exposed to the clinically relevant GA combination. We found that in vitro exposure to a combination of midazolam, 0.75% Iso, and 70% N2O for 6 h leads to lasting increase in frequency of mIPSCs, while amplitudes and kinetics of the events were spared. Importantly, co-application of entinostat (MS-275), a selective inhibitor of class I histone deacetylases (HDAC), completely reversed GA-induced synaptic plasticity. Furthermore, when given in vivo to P7 pups exposed to GA with midazolam, Iso and N2O for 6 h, MS-275 reversed GA-induced histone-3 hypoacetylation as shown by an increase in Ac-H3 protein expression in the hippocampus. We conclude that exposure to a combination of Iso with N2O and midazolam causes plasticity of mIPSCs in hippocampal neurons by epigenetic mechanisms that target presynaptic sites. We hypothesize that GA-induced epigenetic alterations in inhibitory synaptic transmission in the hippocampus may contribute to altered neuronal excitability and consequently abnormal learning and memory later in life.

Unexpected role of interferon-γ in regulating neuronal connectivity and social behaviour.

Immune dysfunction is commonly associated with several neurological and mental disorders. Although the mechanisms by which peripheral immunity may influence neuronal function are largely unknown, recent findings implicate meningeal immunity influencing behaviour, such as spatial learning and memory. Here we show that meningeal immunity is also critical for social behaviour; mice deficient in adaptive immunity exhibit social deficits and hyper-connectivity of fronto-cortical brain regions. Associations between rodent transcriptomes from brain and cellular transcriptomes in response to T-cell-derived cytokines suggest a strong interaction between social behaviour and interferon-γ (IFN-γ)-driven responses. Concordantly, we demonstrate that inhibitory neurons respond to IFN-γ and increase GABAergic (γ-aminobutyric-acid) currents in projection neurons, suggesting that IFN-γ is a molecular link between meningeal immunity and neural circuits recruited for social behaviour. Meta-analysis of the transcriptomes of a range of organisms reveals that rodents, fish, and flies elevate IFN-γ/JAK-STAT-dependent gene signatures in a social context, suggesting that the IFN-γ signalling pathway could mediate a co-evolutionary link between social/aggregation behaviour and an efficient anti-pathogen response. This study implicates adaptive immune dysfunction, in particular IFN-γ, in disorders characterized by social dysfunction and suggests a co-evolutionary link between social behaviour and an anti-pathogen immune response driven by IFN-γ signalling.

Genetically targeted magnetic control of the nervous system.

Optogenetic and chemogenetic actuators are critical for deconstructing the neural correlates of behavior. However, these tools have several limitations, including invasive modes of stimulation or slow on/off kinetics. We have overcome these disadvantages by synthesizing a single-component, magnetically sensitive actuator, "Magneto," comprising the cation channel TRPV4 fused to the paramagnetic protein ferritin. We validated noninvasive magnetic control over neuronal activity by demonstrating remote stimulation of cells using in vitro calcium imaging assays, electrophysiological recordings in brain slices, in vivo electrophysiological recordings in the brains of freely moving mice, and behavioral outputs in zebrafish and mice. As proof of concept, we used Magneto to delineate a causal role of striatal dopamine receptor 1 neurons in mediating reward behavior in mice. Together our results present Magneto as an actuator capable of remotely controlling circuits associated with complex animal behaviors.

Role of voltage-gated calcium channels in the regulation of aldosterone production from zona glomerulosa cells of the adrenal cortex.

Zona glomerulosa cells (ZG) of the adrenal gland constantly integrate fluctuating ionic, hormonal and paracrine signals to control the synthesis and secretion of aldosterone. These signals modulate Ca2+ levels, which provide the critical second messenger to drive steroid hormone production. Angiotensin II is a hormone known to modulate the activity of voltage-dependent L- and T-type Ca2+ channels that are expressed on the plasma membrane of ZG cells in many species. Because the ZG cell maintains a resting membrane voltage of approximately -85 mV and has been considered electrically silent, low voltage-activated T-type Ca2+ channels are assumed to provide the primary Ca2+ signal that drives aldosterone production. However, this view has recently been challenged by human genetic studies identifying somatic gain-of-function mutations in L-type CaV 1.3 channels in aldosterone-producing adenomas of patients with primary hyperaldosteronism. We provide a review of these assumptions and challenges, and update our understanding of the state of the ZG cell in a layer in which native cellular associations are preserved. This updated view of Ca2+ signalling in ZG cells provides a unifying mechanism that explains how transiently activating CaV 3.2 channels can generate a significant and recurring Ca2+ signal, and how CaV 1.3 channels may contribute to the Ca2+ signal that drives aldosterone production.