Large and long-lasting cytosolic calcium surges in astrocytes have been described in cultured cells and acute slice preparations. The mechanisms that give rise to these calcium events have been extensively studied in vitro. However, their existence and functions in the intact brain are unknown. We have topically applied Fluo-4 AM on the cerebral cortex of anesthetized rats, and imaged cytosolic calcium fluctuation in astrocyte populations of superficial cortical layers in vivo, using two-photon laser scanning microscopy. Spontaneous [Ca(2+)](i) events in individual astrocytes were similar to those observed in vitro. Coordination of [Ca(2+)](i) events among astrocytes was indicated by the broad cross-correlograms. Increased neuronal discharge was associated with increased astrocytic [Ca(2+)](i) activity in individual cells and a robust coordination of [Ca(2+)](i) signals in neighboring astrocytes. These findings indicate potential neuron-glia communication in the intact brain.

The Neurodata Without Borders (NWB) initiative promotes data standardization in neuroscience to increase research reproducibility and opportunities. In the first NWB pilot project, neurophysiologists and software developers produced a common data format for recordings and metadata of cellular electrophysiology and optical imaging experiments. The format specification, application programming interfaces, and sample datasets have been released.

Animal models have been developed to investigate aspects of stress, anxiety, and depression, but our understanding of the circuitry underlying these models remains incomplete. Prior studies of the habenula, a poorly understood nucleus in the dorsal diencephalon, suggest that projections to the medial habenula (MHb) regulate fear and anxiety responses, whereas the lateral habenula (LHb) is involved in the expression of learned helplessness, a model of depression. Tissue-specific deletion of the transcription factor Pou4f1 in the dorsal MHb (dMHb) results in a developmental lesion of this subnucleus. These dMHb-ablated mice show deficits in voluntary exercise, a possible correlate of depression. Here we explore the role of the dMHb in mood-related behaviors and intrinsic reinforcement. Lesions of the dMHb do not elicit changes in contextual conditioned fear. However, dMHb-lesioned mice exhibit shorter immobility time in the tail suspension test, another model of depression. dMHb-lesioned mice also display increased vulnerability to the induction of learned helplessness. However, this effect is not due specifically to the dMHb lesion, but appears to result from Pou4f1 haploinsufficiency elsewhere in the nervous system. Pou4f1 haploinsufficiency does not produce the other phenotypes associated with dMHb lesions. Using optogenetic intracranial self-stimulation, intrinsic reinforcement by the dMHb can be mapped to a specific population of neurokinin-expressing habenula neurons. Together, our data show that the dMHb is involved in the regulation of multiple mood-related behaviors, but also support the idea that these behaviors do not reflect a single functional pathway.

Hmx1 is a variant homeodomain transcription factor expressed in the developing sensory nervous system, retina, and craniofacial mesenchyme. Recently, mutations at the Hmx1 locus have been linked to craniofacial defects in humans, rats, and mice, but its role in nervous system development is largely unknown. Here we show that Hmx1 is expressed in a subset of sensory neurons in the cranial and dorsal root ganglia which does not correspond to any specific sensory modality. Sensory neurons in the dorsal root and trigeminal ganglia of Hmx1dm/dm mouse embryos have no detectable Hmx1 protein, yet they undergo neurogenesis and express sensory subtype markers normally, demonstrating that Hmx1 is not globally required for the specification of sensory neurons from neural crest precursors. Loss of Hmx1 expression has no obvious effect on the early development of the trigeminal (V), superior (IX/X), or dorsal root ganglia neurons in which it is expressed, but results in marked defects in the geniculate (VII) ganglion. Hmx1dm/dm mouse embryos possess only a vestigial posterior auricular nerve, and general somatosensory neurons in the geniculate ganglion are greatly reduced by mid-gestation. Although Hmx1 is expressed in geniculate neurons prior to cell cycle exit, it does not appear to be required for neurogenesis, and the loss of geniculate neurons is likely to be the result of increased cell death. Fate mapping of neural crest-derived tissues indicates that Hmx1-expressing somatosensory neurons at different axial levels may be derived from either the neural crest or the neurogenic placodes.

Conditioned place preference (CPP) is a widely used model of addiction-related behavior whose underlying mechanisms are not understood. In this study, we used dual site silicon probe recordings in freely moving mice to examine interactions between the hippocampus and nucleus accumbens in cocaine CPP. We found that CPP was associated with recruitment of D2-positive nucleus accumbens medium spiny neurons to fire in the cocaine-paired location, and this recruitment was driven predominantly by selective strengthening of coupling with hippocampal place cells that encode the cocaine-paired location. These findings provide in vivo evidence suggesting that the synaptic potentiation in the accumbens caused by repeated cocaine administration preferentially affects inputs that were active at the time of drug exposure. This provides a potential physiological mechanism by which drug use becomes associated with specific environmental contexts.

Excitatory control of inhibitory neurons is poorly understood due to the difficulty of studying synaptic connectivity in vivo. We inferred such connectivity through analysis of spike timing and validated this inference using juxtacellular and optogenetic control of presynaptic spikes in behaving mice. We observed that neighboring CA1 neurons had stronger connections and that superficial pyramidal cells projected more to deep interneurons. Connection probability and strength were skewed, with a minority of highly connected hubs. Divergent presynaptic connections led to synchrony between interneurons. Synchrony of convergent presynaptic inputs boosted postsynaptic drive. Presynaptic firing frequency was read out by postsynaptic neurons through short-term depression and facilitation, with individual pyramidal cells and interneurons displaying a diversity of spike transmission filters. Additionally, spike transmission was strongly modulated by prior spike timing of the postsynaptic cell. These results bridge anatomical structure with physiological function.

Bouts of high frequency activity known as sharp wave ripples (SPW-Rs) facilitate communication between the hippocampus and neocortex. However, the paths and mechanisms by which SPW-Rs broadcast their content are not well understood. Due to its anatomical positioning, the granular retrosplenial cortex (gRSC) may be a bridge for this hippocampo-cortical dialogue. Using silicon probe recordings in awake, head-fixed mice, we show the existence of SPW-R analogues in gRSC and demonstrate their coupling to hippocampal SPW-Rs. gRSC neurons reliably distinguished different subclasses of hippocampal SPW-Rs according to ensemble activity patterns in CA1. We demonstrate that this coupling is brain state-dependent, and delineate a topographically-organized anatomical pathway via VGlut2-expressing, bursty neurons in the subiculum. Optogenetic stimulation or inhibition of bursty subicular cells induced or reduced responses in superficial gRSC, respectively. These results identify a specific path and underlying mechanisms by which the hippocampus can convey neuronal content to the neocortex during SPW-Rs.

Uncovering spatial representations from large-scale ensemble spike activity in specific brain circuits provides valuable feedback in closed-loop experiments. We develop a graphics processing unit (GPU)-powered population-decoding system for ultrafast reconstruction of spatial positions from rodents' unsorted spatiotemporal spiking patterns, during run behavior or sleep. In comparison with an optimized quad-core central processing unit (CPU) implementation, our approach achieves an ∼20- to 50-fold increase in speed in eight tested rat hippocampal, cortical, and thalamic ensemble recordings, with real-time decoding speed (approximately fraction of a millisecond per spike) and scalability up to thousands of channels. By accommodating parallel shuffling in real time (computation time <15 ms), our approach enables assessment of the statistical significance of online-decoded "memory replay" candidates during quiet wakefulness or sleep. This open-source software toolkit supports the decoding of spatial correlates or content-triggered experimental manipulation in closed-loop neuroscience experiments.

Hippocampal theta oscillations coordinate neuronal firing to support memory and spatial navigation. The medial septum (MS) is critical in theta generation by two possible mechanisms: either a unitary "pacemaker" timing signal is imposed on the hippocampal system, or it may assist in organizing target subcircuits within the phase space of theta oscillations. We used temperature manipulation of the MS to test these models. Cooling of the MS reduced both theta frequency and power and was associated with an enhanced incidence of errors in a spatial navigation task, but it did not affect spatial correlates of neurons. MS cooling decreased theta frequency oscillations of place cells and reduced distance-time compression but preserved distance-phase compression of place field sequences within the theta cycle. Thus, the septum is critical for sustaining precise theta phase coordination of cell assemblies in the hippocampal system, a mechanism needed for spatial memory.

The large diversity of neuron types provides the means by which cortical circuits perform complex operations. Neuron can be described by biophysical and molecular characteristics, afferent inputs, and neuron targets. To quantify, visualize, and standardize those features, we developed the open-source, MATLAB-based framework CellExplorer. It consists of three components: a processing module, a flexible data structure, and a powerful graphical interface. The processing module calculates standardized physiological metrics, performs neuron-type classification, finds putative monosynaptic connections, and saves them to a standardized, yet flexible, machine-readable format. The graphical interface makes it possible to explore the computed features at the speed of a mouse click. The framework allows users to process, curate, and relate their data to a growing public collection of neurons. CellExplorer can link genetically identified cell types to physiological properties of neurons collected across laboratories and potentially lead to interlaboratory standards of single-cell metrics.

During periods of disengagement from the environment, transient population bursts, known as sharp wave ripples (SPW-Rs), occur sporadically. While numerous experiments have characterized the bidirectional relationship between SPW-Rs and activity in chosen brain areas, the topographic relationship between different segments of the hippocampus and brain-wide target areas has not been studied at high temporal and spatial resolution. Yet, such knowledge is necessary to infer the direction of communication. We analyzed two publicly available datasets with simultaneous high-density silicon probe recordings from across the mouse forebrain. We found that SPW-Rs coincide with a transient brain-wide increase in functional connectivity. In addition, we show that the diversity in SPW-R features, such as their incidence, magnitude, and intrahippocampal topography in the septotemporal axis, are correlated with slower excitability fluctuations in cortical and subcortical areas. Further, variations in SPW-R features correlated with the timing, sign, and magnitude of downstream responses with large-amplitude SPW-Rs followed by transient silence in extrahippocampal structures. Our findings expand on previous results and demonstrate that the activity patterns in extrahippocampal structures depend both on the intrahippocampal topographic origin and magnitude of hippocampal SPW-Rs.

Dysregulated fear reactions can result from maladaptive processing of trauma-related memories. In post-traumatic stress disorder (PTSD) and other psychiatric disorders, dysfunctional extinction learning prevents discretization of trauma-related memory engrams and generalizes fear responses. Although PTSD may be viewed as a memory-based disorder, no approved treatments target pathological fear memory processing. Hippocampal sharp wave-ripples (SWRs) and concurrent neocortical oscillations are scaffolds to consolidate contextual memory, but their role during fear processing remains poorly understood. Here, we show that closed-loop, SWR triggered neuromodulation of the medial forebrain bundle (MFB) can enhance fear extinction consolidation in male rats. The modified fear memories became resistant to induced recall (i.e., 'renewal' and 'reinstatement') and did not reemerge spontaneously. These effects were mediated by D2 receptor signaling-induced synaptic remodeling in the basolateral amygdala. Our results demonstrate that SWR-triggered closed-loop stimulation of the MFB reward system enhances extinction of fearful memories and reducing fear expression across different contexts and preventing excessive and persistent fear responses. These findings highlight the potential of neuromodulation to augment extinction learning and provide a new avenue to develop treatments for anxiety disorders.

Cortical GABAergic interneurons (INs) represent a diverse population of mainly locally projecting cells that provide specialized forms of inhibition to pyramidal neurons and other INs. Most recent work on INs has focused on subtypes distinguished by expression of Parvalbumin (PV), Somatostatin (SST), or Vasoactive Intestinal Peptide (VIP). However, a fourth group that includes neurogliaform cells (NGFCs) has been less well characterized due to a lack of genetic tools. Here, we show that these INs can be accessed experimentally using intersectional genetics with the gene Id2. We find that outside of layer 1 (L1), the majority of Id2 INs are NGFCs that express high levels of neuropeptide Y (NPY) and exhibit a late-spiking firing pattern, with extensive local connectivity. While much sparser, non-NGFC Id2 INs had more variable properties, with most cells corresponding to a diverse group of INs that strongly expresses the neuropeptide CCK. In vivo, using silicon probe recordings, we observed several distinguishing aspects of NGFC activity, including a strong rebound in activity immediately following the cortical down state during NREM sleep. Our study provides insights into IN diversity and NGFC distribution and properties, and outlines an intersectional genetics approach for further study of this underappreciated group of INs.

As the use of Radio Frequency (RF) technologies increases, the impact of RF radiation on neurological function continues to receive attention. Whether RF radiation can modulate ongoing neuronal activity by non-thermal mechanisms has been debated for decades. However, the interactions between radiated energy and metal-based neural probes during experimentation could impact neural activity, making interpretation of the results difficult. To address this problem, we modified a miniature 1-photon Ca2+ imaging device to record interference-free neural activity and compared the results to those acquired using metal-containing silicon probes. We monitored the neuronal activity of awake rodent-brains under RF energy exposure (at 950 MHz) and in sham control paradigms. Spiking activity was reliably affected by RF energy in metal containing systems. However, we did not observe neuronal responses using metal-free optical recordings at induced local electric field strengths up to 230 V/m. Our results suggest that RF exposure higher than levels that are allowed by regulatory limits in real-life scenarios do not affect neuronal activity.

Using silicon-based recording electrodes, we recorded neuronal activity of the dorsal hippocampus and dorsomedial entorhinal cortex from behaving rats. The entorhinal neurons were classified as principal neurons and interneurons based on monosynaptic interactions and wave-shapes. The hippocampal neurons were classified as principal neurons and interneurons based on monosynaptic interactions, wave-shapes and burstiness. The data set contains recordings from 7,736 neurons (6,100 classified as principal neurons, 1,132 as interneurons, and 504 cells that did not clearly fit into either category) obtained during 442 recording sessions from 11 rats (a total of 204.5 hours) while they were engaged in one of eight different behaviours/tasks. Both original and processed data (time stamp of spikes, spike waveforms, result of spike sorting and local field potential) are included, along with metadata of behavioural markers. Community-driven data sharing may offer cross-validation of findings, refinement of interpretations and facilitate discoveries.

The relationship between mesoscopic local field potentials (LFPs) and single-neuron firing in the multi-layered neocortex is poorly understood. Simultaneous recordings from all layers in the primary visual cortex (V1) of the behaving mouse revealed functionally defined layers in V1. The depth of maximum spike power and sink-source distributions of LFPs provided consistent laminar landmarks across animals. Coherence of gamma oscillations (30-100 Hz) and spike-LFP coupling identified six physiological layers and further sublayers. Firing rates, burstiness, and other electrophysiological features of neurons displayed unique layer and brain state dependence. Spike transmission strength from layer 2/3 cells to layer 5 pyramidal cells and interneurons was stronger during waking compared with non-REM sleep but stronger during non-REM sleep among deep-layer excitatory neurons. A subset of deep-layer neurons was active exclusively in the DOWN state of non-REM sleep. These results bridge mesoscopic LFPs and single-neuron interactions with laminar structure in V1.

Interactions between the hippocampus and the cortex are critical for memory. Interictal epileptiform discharges (IEDs) identify epileptic brain regions and can impair memory, but the mechanisms by which they interact with physiological patterns of network activity are mostly undefined. We show in a rat model of temporal lobe epilepsy that spontaneous hippocampal IEDs correlate with impaired memory consolidation, and that they are precisely coordinated with spindle oscillations in the prefrontal cortex during nonrapid-eye-movement (NREM) sleep. This coordination surpasses the normal physiological ripple-spindle coupling and is accompanied by decreased ripple occurrence. IEDs also induce spindles during rapid-eye movement (REM) sleep and wakefulness-behavioral states that do not naturally express these oscillations-by generating a cortical 'down' state. In a pilot clinical examination of four subjects with focal epilepsy, we confirm a similar correlation of temporofrontal IEDs with spindles over anatomically restricted cortical regions. These findings imply that IEDs may impair memory via the misappropriation of physiological mechanisms for hippocampal-cortical coupling, which suggests a target for the treatment of memory impairment in epilepsy.

To understand how function arises from the interactions between neurons, it is necessary to use methods that allow the monitoring of brain activity at the single-neuron, single-spike level and the targeted manipulation of the diverse neuron types selectively in a closed-loop manner. Large-scale recordings of neuronal spiking combined with optogenetic perturbation of identified individual neurons has emerged as a suitable method for such tasks in behaving animals. To fully exploit the potential power of these methods, multiple steps of technical innovation are needed. We highlight the current state of the art in electrophysiological recording methods, combined with optogenetics, and discuss directions for progress. In addition, we point to areas where rapid development is in progress and discuss topics where near-term improvements are possible and needed.

Cell type-specific expression of optogenetic molecules allows temporally precise manipulation of targeted neuronal activity. Here we present a toolbox of four knock-in mouse lines engineered for strong, Cre-dependent expression of channelrhodopsins ChR2-tdTomato and ChR2-EYFP, halorhodopsin eNpHR3.0 and archaerhodopsin Arch-ER2. All four transgenes mediated Cre-dependent, robust activation or silencing of cortical pyramidal neurons in vitro and in vivo upon light stimulation, with ChR2-EYFP and Arch-ER2 demonstrating light sensitivity approaching that of in utero or virally transduced neurons. We further show specific photoactivation of parvalbumin-positive interneurons in behaving ChR2-EYFP reporter mice. The robust, consistent and inducible nature of our ChR2 mice represents a significant advance over previous lines, and the Arch-ER2 and eNpHR3.0 mice are to our knowledge the first demonstration of successful conditional transgenic optogenetic silencing. When combined with the hundreds of available Cre driver lines, this optimized toolbox of reporter mice will enable widespread investigations of neural circuit function with unprecedented reliability and accuracy.

The mnemonic functions of hippocampal sharp wave ripples (SPW-Rs) have been studied extensively. Because hippocampal outputs affect not only cortical but also subcortical targets, we examined the impact of SPW-Rs on the firing patterns of lateral septal (LS) neurons in behaving rats. A large fraction of SPW-Rs were temporally locked to high-frequency oscillations (HFOs) (120-180 Hz) in LS, with strongest coupling during non-rapid eye movement (NREM) sleep, followed by waking immobility. However, coherence and spike-local field potential (LFP) coupling between the two structures were low, suggesting that HFOs are generated locally within the LS GABAergic population. This hypothesis was supported by optogenetic induction of HFOs in LS. Spiking of LS neurons was largely independent of the sequential order of spiking in SPW-Rs but instead correlated with the magnitude of excitatory synchrony of the hippocampal output. Thus, LS is strongly activated by SPW-Rs and may convey hippocampal population events to its hypothalamic and brainstem targets.