Protocol for calcium imaging and analysis of hippocampal CA1 activity evoked by non-spatial stimuli.

The hippocampus has a major role in processing spatial information but has been found to encode non-spatial information from multisensory modalities in recent studies. Here, we present a protocol for recording non-spatial stimuli (visual, auditory, and a combination) that evoked calcium activity of hippocampal CA1 neuronal ensembles in C57BL/6 mice using a miniaturized fluorescence microscope. We describe steps for experimental apparatus setup, surgical procedures, software development, and neuronal population activity analysis. For complete details on the use and execution of this protocol, please refer to Sun et al.1.

Dual Extracellular Recordings in the Mouse Hippocampus and Prefrontal Cortex.

The technique of recording local field potentials (LFPs) is an electrophysiological method used to measure the electrical activity of localized neuronal populations. It serves as a crucial tool in cognitive research, particularly in brain regions like the hippocampus and prefrontal cortex. Dual LFP recordings between these areas are of particular interest as they allow the exploration of interregional signal communication. However, methods for performing these recordings are rarely described, and most commercial recording devices are either expensive or lack adaptability to accommodate specific experimental designs. This study presents a comprehensive protocol for performing dual-electrode LFP recordings in the mouse hippocampus and the prefrontal cortex to investigate the effects of antipsychotic drugs and potassium channel modulators on LFP properties in these areas. The technique enables the measurement of LFP properties, including power spectra within each brain region and coherence between the two. Additionally, a low-cost, custom-designed recording device has been developed for these experiments. In summary, this protocol provides a means to record signals with high signal-to-noise ratios in different brain regions, facilitating the investigation of interregional information communication within the brain.

Large Clusters of Invasive emm49 Group A Streptococcus Identified within and across Arizona Healthcare Facilities through Statewide Genomic Surveillance System, 2019-2021.

A statewide genomic surveillance system for invasive Group A Streptococcus was implemented in Arizona in June 2019, resulting in 1,046 isolates being submitted for genomic analysis to characterize emm-types and identify transmission clusters. Eleven of the 32 identified distinct emm-types comprised >80% of samples, with 29.7% of all isolates being typed as emm49 (and its genetic derivative emm151). Phylogenetic analysis initially identified an emm49 genomic cluster of four isolates that rapidly expanded over subsequent months (June 2019-February 2020). Public health investigations identified epidemiologic links with three different long-term care facilities, resulting in specific interventions. Unbiased genomic surveillance allowed for identification and response to clusters that would have otherwise remained undetected.

Hippocampal cognitive and relational map paradigms explored by multisensory encoding recording with wide-field calcium imaging.

Two major theories have been proposed to explain hippocampal function: cognitive map and the relational theories. They differ in their views on whether hippocampal neurons can process non-spatial information independently. However, the explanatory power of these theories remains unresolved. Additionally, more complex aspects of hippocampal neural population responses to non-spatial stimuli have not been investigated. Here, we used miniaturized fluorescence microscopy to investigate mouse CA1 responses to spatial, visual, auditory modalities, and combinations. We found that while neuronal populations primarily processed spatial information, they also showed strong sensitivity to non-spatial modalities independent of spatial inputs, exhibiting distinct neuronal dynamics and coding patterns. These results provide strong support for the relational theories.

An In Vitro Brain Tumour Model in Organotypic Slice Cultures Displaying Epileptiform Activity.

Brain tumours have significant impacts on patients' quality of life, and current treatments have limited effectiveness. To improve understanding of tumour development and explore new therapies, researchers rely on experimental models. However, reproducing tumour-associated epilepsy (TAE) in these models has been challenging. Existing models vary from cell lines to in vivo studies, but in vivo models are resource-intensive and often fail to mimic crucial features like seizures. In this study, we developed a technique in which normal rat organotypic brain tissue is implanted with an aggressive brain tumour. This method produces a focal invasive lesion that preserves neural responsiveness and exhibits epileptiform hyperexcitability. It allows for real-time imaging of tumour growth and invasion for up to four weeks and microvolume fluid sampling analysis of different regions, including the tumour, brain parenchyma, and peritumoral areas. The tumour cells expand and infiltrate the organotypic slice, resembling in vivo behaviour. Spontaneous seizure-like events occur in the tumour slice preparation and can be induced with stimulation or high extracellular potassium. Furthermore, we assess extracellular fluid composition in various regions of interest. This technique enables live cell confocal microscopy to record real-time tumour invasion properties, whilst maintaining neural excitability, generating field potentials, and epileptiform discharges, and provides a versatile preparation for the study of major clinical problems of tumour-associated epilepsy.

Critical dynamics arise during structured information presentation within embodied in vitro neuronal networks.

Understanding how brains process information is an incredibly difficult task. Amongst the metrics characterising information processing in the brain, observations of dynamic near-critical states have generated significant interest. However, theoretical and experimental limitations associated with human and animal models have precluded a definite answer about when and why neural criticality arises with links from attention, to cognition, and even to consciousness. To explore this topic, we used an in vitro neural network of cortical neurons that was trained to play a simplified game of 'Pong' to demonstrate Synthetic Biological Intelligence (SBI). We demonstrate that critical dynamics emerge when neural networks receive task-related structured sensory input, reorganizing the system to a near-critical state. Additionally, better task performance correlated with proximity to critical dynamics. However, criticality alone is insufficient for a neuronal network to demonstrate learning in the absence of additional information regarding the consequences of previous actions. These findings offer compelling support that neural criticality arises as a base feature of incoming structured information processing without the need for higher order cognition.

Unique Genomic Epidemiology of COVID-19 in the White Mountain Apache Tribe, April to August 2020, Arizona.

The first case of coronavirus disease 2019 (COVID-19) within the White Mountain Apache Tribe (WMAT) in Arizona was diagnosed almost 1 month after community transmission was recognized in the state. Aggressive contact tracing allowed for robust genomic epidemiology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and subsequent phylogenetic analyses implicated only two virus introductions, which resulted in the spread of two unique viral lineages on the reservation. The phylogenies of these lineages reflect the nature of the introductions, the remoteness of the community, and the extraordinarily high attack rates. The timing and space-limited nature of the outbreaks validate the public health tracing efforts involved, which were illustrated by multiple short transmission chains over a period of several weeks, eventually resulting in extinction of the lineages. Comprehensive sampling and successful infection control efforts are illustrated in both the effective population size analyses and the limited mortality outcomes. The rapid spread and high attack rates of the two lineages may be due to a combination of sociological determinants of the WMAT and a seemingly enhanced transmissibility. The SARS-CoV-2 genomic epidemiology of the WMAT demonstrates a unique local history of the pandemic and highlights the extraordinary and successful efforts of their public health response. IMPORTANCE This article discusses the introduction and spread of two unique viral lineages of SARS-CoV-2 within the White Mountain Apache Tribe in Arizona. Both genomic sequencing and traditional epidemiological strategies (e.g., contract tracing) were used to understand the nature of the spread of both lineages. Beyond providing a robust genomic analysis of the epidemiology of the outbreaks, this work also highlights the successful efforts of the local public health response.

High synaptic threshold for dendritic NMDA spike generation in human layer 2/3 pyramidal neurons.

Neurons receive synaptic input primarily onto their dendrites. While we know much about the electrical properties of dendrites in rodents, we have only just started to describe their properties in the human brain. Here, we investigate the capacity of human dendrites to generate NMDA-receptor-dependent spikes (NMDA spikes). Using dendritic glutamate iontophoresis, as well as local dendritic synaptic stimulation, we find that human layer 2/3 pyramidal neurons can generate dendritic NMDA spikes. The capacity to evoke NMDA spikes in human neurons, however, was significantly reduced compared with that in rodents. Simulations in morphologically realistic and simplified models indicated that human neurons have a higher synaptic threshold for NMDA spike generation primarily due to the wider diameter of their dendrites. In summary, we find reduced NMDA spike generation in human compared with rodent layer 2/3 pyramidal neurons and provide evidence that this is due to the wider diameter of human dendrites.

A Raman spectral reference library of potential anthropogenic and biological ocean polymers.

Microplastics have been extensively documented in marine ecosystems and food webs with devastating impacts. To solve this global crisis, identifying the polymer composition is key for resolving the material origin, geographic source, and ecosystem life cycle of ocean plastics. Visually based techniques, importantly, are not diagnostic. Raman spectroscopy is an increasingly preferred identification method for its accuracy and reduced likelihood of misinterpretation, though it can be inaccessible due to cost of paywalled spectral libraries and availability of relevant polymer spectra for comparison. Here, we provide an open-access reference library of high-quality, broad-spectrum Raman spectra of major polymer categories germane to marine environments. The library includes high-quality spectra from: (a) pristine anthropogenic polymers newly sourced from manufacturers (n = 40), (b) weathered anthropogenic polymers collected from used consumer, beachcast, agricultural, and fishery sources (n = 22), and (c) biological polymers representing diverse marine taxa, trophic levels, and tissues (n = 17). We hope this reference library can help this rapidly expanding scientific community and facilitate progress in the global plastic pollution crisis.

Alpha-lipoic acid analogues in the regulation of redox balance in epilepsy: A molecular docking and simulation study.

OBJECTIVE: Oxidative stress is one of the pathophysiological mechanisms implicated in drug-resistant epilepsy. Recurrent seizures and prolonged treatment with anti-seizure medicines (ASMs) can produce reactive oxygen species (ROS) resulting in neuronal cell damage, cell toxicity, and cell death. This damage may contribute to the loss of efficacy of anti-seizure medicines. Add-on therapy with antioxidants, neuroimmunophilins, and polyphenols may thus be beneficial in drug-resistant epilepsy. In vitro and in vivo studies have shown a significant improvement in drug efficacy and seizure suppression using co-treatment of anti-seizure medication with naturally available antioxidants including alpha-lipoic acid (α-lipoic acid) from walnut; however, the underlying mechanisms of action remain to be fully understood. METHODS: We undertook molecular docking and molecular dynamics simulations to determine whether alpha-lipoic acid and related analogues interacted with the human manganese superoxide dismutase (MnSOD) protein, a member of the oxidative metabolic pathway. The 3D structure of the compounds and the protein were retrieved from protein and chemical databases, binding sites were identified and ligand-protein interactions were performed. RESULTS: Alpha-lipoic acid and various analogues docked within a human MnSOD binding region. Docking results were validated by molecular dynamic simulation. The CMX-2043 analogue showed strong binding with MnSOD compared to alpha-lipoic acid and other analogues. SIGNIFICANCE: Our findings provide new insights into additional mechanisms of action, which may in part, account for the antioxidant properties associated with alpha-lipoic acid and related analogues. The results support further in vitro and in vivo evaluation of these compounds to better understand their potential as add-on therapy for ASM treatment in epilepsy.

Applying Genomic Epidemiology to Characterize a COVID-19 Outbreak in a Developmentally Disabled Adult Group Home Setting, Arizona.

Individuals living in congregate settings, including those in group homes, have been disproportionately impacted by COVID-19 and may be at increased risk of exposure or infection due to underlying illness. In mid-May 2020, local public health officials responded to an outbreak of COVID-19 among staff and residents associated with a multi-residential group home that provides care for adults with intellectual and developmental disabilities. Samples were collected at 16 of the homes. In four of the homes all the residents tested positive, and in the remaining 12 houses where samples were collected, all residents tested negative. Of the 152 individuals tested, 15/58 (25.9%) residents and 27/94 (28.7%) staff were positive for SARS-CoV-2, including eight hospitalizations and four deaths. Phylogenetic analysis of genomes from this outbreak in the context of genomes from Northern Arizona shows that very few mutations separate the samples from this outbreak. A potential transmission network was developed to illustrate person-place epidemiologic linkages and further demonstrates the dynamic connections between staff and residents with respect to each group home location. Epidemiologic and genomic evidence correlate, and suggest that asymptomatic infected staff likely introduced and spread COVID-19 in this setting. Implementation of public health prevention measures alongside rapid genomic analysis can help guide policy development and guide management efforts to prevent and mitigate future outbreaks.

Scopolamine Impairs Spatial Information Recorded With "Miniscope" Calcium Imaging in Hippocampal Place Cells.

The hippocampus and associated cholinergic inputs have important roles in spatial memory in rodents. Muscarinic acetylcholine receptors (mAChRs) are involved in the communication of cholinergic signals and regulate spatial memory. They have been found to impact the memory encoding process, but the effect on memory retrieval is controversial. Previous studies report that scopolamine (a non-selective antagonist of mAChR) induces cognitive deficits on animals, resulting in impaired memory encoding, but the effect on memory retrieval is less certain. We tested the effects of blocking mAChRs on hippocampal network activity and neural ensembles that had previously encoded spatial information. The activity of hundreds of neurons in mouse hippocampal CA1 was recorded using calcium imaging with a miniaturised fluorescent microscope and properties of place cells and neuronal ensemble behaviour in a linear track environment were observed. We found that the decoding accuracy and the stability of spatial representation revealed by hippocampal neural ensemble were significantly reduced after the administration of scopolamine. Several other parameters, including neural firing rate, total number of active neurons, place cell number and spatial information content were affected. Similar results were also observed in a simulated hippocampal network model. This study enhances the understanding of the function of mAChRs on spatial memory impairment.

Effects of Several Classes of Voltage-Gated Ion Channel Conductances on Gamma and Theta Oscillations in a Hippocampal Microcircuit Model.

Gamma and theta oscillations have been functionally associated with cognitive processes, such as learning and memory. Synaptic conductances play an important role in the generation of intrinsic network rhythmicity, but few studies have examined the effects of voltage-gated ion channels (VGICs) on these rhythms. In this report, we have used a pyramidal-interneuron-gamma (PING) network consisting of excitatory pyramidal cells and two types of inhibitory interneurons. We have constructed a conductance-based neural network incorporating a persistent sodium current (I NaP ), a delayed rectifier potassium current (I KDR ), a inactivating potassium current (I A ) and a hyperpolarization-activated current (I H ). We have investigated the effects of several conductances on network theta and gamma frequency oscillations. Variation of all conductances of interest changed network rhythmicity. Theta power was altered by all conductances tested. Gamma rhythmogenesis was dependent on I A and I H . The I KDR currents in excitatory pyramidal cells as well as both types of inhibitory interneurons were essential for theta rhythmogenesis and altered gamma rhythm properties. Increasing I NaP suppressed both gamma and theta rhythms. Addition of noise did not alter these patterns. Our findings suggest that VGICs strongly affect brain network rhythms. Further investigations in vivo will be of great interest, including potential effects on neural function and cognition.

Effects of antipsychotic drugs and potassium channel modulators on spectral properties of local field potentials in mouse hippocampus and pre-frontal cortex.

Local field potentials (LFPs) recorded intracranially display a range of location-specific oscillatory spectra which have been related to cognitive processes. Although the mechanisms producing LFPs are not completely understood, it is likely that voltage-gated ion channels which produce action potentials and patterned discharges play a significant role. It is also known that antipsychotic drugs (APDs) affect LFP spectra and a direct inhibitory effect on voltage-gated potassium channels has been reported. Additionally, voltage-gated potassium channels have been implicated in the pathophysiology of schizophrenia, a disorder for which APDs are primary therapies. In this study we sought to: i) better characterise the effects of two APDs on LFPs spectra and connectivity measures and ii) examine the effects of potassium channel modulators on LFPs and potential overlap of effects with APDs. Intracranial electrodes were implanted in hippocampus (HIP) and pre-frontal cortex (PFC) of C57BL/6J mice; power spectra, coherence and phase-amplitude cross-frequency coupling were measured. Drugs tested were APDs haloperidol and clozapine as well as voltage-gated potassium channel modulators (KVMs) 4-aminopyridine (4-AP), tetraethylammonium, retigabine and E-4031. Both APDs and KVMs significantly reduced gamma power except 4-AP, which conversely increased gamma power. Clozapine and retigabine additionally reduced gamma coherence between HIP and PFC, while 4-AP demonstrated the opposite effect. Phase-amplitude coupling between theta and gamma oscillations in HIP was significantly reduced by the administration of haloperidol and retigabine. These results provide previously undescribed effects of APDs on LFP properties and demonstrate novel modulation of LFP characteristics by KVMs that intriguingly overlap with the APD effects.

Spatiotemporal Patterns of High-Frequency Activity (80-170 Hz) in Long-Term Intracranial EEG.

OBJECTIVE: To determine the utility of high-frequency activity (HFA) and epileptiform spikes as biomarkers for epilepsy, we examined the variability in their rates and locations using long-term ambulatory intracranial EEG (iEEG) recordings. METHODS: This study used continuous iEEG recordings obtained over an average of 1.4 years from 15 patients with drug-resistant focal epilepsy. HFA was defined as 80- to 170-Hz events with amplitudes clearly larger than the background, which was automatically detected with a custom algorithm. The automatically detected HFA was compared with visually annotated high-frequency oscillations (HFOs). The variations of HFA rates were compared with spikes and seizures on patient-specific and electrode-specific bases. RESULTS: HFA included manually annotated HFOs and high-amplitude events occurring in the 80- to 170-Hz range without observable oscillatory behavior. HFA and spike rates had high amounts of intrapatient and interpatient variability. Rates of HFA and spikes had large variability after electrode implantation in most of the patients. Locations of HFA and spikes varied up to weeks in more than one-third of the patients. Both HFA and spike rates showed strong circadian rhythms in all patients, and some also showed multiday cycles. Furthermore, the circadian patterns of HFA and spike rates had patient-specific correlations with seizures, which tended to vary across electrodes. CONCLUSION: Analysis of HFA and epileptiform spikes should consider postimplantation variability. HFA and epileptiform spikes, like seizures, show circadian rhythms. However, the circadian profiles can vary spatially within patients, and their correlations to seizures are patient-specific.

Methods for sequencing the pandemic: benefits of rapid or high-throughput processing.

Genomic epidemiology has proven successful for real-time and retrospective monitoring of small and large-scale outbreaks. Here, we report two genomic sequencing and analysis strategies for rapid-turnaround or high-throughput processing of metagenomic samples. The rapid-turnaround method was designed to provide a quick phylogenetic snapshot of samples at the heart of active outbreaks, and has a total turnaround time of <48 hours from raw sample to analyzed data. The high-throughput method was designed for semi-retrospective data analysis, and is both cost effective and highly scalable. Though these methods were developed and utilized for the SARS-CoV-2 pandemic response in Arizona, U.S, and we envision their use for infectious disease epidemiology in the 21 st Century.

Rhizopus microsporus Infections Associated with Surgical Procedures, Argentina, 2006-2014.

Rhizopus spp. fungi are ubiquitous in the environment and a rare but substantial cause of infection in immunosuppressed persons and surgery patients. During 2005-2017, an abnormally high number of Rhizopus infections in surgery patients, with no apparent epidemiologic links, were reported in Argentina. To determine the likelihood of a common source of the cluster, we performed whole-genome sequencing on samples collected during 2006-2014. Most isolates were separated by >60 single-nucleotide polymorphisms, and we found no evidence for recombination or nonneutral mutation accumulation; these findings do not support common source or patient-to-patient transmission. Assembled genomes of most isolates were ≈25 Mbp, and multiple isolates had substantially larger assembled genomes (43-51 Mbp), indicative of infections with strain types that underwent genome expansion. Whole-genome sequencing has become an essential tool for studying epidemiology of fungal infections. Less discriminatory techniques may miss true relationships, possibly resulting in inappropriate attribution of point source.

Cascaded amplifying circuits enable ultrasensitive cellular sensors for toxic metals.

Cell-based biosensors have great potential to detect various toxic and pathogenic contaminants in aqueous environments. However, frequently they cannot meet practical requirements due to insufficient sensing performance. To address this issue, we investigated a modular, cascaded signal amplifying methodology. We first tuned intracellular sensory receptor densities to increase sensitivity, and then engineered multi-layered transcriptional amplifiers to sequentially boost output expression level. We demonstrated these strategies by engineering ultrasensitive bacterial sensors for arsenic and mercury, and improved detection limit and output up to 5,000-fold and 750-fold, respectively. Coupled by leakage regulation approaches, we developed an encapsulated microbial sensor cell array for low-cost, portable and precise field monitoring, where the analyte can be readily quantified via displaying an easy-to-interpret volume bar-like pattern. The ultrasensitive signal amplifying methodology along with the background regulation and the sensing platform will be widely applicable to many other cell-based sensors, paving the way for their real-world applications.