A-SOiD, an active-learning platform for expert-guided, data-efficient discovery of behavior.

To identify and extract naturalistic behavior, two methods have become popular: supervised and unsupervised. Each approach carries its own strengths and weaknesses (for example, user bias, training cost, complexity and action discovery), which the user must consider in their decision. Here, an active-learning platform, A-SOiD, blends these strengths, and in doing so, overcomes several of their inherent drawbacks. A-SOiD iteratively learns user-defined groups with a fraction of the usual training data, while attaining expansive classification through directed unsupervised classification. In socially interacting mice, A-SOiD outperformed standard methods despite requiring 85% less training data. Additionally, it isolated ethologically distinct mouse interactions via unsupervised classification. We observed similar performance and efficiency using nonhuman primate and human three-dimensional pose data. In both cases, the transparency in A-SOiD's cluster definitions revealed the defining features of the supervised classification through a game-theoretic approach. To facilitate use, A-SOiD comes as an intuitive, open-source interface for efficient segmentation of user-defined behaviors and discovered sub-actions.

Induced Remodelling of Astrocytes In Vitro and In Vivo by Manipulation of Astrocytic RhoA Activity.

Structural changes of astrocytes and their perisynaptic processes occur in response to various physiological and pathophysiological stimuli. They are thought to profoundly affect synaptic signalling and neuron-astrocyte communication. Understanding the causal relationship between astrocyte morphology changes and their functional consequences requires experimental tools to selectively manipulate astrocyte morphology. Previous studies indicate that RhoA-related signalling can play a major role in controlling astrocyte morphology, but the direct effect of increased RhoA activity has not been documented in vitro and in vivo. Therefore, we established a viral approach to manipulate astrocytic RhoA activity. We tested if and how overexpression of wild-type RhoA, of a constitutively active RhoA mutant (RhoA-CA), and of a dominant-negative RhoA variant changes the morphology of cultured astrocytes. We found that astrocytic expression of RhoA-CA induced robust cytoskeletal changes and a withdrawal of processes in cultured astrocytes. In contrast, overexpression of other RhoA variants led to more variable changes of astrocyte morphology. These induced morphology changes were reproduced in astrocytes of the hippocampus in vivo. Importantly, astrocytic overexpression of RhoA-CA did not alter the branching pattern of larger GFAP-positive processes of astrocytes. This indicates that a prolonged increase of astrocytic RhoA activity leads to a distinct morphological phenotype in vitro and in vivo, which is characterized by an isolated reduction of fine peripheral astrocyte processes in vivo. At the same time, we identified a promising experimental approach for investigating the functional consequences of astrocyte morphology changes.

Deletion of the P/Q-type calcium channel from serotonergic neurons drives male aggression in mice.

Aggressive behavior is one of the most conserved social interactions in nature and serves as a crucial evolutionary trait. Serotonin (5-HT) plays a key role in the regulation of our emotions such as anxiety and aggression, but which molecules and mechanisms in the serotonergic system are involved in violent behavior is still unknown. In this study we show that deletion of the P/Q-type calcium channel selectively from serotonergic neurons in the dorsal raphe nuclei (DRN) augments aggressive behavior in male mice, while anxiety is not affected. These mice demonstrated increased induction of the immediate early gene c-fos and in vivo serotonergic firing activity in the DRN. The ventrolateral part of the ventromedial hypothalamus (VHMvl) is also a prominent region of the brain mediating aggression. We confirmed a monosynaptic projection from the DRN to the VHMvl and silencing these projections with an inhibitory designer receptor exclusively activated by a designer drug (DREADD) effectively reduced aggressive behavior. Overall, our findings show that deletion of the P/Q-type calcium channel from DRN neurons is sufficient to induce male aggression in mice and regulating its activity may serve as a therapeutic approach to treat violent behavior.SIGNIFICANCE STATEMENTIn this study we show that P/Q-type calcium channel is mediating aggression in serotonergic neurons from the dorsal raphe nucleus via monosynaptic projections to the ventrolateral part of the ventromedial hypothalamus. More importantly, silencing these projections reduced aggressive behavior in mice and may serve as a therapeutic approach for treating aggression in humans.

DeepLabStream enables closed-loop behavioral experiments using deep learning-based markerless, real-time posture detection.

In general, animal behavior can be described as the neuronal-driven sequence of reoccurring postures through time. Most of the available current technologies focus on offline pose estimation with high spatiotemporal resolution. However, to correlate behavior with neuronal activity it is often necessary to detect and react online to behavioral expressions. Here we present DeepLabStream, a versatile closed-loop tool providing real-time pose estimation to deliver posture dependent stimulations. DeepLabStream has a temporal resolution in the millisecond range, can utilize different input, as well as output devices and can be tailored to multiple experimental designs. We employ DeepLabStream to semi-autonomously run a second-order olfactory conditioning task with freely moving mice and optogenetically label neuronal ensembles active during specific head directions.

Epothilones Improve Axonal Growth and Motor Outcomes after Stroke in the Adult Mammalian CNS.

Stroke leads to the degeneration of short-range and long-range axonal connections emanating from peri-infarct tissue, but it also induces novel axonal projections. However, this regeneration is hampered by growth-inhibitory properties of peri-infarct tissue and fibrotic scarring. Here, we tested the effects of epothilone B and epothilone D, FDA-approved microtubule-stabilizing drugs that are powerful modulators of axonal growth and scar formation, on neuroplasticity and motor outcomes in a photothrombotic mouse model of cortical stroke. We find that both drugs, when administered systemically 1 and 15 days after stroke, augment novel peri-infarct projections connecting the peri-infarct motor cortex with neighboring areas. Both drugs also increase the magnitude of long-range motor projections into the brainstem and reduce peri-infarct fibrotic scarring. Finally, epothilone treatment induces an improvement in skilled forelimb motor function. Thus, pharmacological microtubule stabilization represents a promising target for therapeutic intervention with a wide time window to ameliorate structural and functional sequelae after stroke.

Local Efficacy of Glutamate Uptake Decreases with Synapse Size.

Synaptically released glutamate is largely cleared by glutamate transporters localized on perisynaptic astrocyte processes. Therefore, the substantial variability of astrocyte coverage of individual hippocampal synapses implies that the efficacy of local glutamate uptake and thus the spatial fidelity of synaptic transmission is synapse dependent. By visualization of sub-diffraction-limit perisynaptic astrocytic processes and adjacent postsynaptic spines, we show that, relative to their size, small spines display a stronger coverage by astroglial transporters than bigger neighboring spines. Similarly, glutamate transients evoked by synaptic stimulation are more sensitive to pharmacological inhibition of glutamate uptake at smaller spines, whose high-affinity N-methyl-D-aspartate receptors (NMDARs) are better shielded from remotely released glutamate. At small spines, glutamate-induced and NMDAR-dependent Ca2+ entry is also more strongly increased by uptake inhibition. These findings indicate that spine size inversely correlates with the efficacy of local glutamate uptake and thereby likely determines the probability of synaptic crosstalk.

Cortical layer 6 control of sensory responses in higher-order thalamus.

KEY POINTS: Thalamic activity is regulated by corticothalamic feedback from layers 5B and 6. To selectively study the importance of the layer 6 corticothalamic (L6 CT) projection, a transgenic mouse line was used in which layer 6 cells projecting to posterior medial thalamus (POm) were targeted for expression of channelrhodopsin-2. Repetitive optogenetic stimulation of this sub-type of L6 cells caused a rapid adaptation in POm spiking output, but had little effect on the spiking activity in the other cortical layers. L6 photoactivation increased POm spiking to the first, but not to subsequent whisker deflections in a 4 Hz train. A sub-population of L6 CT cells that can cause an initial increase in POm activity, that is not sustained with repetitive stimulation, could indicate that this L6 projection does not modulate ongoing sensory processing, but rather serves to briefly increase POm activity in specific behavioural contexts. ABSTRACT: Thalamic activity is regulated by corticothalamic feedback from layers 5B and 6. The nature of these feedback systems differs, one difference being that whereas layer 5 provides 'driver' input, the layer 6 input is thought to be 'modulatory'. To selectively study the importance of the layer 6 corticothalamic (L6 CT) projection, a transgenic mouse line was used in which layer 6 cells projecting to posterior medial thalamus (POm) were targeted for expression of channelrhodopsin-2 and in vivo electrophysiology recordings were done in urethane-anaesthetized mice. Pre- and postsynaptic targets were identified using tracing techniques and light-sheet microscopy in cleared intact brains. We find that optogenetic activation of this subtype of L6 CT cells (L6-Drd1) has little effect on cortical activity, but activates POm. Repetitive photoactivation of L6-Drd1 cells evoked a reliable response following every photoactivation, whereas in the connected POm area spiking was only initially increased. The response to repetitive whisker stimulation showed a similar pattern with only an initial increase in whisker-evoked spiking. Furthermore, the increase in whisker-evoked spiking with optogenetic activation of L6-Drd1 cells is additive, rather than multiplicative, causing even cells that in the absence of L6 activation produce relatively few spikes to increase their spiking substantially. We show that layer 6 corticothalamic cells can provide a strong, albeit rapidly depressing, input to POm. This type of cortical L6 activity could be important for rapid gain control in POm, rather than providing a modulation in phase with the whisking cycle.

Author Correction: Shifted pallidal co-release of GABA and glutamate in habenula drives cocaine withdrawal and relapse.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Enhanced adenosine A1 receptor and Homer1a expression in hippocampus modulates the resilience to stress-induced depression-like behavior.

Resilience to stress is critical for the development of depression. Enhanced adenosine A1 receptor (A1R) signaling mediates the antidepressant effects of acute sleep deprivation (SD). However, chronic SD causes long-lasting upregulation of brain A1R and increases the risk of depression. To investigate the effects of A1R on mood, we utilized two transgenic mouse lines with inducible A1R overexpression in forebrain neurons. These two lines have identical levels of A1R increase in the cortex, but differ in the transgenic A1R expression in the hippocampus. Switching on the transgene promotes robust antidepressant and anxiolytic effects in both lines. The mice of the line without transgenic A1R overexpression in the hippocampus (A1Hipp-) show very strong resistance towards development of stress-induced chronic depression-like behavior. In contrast, the mice of the line in which A1R upregulation extends to the hippocampus (A1Hipp+), exhibit decreased resilience to depression as compared to A1Hipp-. Similarly, automatic analysis of reward behavior of the two lines reveals that depression resistant A1Hipp-transgenic mice exhibit high sucrose preference, while mice of the vulnerable A1Hipp + line developed stress-induced anhedonic phenotype. The A1Hipp + mice have increased Homer1a expression in hippocampus, correlating with impaired long-term potentiation in the CA1 region, mimicking the stressed mice. Furthermore, virus-mediated overexpression of Homer1a in the hippocampus decreases stress resilience. Taken together our data indicate for first time that increased expression of A1R and Homer1a in the hippocampus modulates the resilience to stress-induced depression and thus might potentially mediate the detrimental effects of chronic sleep restriction on mood.

A New Projection From the Deep Cerebellar Nuclei to the Hippocampus via the Ventrolateral and Laterodorsal Thalamus in Mice.

The cerebellar involvement in cognitive functions such as attention, language, working memory, emotion, goal-directed behavior and spatial navigation is constantly growing. However, an exact connectivity map between the hippocampus and cerebellum in mice is still unknown. Here, we conducted a tracing study to identify the sequence of transsynaptic, cerebellar-hippocampal connections in the mouse brain using combinations of Recombinant adeno-associated virus (rAAV) and pseudotyped deletion-mutant rabies (RABV) viruses. Stereotaxic injection of a primarily anterograde rAAV-WGA (wheat germ agglutinin)-Cre tracer virus in the deep cerebellar nuclei (DCN) of a Cre-dependent tdTomato reporter mouse resulted in strong tdTomato labeling in hippocampal CA1 neurons, retrosplenial cortex (RSC), rhinal cortex (RC) as well as thalamic and cerebellar areas. Whereas hippocampal injections with the retrograde tracer virus rAAV-TTC (tetanus toxin C fragment)-eGFP, displayed eGFP positive cells in the rhinal cortex and subiculum. To determine the sequence of mono-transsynaptic connections between the cerebellum and hippocampus, we used the retrograde tracer RABVΔG-eGFP(EnvA). The tracing revealed a direct connection from the dentate gyrus (DG) in the hippocampus to the RSC, RC and subiculum (S), which are monosynaptically connected to thalamic laterodorsal and ventrolateral areas. These thalamic nuclei are directly connected to cerebellar fastigial (FN), interposed (IntP) and lateral (Lat) nuclei, discovering a new projection route from the fastigial to the laterodorsal thalamic nucleus in the mouse brain. Collectively, our findings suggest a new cerebellar-hippocampal connection via the laterodorsal and ventrolateral thalamus to RSC, RC and S. These results strengthen the notion of the cerebellum's involvement in cognitive functions such as spatial navigation via a polysynaptic circuitry.

Enhanced mGlu5 Signaling in Excitatory Neurons Promotes Rapid Antidepressant Effects via AMPA Receptor Activation.

Conventional antidepressants have limited efficacy and many side effects, highlighting the need for fast-acting and specific medications. Induction of the synaptic protein Homer1a mediates the effects of different antidepressant treatments, including the rapid action of ketamine and sleep deprivation (SD). We show here that mimicking Homer1a upregulation via intravenous injection of cell-membrane-permeable TAT-Homer1a elicits rapid antidepressant effects in various tests. Similar to ketamine and SD, in vitro and in vivo application of TAT-Homer1a enhances mGlu5 signaling, resulting in increased mTOR pathway phosphorylation, and upregulates synaptic AMPA receptor expression and activity. The antidepressant action of SD and Homer1a induction depends on mGlu5 activation specifically in excitatory CaMK2a neurons and requires enhanced AMPA receptor activity, translation, and trafficking. Moreover, our data demonstrate a pronounced therapeutic potential of different TAT-fused peptides that directly modulate mGlu5 and AMPA receptor activity and thus might provide a novel strategy for rapid and effective antidepressant treatment.

Rabies virus-mediated connectivity tracing from single neurons.

An understanding of how the brain processes information requires knowledge of its underlying wiring diagrams, as well as insights into the relationship between circuit architecture and physiological function. Notably, rabies virus based single-cell genetic manipulations that can facilitate an experimental link between physiology and genetics have recently advanced the field of systems neuroscience. It allows capturing the synaptic and the anatomical receptive fields of individual neurons. Recently, the methodological portfolio has been upgraded by two novel approaches, single cell electroporation with genetically encoded Ca2+ sensors allowing for functionalized transsynaptic tracing and single cell targeted virus stamping. Especially the development of virus stamping provides a versatile solution for targeted single-cell infection of diverse cell types with different viruses at once, both in vitro and in vivo. Here we will summarize the latest developments in this rapidly moving field and provide a perspective for automated, quantitative analysis of single cell initiated connectomes.

The in vivo timeline of differentiation of engrafted human neural progenitor cells.

Understanding the individual timeline of stem cell differentiation in vivo is critical for evaluating stem cell properties in animal models. However, with conventional ex vivo techniques, such as histology, the individual timeline of differentiation is not accessible. Therefore, we designed lentiviral plasmids with cell-specific promoters to control the expression of bioluminescence and fluorescence imaging reporters. Promoter-dependent reporter expression in transduced human induced pluripotent stem cell-derived neural progenitor cells (hNPCs) was an effective indicator of differentiation in cell culture. A 12-week in vivo imaging observation period revealed the time profile of differentiation of engrafted hNPCs in the mouse brain into astrocytes and mature neurons which was verified by immunostainings, patch-clamp electrophysiology, and light-sheet fluorescence microscopy. The lentiviral vectors validated in this study provide an efficient imaging toolbox for non-invasive and longitudinal characterization of stem cell differentiation, in vitro screenings, and in vivo studies of cell therapy in animal models.

Light-sheet fluorescence expansion microscopy: fast mapping of neural circuits at super resolution.

The goal of understanding the architecture of neural circuits at the synapse level with a brain-wide perspective has powered the interest in high-speed and large field-of-view volumetric imaging at subcellular resolution. Here, we developed a method combining tissue expansion and light-sheet fluorescence microscopy to allow extended volumetric super resolution high-speed imaging of large mouse brain samples. We demonstrate the capabilities of this method by performing two color fast volumetric super resolution imaging of mouse CA1 and dentate gyrus molecular-, granule cell-, and polymorphic layers. Our method enables an exact evaluation of granule cell and neurite morphology within the context of large cell ensembles spanning several orders of magnitude in resolution. We found that imaging a brain region of 1 mm 3 in super resolution using light-sheet fluorescence expansion microscopy is about 17-fold faster than imaging the same region by a current state-of-the-art high-resolution confocal laser scanning microscope.

The structural and functional evidence for vesicular release from astrocytes in situ.

The concept of the tripartite synapse states that bi-directional signalling between perisynaptic astrocyte processes, presynaptic axonal boutons and postsynaptic neuronal structures defines the properties of synaptic information processing. Ca2+-dependent vesicular release from astrocytes, as one of the mechanisms of astrocyte-neuron communication, has attracted particular attention but has also been the subject of intense debate. In neurons, regulated vesicular release is a strongly coordinated process. It requires a complex release machinery comprised of many individual components ranging from vesicular neurotransmitter transporters and soluble NSF attachment protein receptors (SNARE) proteins to Ca2+-sensors and the proteins that spatially and temporally control exocytosis of synaptic vesicles. If astrocytes employ similar mechanisms to release neurotransmitters is less well understood. The aim of this review is therefore to discuss recent experimental evidence that sheds light on the central structural components responsible for vesicular release from astrocytes in situ.

Subcellular reorganization and altered phosphorylation of the astrocytic gap junction protein connexin43 in human and experimental temporal lobe epilepsy.

Dysfunctional astrocytes are increasingly recognized as key players in the development and progression of mesial temporal lobe epilepsy (MTLE). One of the dramatic changes astrocytes undergo in MTLE with hippocampal sclerosis (HS) is loss of gap junction coupling. To further elucidate molecular mechanism(s) underlying this alteration, we assessed expression, cellular localization and phosphorylation status of astrocytic gap junction proteins in human and experimental MTLE-HS. In addition to conventional confocal analysis of immunohistochemical staining we employed expansion microscopy, which allowed visualization of blood-brain-barrier (BBB) associated cellular elements at a sub-µm scale. Western Blot analysis showed that plasma membrane expression of connexin43 (Cx43) and Cx30 were not significantly different in hippocampal specimens with and without sclerosis. However, we observed a pronounced subcellular redistribution of Cx43 toward perivascular endfeet in HS, an effect that was accompanied by increased plaque size. Furthermore, in HS Cx43 was characterized by enhanced C-terminal phosphorylation of sites affecting channel permeability. Prominent albumin immunoreactivity was found in the perivascular space of HS tissue, indicating that BBB damage and consequential albumin extravasation was involved in Cx43 dysregulation. Together, our results suggest that subcellular reorganization and/or abnormal posttranslational processing rather than transcriptional downregulation of astrocytic gap junction proteins account for the loss of coupling reported in human and experimental TLE. The observations of the present study provide new insights into pathological alterations of astrocytes in HS, which may aid in the identification of novel therapeutic targets and development of alternative anti-epileptogenic strategies.

Adrenergic Gate Release for Spike Timing-Dependent Synaptic Potentiation.

Spike timing-dependent synaptic plasticity (STDP) serves as a key cellular correlate of associative learning, which is facilitated by elevated attentional and emotional states involving activation of adrenergic signaling. At cellular levels, adrenergic signaling increases dendrite excitability, but the underlying mechanisms remain elusive. Here we show that activation of ß2-adrenoceptors promoted STD long-term synaptic potentiation at mouse hippocampal excitatory synapses by inactivating dendritic Kv1.1-containing potassium channels, which increased dendrite excitability and facilitated dendritic propagation of postsynaptic depolarization, potentially improving coincidental activation of pre- and postsynaptic terminals. We further demonstrate that adrenergic modulation of Kv1.1 was mediated by the signaling scaffold SAP97, which, through direct protein-protein interactions, escorts ß2 signaling to remove Kv1.1 from the dendrite surface. These results reveal a mechanism through which the postsynaptic signaling scaffolds bridge the aroused brain state to promote induction of synaptic plasticity and potentially to enhance spike timing and memory encoding.

Dysfunction of Somatostatin-Positive Interneurons Associated with Memory Deficits in an Alzheimer's Disease Model.

Alzheimer's disease (AD) is characterized by cognitive decline and neuronal network dysfunction, but the underlying mechanisms remain unknown. In the hippocampus, microcircuit activity during learning and memory processes is tightly controlled by O-LM interneurons. Here, we investigated the effect of beta-amyloidosis on O-LM interneuron structural and functional connectivity, combining two-photon in vivo imaging of synaptic morphology, awake Ca2+ imaging, and retrograde mono-transsynaptic rabies tracing. We find severely impaired synaptic rewiring that occurs on the O-LM interneuron input and output level in a mouse model of AD. Synaptic rewiring that occurs upon fear learning on O-LM interneuron input level is affected in mice with AD-like pathology. This process requires the release of acetylcholine from septo-hippocampal projections. We identify decreased cholinergic action on O-LM interneurons in APP/PS1 mice as a key pathomechanism that contributes to memory impairment in a mouse model, with potential relevance for human AD.

Shifted pallidal co-release of GABA and glutamate in habenula drives cocaine withdrawal and relapse.

Cocaine withdrawal produces aversive states and vulnerability to relapse, hallmarks of addiction. The lateral habenula (LHb) encodes negative stimuli and contributes to aversive withdrawal symptoms. However, it remains unclear which inputs to LHb promote this and what the consequences are for relapse susceptibility. We report, using rabies-based retrolabeling and optogenetic mapping, that the entopeduncular nucleus (EPN, the mouse equivalent of the globus pallidus interna) projects to an LHb neuronal subset innervating aversion-encoding midbrain GABA neurons. EPN-to-LHb excitatory signaling is limited by GABAergic cotransmission. This inhibitory component decreases during cocaine withdrawal as a result of reduced presynaptic vesicular GABA transporter (VGAT). This shifts the EPN-to-LHb GABA/glutamate balance, disinhibiting EPN-driven LHb activity. Selective virally mediated VGAT overexpression at EPN-to-LHb terminals during withdrawal normalizes GABAergic neurotransmission. This intervention rescues cocaine-evoked aversive states and prevents stress-induced reinstatement, used to model relapse. This identifies diminished inhibitory transmission at EPN-to-LHb GABA/glutamate synapses as a mechanism contributing to the relapsing feature of addictive behavior.