Cholinergic suppression of hippocampal sharp-wave ripples impairs working memory.

Learning and memory are assumed to be supported by mechanisms that involve cholinergic transmission and hippocampal theta. Using G protein-coupled receptor-activation-based acetylcholine sensor (GRABACh3.0) with a fiber-photometric fluorescence readout in mice, we found that cholinergic signaling in the hippocampus increased in parallel with theta/gamma power during walking and REM sleep, while ACh3.0 signal reached a minimum during hippocampal sharp-wave ripples (SPW-R). Unexpectedly, memory performance was impaired in a hippocampus-dependent spontaneous alternation task by selective optogenetic stimulation of medial septal cholinergic neurons when the stimulation was applied in the delay area but not in the central (choice) arm of the maze. Parallel with the decreased performance, optogenetic stimulation decreased the incidence of SPW-Rs. These findings suggest that septo-hippocampal interactions play a task-phase-dependent dual role in the maintenance of memory performance, including not only theta mechanisms but also SPW-Rs.

Novel expression system based on enhanced permeability of Vibrio natriegens cells induced by D,D- carboxypeptidase overexpression.

Vibrio natriegens is a fast-growing, non-pathogenic marine bacterium with promising features for biotechnological applications such as high-level recombinant protein production or fast DNA propagation. A remarkable short generation time (< 10 min), robust proteosynthetic activity and versatile metabolism with abilities to utilise wide range of substrates contribute to its establishment as a future industrial platform for fermentation processes operating with high productivity.D,D-carboxypeptidases are membrane-associated enzymes involved in peptidoglycan biosynthesis and cell wall formation. This study investigates the impact of overexpressed D,D-carboxypeptidases on membrane integrity and the increased leakage of intracellular proteins into the growth medium in V. natriegens. Our findings confirm that co-expression of these enzymes can enhance membrane permeability, thereby facilitating the transport of target proteins into the extracellular environment, without the need for secretion signals, tags, or additional permeabilization methods. Using only a single step IMAC chromatography, we were able to purify AfKatG, MDBP or Taq polymerase in total yields of 117.9 ± 56.0 mg/L, 36.5 ± 12.9 mg/L and 26.5 ± 6.0 mg/L directly from growth medium, respectively. These results demonstrate the feasibility of our V. natriegens based system as a broadly applicable extracellular tag-less recombinant protein producer.

Optogenetic activation of septal cholinergic neurons suppresses sharp wave ripples and enhances theta oscillations in the hippocampus.

Theta oscillations in the limbic system depend on the integrity of the medial septum. The different populations of medial septal neurons (cholinergic and GABAergic) are assumed to affect different aspects of theta oscillations. Using optogenetic stimulation of cholinergic neurons in ChAT-Cre mice, we investigated their effects on hippocampal local field potentials in both anesthetized and behaving mice. Cholinergic stimulation completely blocked sharp wave ripples and strongly suppressed the power of both slow oscillations (0.5-2 Hz in anesthetized, 0.5-4 Hz in behaving animals) and supratheta (6-10 Hz in anesthetized, 10-25 Hz in behaving animals) bands. The same stimulation robustly increased both the power and coherence of theta oscillations (2-6 Hz) in urethane-anesthetized mice. In behaving mice, cholinergic stimulation was less effective in the theta (4-10 Hz) band yet it also increased the ratio of theta/slow oscillation and theta coherence. The effects on gamma oscillations largely mirrored those of theta. These findings show that medial septal cholinergic activation can both enhance theta rhythm and suppress peri-theta frequency bands, allowing theta oscillations to dominate.

Divergent in vivo activity of non-serotonergic and serotonergic VGluT3-neurones in the median raphe region.

KEY POINTS: The median raphe is a key subcortical modulatory centre involved in several brain functions, such as regulation of the sleep-wake cycle, emotions and memory storage. A large proportion of median raphe neurones are glutamatergic and implement a radically different mode of communication compared to serotonergic cells, although their in vivo activity is unknown. We provide the first description of the in vivo, brain state-dependent firing properties of median raphe glutamatergic neurones identified by immunopositivity for the vesicular glutamate transporter type 3 (VGluT3) and serotonin (5-HT). Glutamatergic populations (VGluT3+/5-HT- and VGluT3+/5-HT+) were compared with the purely serotonergic (VGluT3-/5-HT+ and VGluT3-/5-HT-) neurones. VGluT3+/5-HT+ neurones fired similar to VGluT3-/5-HT+ cells, whereas they significantly diverged from the VGluT3+/5-HT- population. Activity of the latter subgroup resembled the spiking of VGluT3-/5-HT- cells, except for their diverging response to sensory stimulation. The VGluT3+ population of the median raphe may broadcast rapidly varying signals on top of a state-dependent, tonic modulation. ABSTRACT: Subcortical modulation is crucial for information processing in the cerebral cortex. Besides the canonical neuromodulators, glutamate has recently been identified as a key cotransmitter of numerous monoaminergic projections. In the median raphe, a pure glutamatergic neurone population projecting to limbic areas was also discovered with a possibly novel, yet undetermined function. In the present study, we report the first functional description of the vesicular glutamate transporter type 3 (VGluT3)-expressing median raphe neurones. Because there is no appropriate genetic marker for the separation of serotonergic (5-HT+) and non-serotonergic (5-HT-) VGluT3+ neurones, we utilized immunohistochemistry after recording and juxtacellular labelling in anaesthetized rats. VGluT3+/5-HT- neurones fired faster, more variably and were permanently activated during sensory stimulation, as opposed to the transient response of the slow firing VGluT3-/5-HT+ subgroup. VGluT3+/5-HT- cells were also more active during hippocampal theta. In addition, the VGluT3-/5-HT- population, comprising putative GABAergic cells, resembled the firing of VGluT3+/5-HT- neurones but without any significant reaction to the sensory stimulus. Interestingly, the VGluT3+/5-HT+ group, spiking slower than the VGluT3+/5-HT- population, exhibited a mixed response (i.e. the initial transient activation was followed by a sustained elevation of firing). Phase coupling to hippocampal and prefrontal slow oscillations was found in VGluT3+/5-HT- neurones, also differentiating them from the VGluT3+/5-HT+ subpopulation. Taken together, glutamatergic neurones in the median raphe may implement multiple, highly divergent forms of modulation in parallel: a slow, tonic mode interrupted by sensory-evoked rapid transients, as well as a fast one capable of conveying complex patterns influenced by sensory inputs.

Working memory features are embedded in hippocampal place fields.

Hippocampal principal neurons display both spatial tuning properties and memory features. Whether this distinction corresponds to separate neuron types or a context-dependent continuum has been debated. We report here that the task-context ("splitter") feature is highly variable along both trial and spatial position axes. Neurons acquire or lose splitter features across trials even when place field features remain unaltered. Multiple place fields of the same neuron can individually encode both past or future run trajectories, implying that splitter fields are under the control of assembly activity. Place fields can be differentiated into subfields by the behavioral choice of the animal, and splitting within subfields evolves across trials. Interneurons also differentiate choices by integrating inputs from pyramidal cells. Finally, bilateral optogenetic inactivation of the medial entorhinal cortex reversibly decreases the fraction of splitter fields. Our findings suggest that place or splitter features are different manifestations of the same hippocampal computation.

Aversive stimulus-tuned responses in the CA1 of the dorsal hippocampus.

Throughout life animals inevitably encounter unforeseen threatening events. Activity of principal cells in the hippocampus is tuned for locations and for salient stimuli in the animals' environment thus forming a map known to be pivotal for guiding behavior. Here, we explored if a code of threatening stimuli exists in the CA1 region of the dorsal hippocampus of mice by recording neuronal response to aversive stimuli delivered at changing locations. We have discovered a rapidly emerging, location independent response to innoxious aversive stimuli composed of the coordinated activation of subgroups of pyramidal cells and connected interneurons. Activated pyramidal cells had higher basal firing rate, more probably participated in ripples, targeted more interneurons than place cells and many of them lacked place fields. We also detected aversive stimulus-coupled assemblies dominated by the activated neurons. Notably, these assemblies could be observed even before the delivery of the first aversive event. Finally, we uncovered the systematic shift of the spatial code from the aversive to, surprisingly, the reward location during the fearful stimulus. Our results uncovered components of the dorsal CA1 circuit possibly key for re-sculpting the spatial map in response to abrupt aversive events.

The medial septum controls hippocampal supra-theta oscillations.

Hippocampal theta oscillations orchestrate faster beta-to-gamma oscillations facilitating the segmentation of neural representations during navigation and episodic memory. Supra-theta rhythms of hippocampal CA1 are coordinated by local interactions as well as inputs from the entorhinal cortex (EC) and CA3 inputs. However, theta-nested gamma-band activity in the medial septum (MS) suggests that the MS may control supra-theta CA1 oscillations. To address this, we performed multi-electrode recordings of MS and CA1 activity in rodents and found that MS neuron firing showed strong phase-coupling to theta-nested supra-theta episodes and predicted changes in CA1 beta-to-gamma oscillations on a cycle-by-cycle basis. Unique coupling patterns of anatomically defined MS cell types suggested that indirect MS-to-CA1 pathways via the EC and CA3 mediate distinct CA1 gamma-band oscillations. Optogenetic activation of MS parvalbumin-expressing neurons elicited theta-nested beta-to-gamma oscillations in CA1. Thus, the MS orchestrates hippocampal network activity at multiple temporal scales to mediate memory encoding and retrieval.

Complex propagation patterns characterize human cortical activity during slow-wave sleep.

Cortical electrical activity during nonrapid eye movement (non-REM) sleep is dominated by slow-wave activity (SWA). At larger spatial scales (∼2-30 cm), investigated by scalp EEG recordings, SWA has been shown to propagate globally over wide cortical regions as traveling waves, which has been proposed to serve as a temporal framework for neural plasticity. However, whether SWA dynamics at finer spatial scales also reflects the orderly propagation has not previously been investigated in humans. To reveal the local, finer spatial scale (∼1-6 cm) patterns of SWA propagation during non-REM sleep, electrocorticographic (ECoG) recordings were conducted from subdurally implanted electrode grids and a nonlinear correlation technique [mutual information (MI)] was implemented. MI analysis revealed spatial maps of correlations between cortical areas demonstrating SWA propagation directions, speed, and association strength. Highest correlations, indicating significant coupling, were detected during the initial positive-going deflection of slow waves. SWA propagated predominantly between adjacent cortical areas, albeit spatial noncontinuities were also frequently observed. MI analysis further uncovered significant convergence and divergence patterns. Areas receiving the most convergent activity were similar to those with high divergence rate, while reciprocal and circular propagation of SWA was also frequent. We hypothesize that SWA is characterized by distinct attributes depending on the spatial scale observed. At larger spatial scales, the orderly SWA propagation dominates; at the finer scale of the ECoG recordings, non-REM sleep is characterized by complex SWA propagation patterns.

Automated high throughput whole slide imaging using area sensors, flash light illumination and solid state light engine.

BACKGROUND: Whole Slide Imagers or digital slide scanners have developed very rapidly in the last 8 years and went through three generations. Third generation instruments have just reached the market which have the stability and throughput to be used for routine clinical work. We describe in this article the technical background and reasoning behind engineering decisions we made during the development of 3DHISTECH's 3rd generation combined brightfield and fluorescent scanner. MATERIALS AND METHODS: The Panoramic 250 FLASH utilizes Plan-Apochromat 20x and 40x objectives, a 2 megapixel 3CCD camera for brightfield and a monochrome scientific CMOS camera for fluorescent scanning. A solid state light engine for fluorescent and a strobe light for bright field illumination are used. RESULTS: The system can scan 1cm2 including focusing at 45x resolution in 1 minute. It can scan a well stained DAPI, FITC, TRIC, 1cm2 fluorescent slide in 11 minutes. It can load and scan 250 slides in walk away mode. CONCLUSION: Using the latest camera technology and electronics, state of the art computer and standard microscope optical components high throughput high quality whole slide imaging is feasible and is sufficient for most of the routine diagnostic work. Extended depth of field and Z-stack scanning is possible with the use of area scan technology.

Tracking Highly Similar Rat Instances under Heavy Occlusions: An Unsupervised Deep Generative Pipeline.

Identity tracking and instance segmentation are crucial in several areas of biological research. Behavior analysis of individuals in groups of similar animals is a task that emerges frequently in agriculture or pharmaceutical studies, among others. Automated annotation of many hours of surveillance videos can facilitate a large number of biological studies/experiments, which otherwise would not be feasible. Solutions based on machine learning generally perform well in tracking and instance segmentation; however, in the case of identical, unmarked instances (e.g., white rats or mice), even state-of-the-art approaches can frequently fail. We propose a pipeline of deep generative models for identity tracking and instance segmentation of highly similar instances, which, in contrast to most region-based approaches, exploits edge information and consequently helps to resolve ambiguity in heavily occluded cases. Our method is trained by synthetic data generation techniques, not requiring prior human annotation. We show that our approach greatly outperforms other state-of-the-art unsupervised methods in identity tracking and instance segmentation of unmarked rats in real-world laboratory video recordings.

Huygens synchronization of medial septal pacemaker neurons generates hippocampal theta oscillation.

Episodic learning and memory retrieval are dependent on hippocampal theta oscillation, thought to rely on the GABAergic network of the medial septum (MS). To test how this network achieves theta synchrony, we recorded MS neurons and hippocampal local field potential simultaneously in anesthetized and awake mice and rats. We show that MS pacemakers synchronize their individual rhythmicity frequencies, akin to coupled pendulum clocks as observed by Huygens. We optogenetically identified them as parvalbumin-expressing GABAergic neurons, while MS glutamatergic neurons provide tonic excitation sufficient to induce theta. In accordance, waxing and waning tonic excitation is sufficient to toggle between theta and non-theta states in a network model of single-compartment inhibitory pacemaker neurons. These results provide experimental and theoretical support to a frequency-synchronization mechanism for pacing hippocampal theta, which may serve as an inspirational prototype for synchronization processes in the central nervous system from Nematoda to Arthropoda to Chordate and Vertebrate phyla.

Uncovering spatial representations from spatiotemporal patterns of rodent hippocampal field potentials.

Spatiotemporal patterns of large-scale spiking and field potentials of the rodent hippocampus encode spatial representations during maze runs, immobility, and sleep. Here, we show that multisite hippocampal field potential amplitude at ultra-high-frequency band (FPAuhf), a generalized form of multiunit activity, provides not only a fast and reliable reconstruction of the rodent's position when awake, but also a readout of replay content during sharp-wave ripples. This FPAuhf feature may serve as a robust real-time decoding strategy from large-scale recordings in closed-loop experiments. Furthermore, we develop unsupervised learning approaches to extract low-dimensional spatiotemporal FPAuhf features during run and ripple periods and to infer latent dynamical structures from lower-rank FPAuhf features. We also develop an optical flow-based method to identify propagating spatiotemporal LFP patterns from multisite array recordings, which can be used as a decoding application. Finally, we develop a prospective decoding strategy to predict an animal's future decision in goal-directed navigation.

Do not waste your electrodes-principles of optimal electrode geometry for spike sorting.

Objective. This study examines how the geometrical arrangement of electrodes influences spike sorting efficiency, and attempts to formalise principles for the design of electrode systems enabling optimal spike sorting performance.Approach. The clustering performance of KlustaKwik, a popular toolbox, was evaluated using semi-artificial multi-channel data, generated from a library of real spike waveforms recorded in the CA1 region of mouse Hippocampusin vivo.Main results. Based on spike sorting results under various channel configurations and signal levels, a simple model was established to describe the efficiency of different electrode geometries. Model parameters can be inferred from existing spike waveform recordings, which allowed quantifying both the cooperative effect between channels and the noise dependence of clustering performance.Significance. Based on the model, analytical and numerical results can be derived for the optimal spacing and arrangement of electrodes for one- and two-dimensional electrode systems, targeting specific brain areas.

Activity and Coupling to Hippocampal Oscillations of Median Raphe GABAergic Cells in Awake Mice.

Ascending serotonergic/glutamatergic projection from the median raphe region (MRR) to the hippocampal formation regulates both encoding and consolidation of memory and the oscillations associated with them. The firing of various types of MRR neurons exhibits rhythmic modulation coupled to hippocampal oscillatory activity. A possible intermediary between rhythm-generating forebrain regions and entrained ascending modulation may be the GABAergic circuit in the MRR, known to be targeted by a diverse array of top-down inputs. However, the activity of inhibitory MRR neurons in an awake animal is still largely unexplored. In this study, we utilized whole cell patch-clamp, single cell, and multichannel extracellular recordings of GABAergic and non-GABAergic MRR neurons in awake, head-fixed mice. First, we have demonstrated that glutamatergic and serotonergic neurons receive both transient, phasic, and sustained tonic inhibition. Then, we observed substantial heterogeneity of GABAergic firing patterns but a marked modulation of activity by brain states and fine timescale coupling of spiking to theta and ripple oscillations. We also uncovered a correlation between the preferred theta phase and the direction of activity change during ripples, suggesting the segregation of inhibitory neurons into functional groups. Finally, we could detect complementary alteration of non-GABAergic neurons' ripple-coupled activity. Our findings support the assumption that the local inhibitory circuit in the MRR may synchronize ascending serotonergic/glutamatergic modulation with hippocampal activity on a subsecond timescale.

Recombinant Enzymatic Redox Systems for Preparation of Aroma Compounds by Biotransformation.

The aim of this study was to develop immobilized enzyme systems that reduce carbonyl compounds to their corresponding alcohols. The demand for natural aromas and food additives has been constantly growing in recent years. However, it can no longer be met by extraction and isolation from natural materials. One way to increase the availability of natural aromas is to prepare them by the enzymatic transformation of suitable precursors. Recombinant enzymes are currently being used for this purpose. We investigated trans-2-hexenal bioreduction by recombinant Saccharomyces cerevisiae alcohol dehydrogenase (ScADH1) with simultaneous NADH regeneration by recombinant Candida boidinii formate dehydrogenase (FDH). In a laboratory bioreactor with two immobilized enzymes, 88% of the trans-2-hexenal was transformed to trans-2-hexenol. The initial substrate concentration was 3.7 mM. The aldehyde destabilized ScADH1 by eluting Zn2+ ions from the enzyme. A fed-batch operation was used and the trans-2-hexenal concentration was maintained at a low level to limit the negative effect of Zn2+ ion elution from the immobilized ScADH1. Another immobilized two-enzyme system was used to reduce acetophenone to (S)-1-phenylethanol. To this end, the recombinant alcohol dehydrogenase (RrADH) from Rhodococcus ruber was used. This biocatalytic system converted 61% of the acetophenone to (S)-1-phenylethanol. The initial substrate concentration was 8.3 mM. All enzymes were immobilized by poly-His tag to Ni2+, which formed strong but reversible bonds that enabled carrier reuse after the loss of enzyme activity.

GABAergic neurons of the medial septum lead the hippocampal network during theta activity.

Information processing in the hippocampus critically relies on its reciprocal interaction with the medial septum (MS). Synchronization of the septo-hippocampal system was demonstrated during both major hippocampal activity states, the regular theta rhythm and the large amplitude irregular activity. Previous experimental and modeling data suggest that the MS provides rhythmic drive to the hippocampus, and hippocampo-septal feedback synchronizes septal pacemaker units. However, this view has recently been questioned based on the possibility of intrahippocampal theta genesis. Previously, we identified putative pacemaker neurons expressing parvalbumin (PV) and/or the pacemaker hyperpolarization-activated and cyclic nucleotide-gated nonselective cation channel (HCN) in the MS. In this study, by analyzing the temporal relationship of activity between the PV/HCN-containing medial septal neurons and hippocampal local field potential, we aimed to uncover whether the sequence of events during theta formation supports the classic view of septal drive or the challenging theory of hippocampal pacing of theta. Importantly, by implementing a circular statistical method, a temporal lead of these septal neurons over the hippocampus was observed on the course of theta synchronization. Moreover, the activity of putative hippocampal interneurons also preceded hippocampal local field theta, but by a shorter time period compared with PV/HCN-containing septal neurons. Using the concept of mutual information, the action potential series of PV/HCN-containing neurons shared higher amount of information with hippocampal field oscillation than PV/HCN-immunonegative cells. Thus, a pacemaker neuron population of the MS leads hippocampal activity, presumably via the synchronization of hippocampal interneurons.

Automated multichannel fluorescent whole slide imaging and its application for cytometry.

Slide-based image cytometry (SBC) has several advantages over flow cytometry but it is not widely used because of its low throughput, complicated workflow, and high price. Fully automated microscopes became affordable with the advent of whole slide imaging (WSI) and they can be transformed into a cytometer. A MIRAX MIDI automated whole slide imager was used with metal-halide and light emitting diode (LED)-based fluorescent illumination, filter block changer, and a cooled monochrome charge coupled device camera. The MIRAX control software was further developed for fluorescent sample detection, autofocusing, multichannel digitization, and signal correction due to nonuniform illumination. Fluorescent calibration beads were used to verify the linearity of the system. The HistoQuant software package of the MIRAX viewer was used for image segmentation and quantitative analysis. The data was displayed by the histogram, scatter plot, and gallery functions of the same program. Fluorescent samples can be reliably detected, focused, and scanned. The measured integrated fluorescence showed linearity with exposure time and staining intensity. Automated fluorescent WSI with stable LED illumination and high-quality homogeneous fluorescent slides can be used conveniently for SBC.

Median raphe controls acquisition of negative experience in the mouse.

Adverse events need to be quickly evaluated and memorized, yet how these processes are coordinated is poorly understood. We discovered a large population of excitatory neurons in mouse median raphe region (MRR) expressing vesicular glutamate transporter 2 (vGluT2) that received inputs from several negative experience-related brain centers, projected to the main aversion centers, and activated the septohippocampal system pivotal for learning of adverse events. These neurons were selectively activated by aversive but not rewarding stimuli. Their stimulation induced place aversion, aggression, depression-related anhedonia, and suppression of reward-seeking behavior and memory acquisition-promoting hippocampal theta oscillations. By contrast, their suppression impaired both contextual and cued fear memory formation. These results suggest that MRR vGluT2 neurons are crucial for the acquisition of negative experiences and may play a central role in depression-related mood disorders.

Brainstem nucleus incertus controls contextual memory formation.

Hippocampal pyramidal cells encode memory engrams, which guide adaptive behavior. Selection of engram-forming cells is regulated by somatostatin-positive dendrite-targeting interneurons, which inhibit pyramidal cells that are not required for memory formation. Here, we found that γ-aminobutyric acid (GABA)-releasing neurons of the mouse nucleus incertus (NI) selectively inhibit somatostatin-positive interneurons in the hippocampus, both monosynaptically and indirectly through the inhibition of their subcortical excitatory inputs. We demonstrated that NI GABAergic neurons receive monosynaptic inputs from brain areas processing important environmental information, and their hippocampal projections are strongly activated by salient environmental inputs in vivo. Optogenetic manipulations of NI GABAergic neurons can shift hippocampal network state and bidirectionally modify the strength of contextual fear memory formation. Our results indicate that brainstem NI GABAergic cells are essential for controlling contextual memories.