A machine learning toolbox for the analysis of sharp-wave ripples reveals common waveform features across species.

The study of sharp-wave ripples has advanced our understanding of memory function, and their alteration in neurological conditions such as epilepsy is considered a biomarker of dysfunction. Sharp-wave ripples exhibit diverse waveforms and properties that cannot be fully characterized by spectral methods alone. Here, we describe a toolbox of machine-learning models for automatic detection and analysis of these events. The machine-learning architectures, which resulted from a crowdsourced hackathon, are able to capture a wealth of ripple features recorded in the dorsal hippocampus of mice across awake and sleep conditions. When applied to data from the macaque hippocampus, these models are able to generalize detection and reveal shared properties across species. We hereby provide a user-friendly open-source toolbox for model use and extension, which can help to accelerate and standardize analysis of sharp-wave ripples, lowering the threshold for its adoption in biomedical applications.

Immunohistochemical field parcellation of the human hippocampus along its antero-posterior axis.

The primate hippocampus includes the dentate gyrus, cornu ammonis (CA), and subiculum. CA is subdivided into four fields (CA1-CA3, plus CA3h/hilus of the dentate gyrus) with specific pyramidal cell morphology and connections. Work in non-human mammals has shown that hippocampal connectivity is precisely patterned both in the laminar and longitudinal axes. One of the main handicaps in the study of neuropathological semiology in the human hippocampus is the lack of clear laminar and longitudinal borders. The aim of this study was to explore a histochemical segmentation of the adult human hippocampus, integrating field (medio-lateral), laminar, and anteroposterior longitudinal patterning. We provide criteria for head-body-tail field and subfield parcellation of the human hippocampus based on immunodetection of Rabphilin3a (Rph3a), Purkinje-cell protein 4 (PCP4), Chromogranin A and Regulation of G protein signaling-14 (RGS-14). Notably, Rph3a and PCP4 allow to identify the border between CA3 and CA2, while Chromogranin A and RGS-14 give specific staining of CA2. We also provide novel histological data about the composition of human-specific regions of the anterior and posterior hippocampus. The data are given with stereotaxic coordinates along the longitudinal axis. This study provides novel insights for a detailed region-specific parcellation of the human hippocampus useful for human brain imaging and neuropathology.

Topological analysis of sharp-wave ripple waveforms reveals input mechanisms behind feature variations.

The reactivation of experience-based neural activity patterns in the hippocampus is crucial for learning and memory. These reactivation patterns and their associated sharp-wave ripples (SWRs) are highly variable. However, this variability is missed by commonly used spectral methods. Here, we use topological and dimensionality reduction techniques to analyze the waveform of ripples recorded at the pyramidal layer of CA1. We show that SWR waveforms distribute along a continuum in a low-dimensional space, which conveys information about the underlying layer-specific synaptic inputs. A decoder trained in this space successfully links individual ripples with their expected sinks and sources, demonstrating how physiological mechanisms shape SWR variability. Furthermore, we found that SWR waveforms segregated differently during wakefulness and sleep before and after a series of cognitive tasks, with striking effects of novelty and learning. Our results thus highlight how the topological analysis of ripple waveforms enables a deeper physiological understanding of SWRs.

From cell types to population dynamics: Making hippocampal manifolds physiologically interpretable.

The study of the hippocampal code is gaining momentum. While the physiological approach targets the contribution of individual cells as determined by genetic, biophysical and circuit factors, the field pushes for a population dynamic approach that considers the representation of behavioural variables by a large number of neurons. In this alternative framework, neuronal activity is projected into low-dimensional manifolds. These manifolds can reveal the structure of population representations, but their physiological interpretation is challenging. Here, we review the recent literature and propose that integrating information regarding behavioral traits, local field potential oscillations and cell-type-specificity into neural manifolds offers strategies to make them interpretable at the physiological level.

A machine learning toolbox for the analysis of sharp-wave ripples reveal common features across species.

The study of sharp-wave ripples (SWRs) has advanced our understanding of memory function, and their alteration in neurological conditions such as epilepsy and Alzheimer's disease is considered a biomarker of dysfunction. SWRs exhibit diverse waveforms and properties that cannot be fully characterized by spectral methods alone. Here, we describe a toolbox of machine learning (ML) models for automatic detection and analysis of SWRs. The ML architectures, which resulted from a crowdsourced hackathon, are able to capture a wealth of SWR features recorded in the dorsal hippocampus of mice. When applied to data from the macaque hippocampus, these models were able to generalize detection and revealed shared SWR properties across species. We hereby provide a user-friendly open-source toolbox for model use and extension, which can help to accelerate and standardize SWR research, lowering the threshold for its adoption in biomedical applications.

Toward Plasmonic Neural Probes: SERS Detection of Neurotransmitters through Gold-Nanoislands-Decorated Tapered Optical Fibers with Sub-10 nm Gaps.

Integration of plasmonic nanostructures with fiber-optics-based neural probes enables label-free detection of molecular fingerprints via surface-enhanced Raman spectroscopy (SERS), and it represents a fascinating technological horizon to investigate brain function. However, developing neuroplasmonic probes that can interface with deep brain regions with minimal invasiveness while providing the sensitivity to detect biomolecular signatures in a physiological environment is challenging, in particular because the same waveguide must be employed for both delivering excitation light and collecting the resulting scattered photons. Here, a SERS-active neural probe based on a tapered optical fiber (TF) decorated with gold nanoislands (NIs) that can detect neurotransmitters down to the micromolar range is presented. To do this, a novel, nonplanar repeated dewetting technique to fabricate gold NIs with sub-10 nm gaps, uniformly distributed on the wide (square millimeter scale in surface area), highly curved surface of TF is developed. It is experimentally and numerically shown that the amplified broadband near-field enhancement of the high-density NIs layer allows for achieving a limit of detection in aqueous solution of 10-7  m for rhodamine 6G and 10-5  m for serotonin and dopamine through SERS at near-infrared wavelengths. The NIs-TF technology is envisioned as a first step toward the unexplored frontier of in vivo label-free plasmonic neural interfaces.

A consensus statement on detection of hippocampal sharp wave ripples and differentiation from other fast oscillations.

Decades of rodent research have established the role of hippocampal sharp wave ripples (SPW-Rs) in consolidating and guiding experience. More recently, intracranial recordings in humans have suggested their role in episodic and semantic memory. Yet, common standards for recording, detection, and reporting do not exist. Here, we outline the methodological challenges involved in detecting ripple events and offer practical recommendations to improve separation from other high-frequency oscillations. We argue that shared experimental, detection, and reporting standards will provide a solid foundation for future translational discovery.

Deep learning-based feature extraction for prediction and interpretation of sharp-wave ripples in the rodent hippocampus.

Local field potential (LFP) deflections and oscillations define hippocampal sharp-wave ripples (SWRs), one of the most synchronous events of the brain. SWRs reflect firing and synaptic current sequences emerging from cognitively relevant neuronal ensembles. While spectral analysis have permitted advances, the surge of ultra-dense recordings now call for new automatic detection strategies. Here, we show how one-dimensional convolutional networks operating over high-density LFP hippocampal recordings allowed for automatic identification of SWR from the rodent hippocampus. When applied without retraining to new datasets and ultra-dense hippocampus-wide recordings, we discovered physiologically relevant processes associated to the emergence of SWR, prompting for novel classification criteria. To gain interpretability, we developed a method to interrogate the operation of the artificial network. We found it relied in feature-based specialization, which permit identification of spatially segregated oscillations and deflections, as well as synchronous population firing typical of replay. Thus, using deep learning-based approaches may change the current heuristic for a better mechanistic interpretation of these relevant neurophysiological events.

Artificial intelligence is finding greater use in society through its ability to process data in new ways. One particularly useful approach known as convolutional neural networks is typically used for image analysis, such as face recognition. This type of artificial intelligence could help neuroscientists analyze data produced by new technologies that record brain activity with higher resolution. Advanced processing could potentially identify events in the brain in real-time. For example, signals called sharp-wave ripples are produced by the hippocampus, a brain region involved in forming memories. Detecting and interacting with these events as they are happening would permit a better understanding of how memory works. However, these signals can vary in form, so it is necessary to detect several distinguishing features to recognize them. To achieve this, Navas-Olive, Amaducci et al. trained convolutional neural networks using signals from electrodes placed in a region of the mouse hippocampus that had already been analyzed, and 'telling' the neural networks whether they got their identifications right or wrong. Once the networks learned to identify sharp-wave ripples from this data, they could then apply this knowledge to analyze other recordings. These included datasets from another part of the mouse hippocampus, the rat brain, and ultra-dense probes that simultaneously assess different brain regions. The convolutional networks were able to recognize sharp-wave ripple events across these diverse circumstances by identifying unique characteristics in the shapes of the waves. These results will benefit neuroscientists by providing new tools to explore brain signals. For instance, this could allow them to analyze the activity of the hippocampus in real-time and potentially discover new aspects of the processes behind forming memories.

Inhibitory conductance controls place field dynamics in the hippocampus.

Hippocampal place cells receive a disparate collection of excitatory and inhibitory currents that endow them with spatially selective discharges and rhythmic activity. Using a combination of in vivo intracellular and extracellular recordings with opto/chemogenetic manipulations and computational modeling, we investigate the influence of inhibitory and excitatory inputs on CA1 pyramidal cell responses. At the cell bodies, inhibition leads and is stronger than excitation across the entire theta cycle. Pyramidal neurons fire on the ascending phase of theta when released from inhibition. Computational models equipped with the observed conductances reproduce these dynamics. In these models, place field properties are favored when the increased excitation is coupled with a reduction of inhibition within the field. As predicted by our simulations, firing rate within place fields and phase locking to theta are impaired by DREADDs activation of interneurons. Our results indicate that decreased inhibitory conductance is critical for place field expression.

Holographic Manipulation of Nanostructured Fiber Optics Enables Spatially-Resolved, Reconfigurable Optical Control of Plasmonic Local Field Enhancement and SERS.

Integration of plasmonic structures on step-index optical fibers is attracting interest for both applications and fundamental studies. However, the possibility to dynamically control the coupling between the guided light fields and the plasmonic resonances is hindered by the turbidity of light propagation in multimode fibers (MMFs). This pivotal point strongly limits the range of studies that can benefit from nanostructured fiber optics. Fortunately, harnessing the interaction between plasmonic modes on the fiber tip and the full set of guided modes can bring this technology to a next generation progress. Here, the intrinsic wealth of information of guided modes is exploited to spatiotemporally control the plasmonic resonances of the coupled system. This concept is shown by employing dynamic phase modulation to structure both the response of plasmonic MMFs on the plasmonic facet and their response in the corresponding Fourier plane, achieving spatial selective field enhancement and direct control of the probe's work point in the dispersion diagram. Such a conceptual leap would transform the biomedical applications of holographic endoscopic imaging by integrating new sensing and manipulation capabilities.

A protocol to extract cell-type-specific signatures from differentially expressed genes in bulk-tissue RNA-seq.

Bulk-tissue RNA-seq is widely used to dissect variation in gene expression levels across tissues and under different experimental conditions. Here, we introduce a protocol that leverages existing single-cell expression data to deconvolve patterns of cell-type-specific gene expression in differentially expressed gene lists from highly heterogeneous tissue. We apply this protocol to interrogate cell-type-specific gene expression and variation in cell type composition between the distinct sublayers of the hippocampal CA1 region of the brain in a rodent model of epilepsy. For complete details on the use and execution of this protocol, please refer to Cid et al. (2021).

Explorers of the cells: Toward cross-platform knowledge integration to evaluate neuronal function.

In this issue of Neuron, Petersen et al. (2021) introduce CellExplorer, an open-source tool to integrate neurophysiological metrics of neuronal activity from circuits to behavior. Together with other neuroinformatic resources, it may facilitate community-based multidisciplinary characterization of brain cell types.

Sublayer- and cell-type-specific neurodegenerative transcriptional trajectories in hippocampal sclerosis.

Hippocampal sclerosis, the major neuropathological hallmark of temporal lobe epilepsy, is characterized by different patterns of neuronal loss. The mechanisms of cell-type-specific vulnerability and their progression and histopathological classification remain controversial. Using single-cell electrophysiology in vivo and immediate-early gene expression, we reveal that superficial CA1 pyramidal neurons are overactive in epileptic rodents. Bulk tissue and single-nucleus expression profiling disclose sublayer-specific transcriptomic signatures and robust microglial pro-inflammatory responses. Transcripts regulating neuronal processes such as voltage channels, synaptic signaling, and cell adhesion are deregulated differently by epilepsy across sublayers, whereas neurodegenerative signatures primarily involve superficial cells. Pseudotime analysis of gene expression in single nuclei and in situ validation reveal separated trajectories from health to epilepsy across cell types and identify a subset of superficial cells undergoing a later stage in neurodegeneration. Our findings indicate that sublayer- and cell-type-specific changes associated with selective CA1 neuronal damage contribute to progression of hippocampal sclerosis.

An update to Hippocampome.org by integrating single-cell phenotypes with circuit function in vivo.

Understanding brain operation demands linking basic behavioral traits to cell-type specific dynamics of different brain-wide subcircuits. This requires a system to classify the basic operational modes of neurons and circuits. Single-cell phenotyping of firing behavior during ongoing oscillations in vivo has provided a large body of evidence on entorhinal-hippocampal function, but data are dispersed and diverse. Here, we mined literature to search for information regarding the phase-timing dynamics of over 100 hippocampal/entorhinal neuron types defined in Hippocampome.org. We identified missing and unresolved pieces of knowledge (e.g., the preferred theta phase for a specific neuron type) and complemented the dataset with our own new data. By confronting the effect of brain state and recording methods, we highlight the equivalences and differences across conditions and offer a number of novel observations. We show how a heuristic approach based on oscillatory features of morphologically identified neurons can aid in classifying extracellular recordings of single cells and discuss future opportunities and challenges towards integrating single-cell phenotypes with circuit function.

Multimodal determinants of phase-locked dynamics across deep-superficial hippocampal sublayers during theta oscillations.

Theta oscillations play a major role in temporarily defining the hippocampal rate code by translating behavioral sequences into neuronal representations. However, mechanisms constraining phase timing and cell-type-specific phase preference are unknown. Here, we employ computational models tuned with evolutionary algorithms to evaluate phase preference of individual CA1 pyramidal cells recorded in mice and rats not engaged in any particular memory task. We applied unbiased and hypothesis-free approaches to identify effects of intrinsic and synaptic factors, as well as cell morphology, in determining phase preference. We found that perisomatic inhibition delivered by complementary populations of basket cells interacts with input pathways to shape phase-locked specificity of deep and superficial pyramidal cells. Somatodendritic integration of fluctuating glutamatergic inputs defined cycle-by-cycle by unsupervised methods demonstrated that firing selection is tuneable across sublayers. Our data identify different mechanisms of phase-locking selectivity that are instrumental for flexible dynamical representations of theta sequences.

Potential factors influencing replay across CA1 during sharp-wave ripples.

Sharp-wave ripples are complex neurophysiological events recorded along the trisynaptic hippocampal circuit (i.e. from CA3 to CA1 and the subiculum) during slow-wave sleep and awake states. They arise locally but scale brain-wide to the hippocampal target regions at cortical and subcortical structures. During these events, neuronal firing sequences are replayed retrospectively or prospectively and in the forward or reverse order as defined by experience. They could reflect either pre-configured firing sequences, learned sequences or an option space to inform subsequent decisions. How can different sequences arise during sharp-wave ripples? Emerging data suggest the hippocampal circuit is organized in different loops across the proximal (close to dentate gyrus) and distal (close to entorhinal cortex) axis. These data also disclose a so-far neglected laminar organization of the hippocampal output during sharp-wave events. Here, I discuss whether by incorporating cell-type-specific mechanisms converging on deep and superficial CA1 sublayers along the proximodistal axis, some novel factors influencing the organization of hippocampal sequences could be unveiled. This article is part of the Theo Murphy meeting issue 'Memory reactivation: replaying events past, present and future'.

Methods for single-cell recording and labeling in vivo.

Targeting individual neurons in vivo is a key method to study the role of single cell types within local and brain-wide microcircuits. While novel technological developments now permit assessing activity from large number of cells simultaneously, there is currently no better solution than glass micropipettes to relate the physiology and morphology of single-cells. Sharp intracellular, juxtacellular, loose-patch and whole-cell approaches are some of the configurations used to record and label individual neurons. Here, we review procedures to establish successful electrophysiological recordings in vivo followed by appropriate labeling for post hoc morphological analysis. We provide operational recommendations for optimizing each configuration and a generic framework for functional, neurochemical and morphological identification of the different cell-types in a given region. Finally, we highlight emerging approaches that are challenging our current paradigms for single-cell recording and labeling in the living brain.