IntelliSleepScorer, a software package with a graphic user interface for automated sleep stage scoring in mice based on a light gradient boosting machine algorithm.

Machine learning has been applied in recent years to categorize sleep stages (NREM, REM, and wake) using electroencephalogram (EEG) recordings; however, a well-validated sleep scoring automatic pipeline in rodent research is still not publicly available. Here, we present IntelliSleepScorer, a software package with a graphic user interface to score sleep stages automatically in mice. IntelliSleepScorer uses the light gradient boosting machine (LightGBM) to score sleep stages for each epoch of recordings. We developed LightGBM models using a large cohort of data, which consisted of 5776 h of sleep EEG and electromyogram (EMG) signals across 519 unique recordings from 124 mice. The LightGBM model achieved an overall accuracy of 95.2% and a Cohen's kappa of 0.91, which outperforms the baseline models such as the logistic regression model (accuracy = 93.3%, kappa = 0.88) and the random forest model (accuracy = 94.3%, kappa = 0.89). The overall performance of the LightGBM model as well as the performance across different sleep stages are on par with that of the human experts. Most importantly, we validated the generalizability of the LightGBM models: (1) The LightGBM model performed well on two publicly available, independent datasets (kappa > = 0.80), which have different sampling frequency and epoch lengths; (2) The LightGBM model performed well on data recorded at a lower sampling frequency (kappa = 0.90); (3) The performance of the LightGBM model is not affected by the light/dark cycle; and (4) A modified LightGBM model performed well on data containing only one EEG and one EMG electrode (kappa > = 0.89). Taken together, the LightGBM models offer state-of-the-art performance for automatic sleep stage scoring in mice. Last, we implemented the IntelliSleepScorer software package based on the validated model to provide an out-of-box solution to sleep researchers (available for download at https://sites.broadinstitute.org/pan-lab/resources ).

Mouse mutants in schizophrenia risk genes GRIN2A and AKAP11 show EEG abnormalities in common with schizophrenia patients.

Schizophrenia is a heterogeneous psychiatric disorder with a strong genetic basis, whose etiology and pathophysiology remain poorly understood. Exome sequencing studies have uncovered rare, loss-of-function variants that greatly increase risk of schizophrenia [1], including loss-of-function mutations in GRIN2A (aka GluN2A or NR2A, encoding the NMDA receptor subunit 2A) and AKAP11 (A-Kinase Anchoring Protein 11). AKAP11 and GRIN2A mutations are also associated with bipolar disorder [2], and epilepsy and developmental delay/intellectual disability [1, 3, 4], respectively. Accessible in both humans and rodents, electroencephalogram (EEG) recordings offer a window into brain activity and display abnormal features in schizophrenia patients. Does loss of Grin2a or Akap11 in mice also result in EEG abnormalities? We monitored EEG in heterozygous and homozygous knockout Grin2a and Akap11 mutant mice compared with their wild-type littermates, at 3- and 6-months of age, across the sleep/wake cycle and during auditory stimulation protocols. Grin2a and Akap11 mutants exhibited increased resting gamma power, attenuated auditory steady-state responses (ASSR) at gamma frequencies, and reduced responses to unexpected auditory stimuli during mismatch negativity (MMN) tests. Sleep spindle density was reduced in a gene dose-dependent manner in Akap11 mutants, whereas Grin2a mutants showed increased sleep spindle density. The EEG phenotypes of Grin2a and Akap11 mutant mice show a variety of abnormal features that overlap considerably with human schizophrenia patients, reflecting systems-level changes caused by Grin2a and Akap11 deficiency. These neurophysiologic findings further substantiate Grin2a and Akap11 mutants as genetic models of schizophrenia and identify potential biomarkers for stratification of schizophrenia patients.

Pathophysiological implication of CaV3.1 T-type Ca2+ channels in trigeminal neuropathic pain.

A crucial pathophysiological issue concerning central neuropathic pain is the modification of sensory processing by abnormally increased low-frequency brain rhythms. Here we explore the molecular mechanisms responsible for such abnormal rhythmicity and its relation to neuropathic pain syndrome. Toward this aim, we investigated the behavioral and electrophysiological consequences of trigeminal neuropathic pain following infraorbital nerve ligations in CaV3.1 T-type Ca(2+) channel knockout and wild-type mice. CaV3.1 knockout mice had decreased mechanical hypersensitivity and reduced low-frequency rhythms in the primary somatosensory cortex and related thalamic nuclei than wild-type mice. Lateral inhibition of gamma rhythm in primary somatosensory cortex layer 4, reflecting intact sensory contrast, was present in knockout mice but severely impaired in wild-type mice. Moreover, cross-frequency coupling between low-frequency and gamma rhythms, which may serve in sensory processing, was pronounced in wild-type mice but not in CaV3.1 knockout mice. Our results suggest that the presence of CaV3.1 channels is a key element in the pathophysiology of trigeminal neuropathic pain.

Altered thalamocortical rhythmicity and connectivity in mice lacking CaV3.1 T-type Ca2+ channels in unconsciousness.

In unconscious status (e.g., deep sleep and anesthetic unconsciousness) where cognitive functions are not generated there is still a significant level of brain activity present. Indeed, the electrophysiology of the unconscious brain is characterized by well-defined thalamocortical rhythmicity. Here we address the ionic basis for such thalamocortical rhythms during unconsciousness. In particular, we address the role of CaV3.1 T-type Ca(2+) channels, which are richly expressed in thalamic neurons. Toward this aim, we examined the electrophysiological and behavioral phenotypes of mice lacking CaV3.1 channels (CaV3.1 knockout) during unconsciousness induced by ketamine or ethanol administration. Our findings indicate that CaV3.1 KO mice displayed attenuated low-frequency oscillations in thalamocortical loops, especially in the 1- to 4-Hz delta band, compared with control mice (CaV3.1 WT). Intriguingly, we also found that CaV3.1 KO mice exhibited augmented high-frequency oscillations during unconsciousness. In a behavioral measure of unconsciousness dynamics, CaV3.1 KO mice took longer to fall into the unconscious state than controls. In addition, such unconscious events had a shorter duration than those of control mice. The thalamocortical interaction level between mediodorsal thalamus and frontal cortex in CaV3.1 KO mice was significantly lower, especially for delta band oscillations, compared with that of CaV3.1 WT mice, during unconsciousness. These results suggest that the CaV3.1 channel is required for the generation of a given set of thalamocortical rhythms during unconsciousness. Further, that thalamocortical resonant neuronal activity supported by this channel is important for the control of vigilance states.

RNS60, a charge-stabilized nanostructure saline alters Xenopus Laevis oocyte biophysical membrane properties by enhancing mitochondrial ATP production.

We have examined the effects of RNS60, a 0.9% saline containing charge-stabilized oxygen nanobubble-based structures. RNS60 is generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. This study, implemented in Xenopus laevis oocytes, addresses both the electrophysiological membrane properties and parallel biological processes in the cytoplasm. Intracellular recordings from defolliculated X. laevis oocytes were implemented in: (1) air oxygenated standard Ringer's solution, (2) RNS60-based Ringer's solution, (3) RNS10.3 (TCP-modified saline without excess oxygen)-based Ringer's, and (4) ONS60 (saline containing high pressure oxygen without TCP modification)-based Ringer's. RNS60-based Ringer's solution induced membrane hyperpolarization from the resting membrane potential. This effect was prevented by: (1) ouabain (a blocker of the sodium/potassium ATPase), (2) rotenone (a mitochondrial electron transfer chain inhibitor preventing usable ATP synthesis), and (3) oligomycin A (an inhibitor of ATP synthase) indicating that RNS60 effects intracellular ATP levels. Increased intracellular ATP levels following RNS60 treatment were directly demonstrated using luciferin/luciferase photon emission. These results indicate that RNS60 alters intrinsic the electrophysiological properties of the X. laevis oocyte membrane by increasing mitochondrial-based ATP synthesis. Ultrastructural analysis of the oocyte cytoplasm demonstrated increased mitochondrial length in the presence of RNS60-based Ringer's solution. It is concluded that the biological properties of RNS60 relate to its ability to optimize ATP synthesis.

Enhanced synaptic transmission at the squid giant synapse by artificial seawater based on physically modified saline.

Superfusion of the squid giant synapse with artificial seawater (ASW) based on isotonic saline containing oxygen nanobubbles (RNS60 ASW) generates an enhancement of synaptic transmission. This was determined by examining the postsynaptic response to single and repetitive presynaptic spike activation, spontaneous transmitter release, and presynaptic voltage clamp studies. In the presence of RNS60 ASW single presynaptic stimulation elicited larger postsynaptic potentials (PSP) and more robust recovery from high frequency stimulation than in control ASW. Analysis of postsynaptic noise revealed an increase in spontaneous transmitter release with modified noise kinetics in RNS60 ASW. Presynaptic voltage clamp demonstrated an increased EPSP, without an increase in presynaptic ICa(++) amplitude during RNS60 ASW superfusion. Synaptic release enhancement reached stable maxima within 5-10 min of RNS60 ASW superfusion and was maintained for the entire recording time, up to 1 h. Electronmicroscopic morphometry indicated a decrease in synaptic vesicle density and the number at active zones with an increase in the number of clathrin-coated vesicles (CCV) and large endosome-like vesicles near junctional sites. Block of mitochondrial ATP synthesis by presynaptic injection of oligomycin reduced spontaneous release and prevented the synaptic noise increase seen in RNS60 ASW. After ATP block the number of vesicles at the active zone and CCV was reduced, with an increase in large vesicles. The possibility that RNS60 ASW acts by increasing mitochondrial ATP synthesis was tested by direct determination of ATP levels in both presynaptic and postsynaptic structures. This was implemented using luciferin/luciferase photon emission, which demonstrated a marked increase in ATP synthesis following RNS60 administration. It is concluded that RNS60 positively modulates synaptic transmission by up-regulating ATP synthesis, thus leading to synaptic transmission enhancement.

Blocking Effects of Human Tau on Squid Giant Synapse Transmission and Its Prevention by T-817 MA.

Filamentous tau inclusions are hallmarks of Alzheimer's disease and related neurodegenerative tauopathies, but the molecular mechanisms involved in tau-mediated changes in neuronal function and their possible effects on synaptic transmission are unknown. We have evaluated the effects of human tau protein injected directly into the presynaptic terminal axon of the squid giant synapse, which affords functional, structural, and biochemical analysis of its action on the synaptic release process. Indeed, we have found that at physiological concentration recombinant human tau (h-tau42) becomes phosphorylated, produces a rapid synaptic transmission block, and induces the formation of clusters of aggregated synaptic vesicles in the vicinity of the active zone. Presynaptic voltage clamp recordings demonstrate that h-tau42 does not modify the presynaptic calcium current amplitude or kinetics. Analysis of synaptic noise at the post-synaptic axon following presynaptic h-tau42 microinjection revealed an initial phase of increase spontaneous transmitter release followed by a marked reduction in noise. Finally, systemic administration of T-817MA, a proposed neuro-protective agent, rescued tau-induced synaptic abnormalities. Our results show novel mechanisms of h-tau42 mediated synaptic transmission failure and identify a potential therapeutic agent to treat tau-related neurotoxicity.

Subthreshold membrane potential oscillations in inferior olive neurons are dynamically regulated by P/Q- and T-type calcium channels: a study in mutant mice.

The role of P/Q- and T-type calcium channels in the rhythmic oscillatory behaviour of inferior olive (IO) neurons was investigated in mutant mice. Mice lacking either the CaV2.1 gene of the pore-forming alpha1A subunit for P/Q-type calcium channel, or the CaV3.1 gene of the pore-forming alpha1G subunit for T-type calcium channel were used. In vitro intracellular recording from IO neurons reveals that the amplitude and frequency of sinusoidal subthreshold oscillations (SSTOs) were reduced in the CaV2.1-/- mice. In the CaV3.1-/- mice, IO neurons also showed altered patterns of SSTOs and the probability of SSTO generation was significantly lower (15%, 5 of 34 neurons) than that of wild-type (78%, 31 of 40 neurons) or CaV2.1-/- mice (73%, 22 of 30 neurons). In addition, the low-threshold calcium spike and the sustained endogenous oscillation following rebound potentials were absent in IO neurons from CaV3.1-/- mice. Moreover, the phase-reset dynamics of oscillatory properties of single neurons and neuronal clusters in IO were remarkably altered in both CaV2.1-/- and CaV3.1-/- mice. These results suggest that both alpha1A P/Q- and alpha1G T-type calcium channels are required for the dynamic control of neuronal oscillations in the IO. These findings were supported by results from a mathematical IO neuronal model that incorporated T and P/Q channel kinetics.

Synaptic transmission block by presynaptic injection of oligomeric amyloid beta.

Early Alzheimer's disease (AD) pathophysiology is characterized by synaptic changes induced by degradation products of amyloid precursor protein (APP). The exact mechanisms of such modulation are unknown. Here, we report that nanomolar concentrations of intraaxonal oligomeric (o)Abeta42, but not oAbeta40 or extracellular oAbeta42, acutely inhibited synaptic transmission at the squid giant synapse. Further characterization of this phenotype demonstrated that presynaptic calcium currents were unaffected. However, electron microscopy experiments revealed diminished docked synaptic vesicles in oAbeta42-microinjected terminals, without affecting clathrin-coated vesicles. The molecular events of this modulation involved casein kinase 2 and the synaptic vesicle rapid endocytosis pathway. These findings open the possibility of a new therapeutic target aimed at ameliorating synaptic dysfunction in AD.

Role of Rab27 in synaptic transmission at the squid giant synapse.

Small GTPase Rab is a member of a large family of Ras-related proteins, highly conserved in eukaryotic cells, and thought to regulate specific type(s) and/or specific step(s) in intracellular membrane trafficking. Given our interest in synaptic transmission, we addressed the possibility that Rab27 (a close isoform of Rab3) could be involved in cytosolic synaptic vesicle mobilization. Indeed, preterminal injection of a specific antibody against squid Rab27 (anti-sqRab27 antibody) combined with confocal microscopy demonstrated that Rab27 is present on squid synaptic vesicles. Electrophysiological study of injected synapses showed that the anti-sqRab27 antibody inhibited synaptic release in a stimulation-dependent manner without affecting presynaptic action potentials or inward Ca(2+) current. This result was confirmed in in vitro synaptosomes by using total internal reflection fluorescence microscopy. Thus, synaptosomal Ca(2+)-stimulated release of FM1-43 dye was greatly impaired by intraterminal anti-sqRab27 antibody. Ultrastructural analysis of the injected giant preterminal further showed a reduced number of docked synaptic vesicles and an increase in nondocked vesicular profiles distant from the active zone. These results, taken together, indicate that Rab27 is primarily involved in the maturation of recycled vesicles and/or their transport to the presynaptic active zone in the squid giant synapse.

Effects of the monomeric, oligomeric, and fibrillar Abeta42 peptides on the proliferation and differentiation of adult neural stem cells from subventricular zone.

The incidence of amyloid plaques, composed mainly of beta-amyloid peptides (Abeta), does not correlate well with the severity of neurodegeneration in patients with Alzheimer's disease (AD). The effects of Abeta(42) on neurons or neural stem cells (NSCs) in terms of the aggregated form remain controversial. We prepared three forms of oligomeric, fibrillar, and monomeric Abeta(42) peptides and investigated their effects on the proliferation and neural differentiation of adult NSCs, according to the degree of aggregation or concentration. A low micromolar concentration (1 micromol/L) of oligomeric Abeta(42) increased the proliferation of adult NSCs remarkably in a neurosphere assay. It also enhanced the neuronal differentiation of adult NSCs and their ability to migrate. These results provide us with valuable information regarding the effects of Abeta(42) on NSCs in the brains of patients with AD.

C-terminal fragments of amyloid precursor protein exert neurotoxicity by inducing glycogen synthase kinase-3beta expression.

The AICD (amyloid precursor protein [APP] intracellular domain) and C31, the caspase-cleaved C-terminal fragment of APP, have been found in the brains of patients with Alzheimer's disease (AD). Here, we demonstrate for the first time that the C-terminal fragments of APP (AICD [C57, C59] and C31) exert neurotoxicity on differentiated PC 12 cells and rat primary cortical neurons by inducing the expression of glycogen synthase kinase 3beta, forming a ternary complex with Fe65 and CP2/LSF/LBP1 in the nucleus, whereas deletion mutants and a point mutant with Y682G of the YENPTY domain, a Fe65 binding domain, do not. Moreover, expression of APP770 and Swedish mutant form of APP increased the levels of C-terminal fragments of APP (APP-CTs) in neuronal cells and also induced the up-regulation of glycogen synthase kinase-3beta at both the mRNA and the protein levels. In addition, we show that CP2/LSF/LBP1 binding site (nt +0 to approximately +10) in human glycogen synthase kinase 3beta promoter region is essential for the induction of the gene transcription by APP-CTs. The neurotoxicities induced by APP-CTs (AICD and C31) were accompanied by an increase in the active form of glycogen synthase kinase-3beta, and by the induction of tau phosphorylation and a reduction in nuclear beta-catenin levels, and led to apoptosis.