Analysis of Functional NMDA Receptors in Astrocytes.

Neuronal N-methyl-D-aspartate (NMDA) receptors are well known for their pivotal role in memory formation. Originally, they were thought to be exclusive to neurons. However, numerous studies revealed their functional expression also on various types of glial cells in the nervous system. Here, the methodology on how to study the physiology of NMDA receptors selectively on astrocytes will be described in detail. Astrocytes are the main class of neuroglia that control transmitter and ion homeostasis, which link cerebral blood flow and neuronal energy demands, but also affect synaptic transmission directly.

A loss-of-function mutation in human Oxidation Resistance 1 disrupts the spatial-temporal regulation of histone arginine methylation in neurodevelopment.

BACKGROUND: Oxidation Resistance 1 (OXR1) gene is a highly conserved gene of the TLDc domain-containing family. OXR1 is involved in fundamental biological and cellular processes, including DNA damage response, antioxidant pathways, cell cycle, neuronal protection, and arginine methylation. In 2019, five patients from three families carrying four biallelic loss-of-function variants in OXR1 were reported to be associated with cerebellar atrophy. However, the impact of OXR1 on cellular functions and molecular mechanisms in the human brain is largely unknown. Notably, no human disease models are available to explore the pathological impact of OXR1 deficiency. RESULTS: We report a novel loss-of-function mutation in the TLDc domain of the human OXR1 gene, resulting in early-onset epilepsy, developmental delay, cognitive disabilities, and cerebellar atrophy. Patient lymphoblasts show impaired cell survival, proliferation, and hypersensitivity to oxidative stress. These phenotypes are rescued by TLDc domain replacement. We generate patient-derived induced pluripotent stem cells (iPSCs) revealing impaired neural differentiation along with dysregulation of genes essential for neurodevelopment. We identify that OXR1 influences histone arginine methylation by activating protein arginine methyltransferases (PRMTs), suggesting OXR1-dependent mechanisms regulating gene expression during neurodevelopment. We model the function of OXR1 in early human brain development using patient-derived brain organoids revealing that OXR1 contributes to the spatial-temporal regulation of histone arginine methylation in specific brain regions. CONCLUSIONS: This study provides new insights into pathological features and molecular underpinnings associated with OXR1 deficiency in patients.

Increased membrane Ca2+ permeability drives astrocytic Ca2+ dynamics during neuronal stimulation at excitatory synapses.

Astrocytes are intricately involved in the activity of neural circuits; however, their basic physiology of interacting with nearby neurons is not well established. Using two-photon imaging of neurons and astrocytes during higher frequency stimulation of hippocampal CA3-CA1 Schaffer collateral (Scc) excitatory synapses, we could show that increasing levels of released glutamate accelerated local astrocytic Ca2+ elevation. However, blockage of glutamate transporters did not abolish this astrocytic Ca2+ response, suggesting that astrocytic Ca2+ elevation is indirectly associated with an uptake of extracellular glutamate. However, during the astrocytic glutamate uptake, the Na+ /Ca2+ exchanger (NCX) reverse mode was activated, and mediated extracellular Ca2+ entry, thereby triggering the internal release of Ca2+ . In addition, extracellular Ca2+ entry via membrane P2X receptors further facilitated astrocytic Ca2+ elevation via ATP binding. These findings suggest a novel mechanism of activity induced Ca2+ permeability increases of astrocytic membranes, which drives astrocytic responses during neuronal stimulation of CA3-CA1 Scc excitatory synapses.

Deletion of aquaporin-4 improves capillary blood flow distribution in brain edema.

Brain edema is a feared complication to disorders and insults affecting the brain. It can be fatal if the increase in intracranial pressure is sufficiently large to cause brain herniation. Moreover, accruing evidence suggests that even slight elevations of intracranial pressure have adverse effects, for instance on brain perfusion. The water channel aquaporin-4 (AQP4), densely expressed in perivascular astrocytic endfeet, plays a key role in brain edema formation. Using two-photon microscopy, we have studied AQP4-mediated swelling of astrocytes affects capillary blood flow and intracranial pressure (ICP) in unanesthetized mice using a mild brain edema model. We found improved regulation of capillary blood flow in mice devoid of AQP4, independently of the severity of ICP increase. Furthermore, we found brisk AQP4-dependent astrocytic Ca2+ signals in perivascular endfeet during edema that may play a role in the perturbed capillary blood flow dynamics. The study suggests that astrocytic endfoot swelling and pathological signaling disrupts microvascular flow regulation during brain edema formation.

Impaired astrocytic Ca2+ signaling in awake-behaving Alzheimer's disease transgenic mice.

Increased astrocytic Ca2+ signaling has been shown in Alzheimer's disease mouse models, but to date no reports have characterized behaviorally induced astrocytic Ca2+ signaling in such mice. Here, we employ an event-based algorithm to assess astrocytic Ca2+ signals in the neocortex of awake-behaving tg-ArcSwe mice and non-transgenic wildtype littermates while monitoring pupil responses and behavior. We demonstrate an attenuated astrocytic Ca2+ response to locomotion and an uncoupling of pupil responses and astrocytic Ca2+ signaling in 15-month-old plaque-bearing mice. Using the genetically encoded fluorescent norepinephrine sensor GRABNE, we demonstrate a reduced norepinephrine signaling during spontaneous running and startle responses in the transgenic mice, providing a possible mechanistic underpinning of the observed reduced astrocytic Ca2+ responses. Our data points to a dysfunction in the norepinephrine-astrocyte Ca2+ activity axis, which may account for some of the cognitive deficits observed in Alzheimer's disease.

Neurodegenerative conditions such as Parkinson's or Alzheimer's disease are characterized by neurons dying and being damaged. Yet neurons are only one type of brain actors; astrocytes, for example, are star-shaped 'companion' cells that have recently emerged as being able to fine-tune neuronal communication. In particular, they can respond to norepinephrine, a signaling molecule that acts to prepare the brain and body for action. This activation results, for instance, in astrocytes releasing chemicals that can act on neurons. Certain cognitive symptoms associated with Alzheimer's disease could be due to a lack of norepinephrine. In parallel, studies in anaesthetized mice have shown perturbed astrocyte signaling in a model of the condition. Disrupted norepinephrine-triggered astrocyte signaling could therefore be implicated in the symptoms of the disease. Experiments in awake mice are needed to investigate this link, especially as anesthesia is known to disrupt the activity of astrocytes. To explore this question, Åbjørsbråten, Skaaraas et al. conducted experiments in naturally behaving mice expressing mutations found in patients with early-onset Alzheimer's disease. These mice develop hallmarks of the disorder. Compared to their healthy counterparts, these animals had reduced astrocyte signaling when running or being startled. Similarly, a fluorescent molecular marker for norepinephrine demonstrated less signaling in the modified mice compared to healthy ones. Over 55 million individuals currently live with Alzheimer's disease. The results by Åbjørsbråten, Skaaraas et al. suggest that astrocyte­norepinephrine communication may be implicated in the condition, an avenue of research that could potentially lead to developing new treatments.

Alternative Anesthesia of Neonatal Mice for Global rAAV Delivery in the Brain With Non-detectable Behavioral Interference in Adults.

Viral-transduced gene expression is the current standard for cell-type-specific labeling and cell tacking in experimental neuroscience. To achieve widespread gene expression, a viral delivery method to neonatal rodents was introduced more than two decades ago. Most of those neonatal viral vector injection-based gene transduction methods in mice used deep hypothermia for anesthesia, which was reported to be associated with behavioral impairments. To explore other options for neonatal viral applications, we applied a combination of Medetomidine, Midazolam, and Fentanyl (MMF), each of which can be antagonized by a specific antagonist. Later in their adulthood, we found that adult mice, that received the MMF-induced anesthesia, combined with virus-injected into the brain at postnatal day 2, showed similar performance in all behavioral tasks tested, including tasks for motor coordination, anxiety-related tasks, and spatial memory when compared to adult naïve littermates. This demonstrates that MMF anesthesia could be safely applied to mice for neonatal viral transduction at P2.

Astrocytic Ca2+ signaling is reduced during sleep and is involved in the regulation of slow wave sleep.

Astrocytic Ca2+ signaling has been intensively studied in health and disease but has not been quantified during natural sleep. Here, we employ an activity-based algorithm to assess astrocytic Ca2+ signals in the neocortex of awake and naturally sleeping mice while monitoring neuronal Ca2+ activity, brain rhythms and behavior. We show that astrocytic Ca2+ signals exhibit distinct features across the sleep-wake cycle and are reduced during sleep compared to wakefulness. Moreover, an increase in astrocytic Ca2+ signaling precedes transitions from slow wave sleep to wakefulness, with a peak upon awakening exceeding the levels during whisking and locomotion. Finally, genetic ablation of an important astrocytic Ca2+ signaling pathway impairs slow wave sleep and results in an increased number of microarousals, abnormal brain rhythms, and an increased frequency of slow wave sleep state transitions and sleep spindles. Our findings demonstrate an essential role for astrocytic Ca2+ signaling in regulating slow wave sleep.

Astroglial endfeet exhibit distinct Ca2+ signals during hypoosmotic conditions.

Astrocytic endfeet cover the brain surface and form a sheath around the cerebral vasculature. An emerging concept is that endfeet control blood-brain water transport and drainage of interstitial fluid and waste along paravascular pathways. Little is known about the signaling mechanisms that regulate endfoot volume and hence the width of these drainage pathways. Here, we used the genetically encoded fluorescent Ca2+ indicator GCaMP6f to study Ca2+ signaling within astrocytic somata, processes, and endfeet in response to an osmotic challenge known to induce cell swelling. Acute cortical slices were subjected to artificial cerebrospinal fluid with 20% reduction in osmolarity while GCaMP6f fluorescence was imaged with two-photon microscopy. Ca2+ signals induced by hypoosmotic conditions were observed in all astrocytic compartments except the soma. The Ca2+ response was most prominent in subpial and perivascular endfeet and included spikes with single peaks, plateau-type elevations, and rapid oscillations, the latter restricted to subpial endfeet. Genetic removal of the type 2 inositol 1,4,5-triphosphate receptor (IP3R2) severely suppressed the Ca2+ responses in endfeet but failed to affect brain water accumulation in vivo after water intoxication. Furthermore, the increase in endfoot Ca2+ spike rate during hypoosmotic conditions was attenuated in mutant mice lacking the aquaporin-4 anchoring molecule dystrophin and after blockage of transient receptor potential vanilloid 4 channels. We conclude that the characteristics and underpinning of Ca2+ responses to hypoosmotic stress differ within the astrocytic territory and that IP3R2 is essential for the Ca2+ signals only in subpial and perivascular endfeet.

Aquaporin-4-independent volume dynamics of astroglial endfeet during cortical spreading depression.

Cortical spreading depression (CSD) is a slowly propagating wave of depolarization of gray matter. This phenomenon is believed to underlie the migraine aura and similar waves of depolarization may exacerbate injury in a number of neurological disease states. CSD is characterized by massive ion dyshomeostasis, cell swelling, and multiphasic blood flow changes. Recently, it was shown that CSD is associated with a closure of the paravascular space (PVS), a proposed exit route for brain interstitial fluid and solutes, including excitatory and inflammatory substances that increase in the wake of CSD. The PVS closure was hypothesized to rely on swelling of astrocytic endfeet due to their high expression of aquaporin-4 (AQP4) water channels. We investigated whether CSD is associated with swelling of endfeet around penetrating arterioles in the cortex of living mice. Endfoot cross-sectional area was assessed by two-photon microscopy of mice expressing enhanced green fluorescent protein in astrocytes and related to the degree of arteriolar constriction. In anesthetized mice CSD triggered pronounced endfoot swelling that was short-lasting and coincided with the initial arteriolar constriction. Mice lacking AQP4 displayed volume changes of similar magnitude. CSD-induced endfoot swelling and arteriolar constriction also occurred in awake mice, albeit with faster kinetics than in anesthetized mice. We conclude that swelling of astrocytic endfeet is a robust event in CSD. The early onset and magnitude of the endfoot swelling is such that it may significantly delay perivascular drainage of interstitial solutes in neurological conditions where CSD plays a pathophysiological role.

Ca2+ Signals in Astrocytes Facilitate Spread of Epileptiform Activity.

Epileptic seizures are associated with increased astrocytic Ca2+ signaling, but the fine spatiotemporal kinetics of the ictal astrocyte-neuron interplay remains elusive. By using 2-photon imaging of awake head-fixed mice with chronic hippocampal windows we demonstrate that astrocytic Ca2+ signals precede neuronal Ca2+ elevations during the initial bout of kainate-induced seizures. On average, astrocytic Ca2+ elevations preceded neuronal activity in CA1 by about 8 s. In subsequent bouts of epileptic seizures, astrocytes and neurons were activated simultaneously. The initial astrocytic Ca2+ elevation was abolished in mice lacking the type 2 inositol-1,4,5-trisphosphate-receptor (Itpr2-/-). Furthermore, we found that Itpr2-/- mice exhibited 60% less epileptiform activity compared with wild-type mice when assessed by telemetric EEG monitoring. In both genotypes we also demonstrate that spreading depression waves may play a part in seizure termination. Our findings imply a role for astrocytic Ca2+ signals in ictogenesis.

A pathway from midcingulate cortex to posterior insula gates nociceptive hypersensitivity.

The identity of cortical circuits mediating nociception and pain is largely unclear. The cingulate cortex is consistently activated during pain, but the functional specificity of cingulate divisions, the roles at distinct temporal phases of central plasticity and the underlying circuitry are unknown. Here we show in mice that the midcingulate division of the cingulate cortex (MCC) does not mediate acute pain sensation and pain affect, but gates sensory hypersensitivity by acting in a wide cortical and subcortical network. Within this complex network, we identified an afferent MCC-posterior insula pathway that can induce and maintain nociceptive hypersensitivity in the absence of conditioned peripheral noxious drive. This facilitation of nociception is brought about by recruitment of descending serotonergic facilitatory projections to the spinal cord. These results have implications for our understanding of neuronal mechanisms facilitating the transition from acute to long-lasting pain.

Deletion of Aquaporin-4 Curtails Extracellular Glutamate Elevation in Cortical Spreading Depression in Awake Mice.

Cortical spreading depression (CSD) is a phenomenon that challenges the homeostatic mechanisms on which normal brain function so critically depends. Analyzing the sequence of events in CSD holds the potential of providing new insight in the physiological processes underlying normal brain function as well as the pathophysiology of neurological conditions characterized by ionic dyshomeostasis. Here, we have studied the sequential progression of CSD in awake wild-type mice and in mice lacking aquaporin-4 (AQP4) or inositol 1,4,5-triphosphate type 2 receptor (IP3R2). By the use of a novel combination of genetically encoded sensors that a novel combination - an unprecedented temporal and spatial resolution, we show that CSD leads to brisk Ca2+ signals in astrocytes and that the duration of these Ca2+ signals is shortened in the absence of AQP4 but not in the absence of IP3R2. The decrease of the astrocytic, AQP4-dependent Ca2+ signals, coincides in time and space with a decrease in the duration of extracellular glutamate overflow but not with the initial peak of the glutamate release suggesting that in CSD, extracellular glutamate accumulation is extended through AQP4-dependent glutamate release from astrocytes. The present data point to a salient glial contribution to CSD and identify AQP4 as a new target for therapy.

Causal Interrogation of Neuronal Networks and Behavior through Virally Transduced Ivermectin Receptors.

The causal interrogation of neuronal networks involved in specific behaviors requires the spatially and temporally controlled modulation of neuronal activity. For long-term manipulation of neuronal activity, chemogenetic tools provide a reasonable alternative to short-term optogenetic approaches. Here we show that virus mediated gene transfer of the ivermectin (IVM) activated glycine receptor mutant GlyRα1 (AG) can be used for the selective and reversible silencing of specific neuronal networks in mice. In the striatum, dorsal hippocampus, and olfactory bulb, GlyRα1 (AG) promoted IVM dependent effects in representative behavioral assays. Moreover, GlyRα1 (AG) mediated silencing had a strong and reversible impact on neuronal ensemble activity and c-Fos activation in the olfactory bulb. Together our results demonstrate that long-term, reversible and re-inducible neuronal silencing via GlyRα1 (AG) is a promising tool for the interrogation of network mechanisms underlying the control of behavior and memory formation.

Dynamics of Ionic Shifts in Cortical Spreading Depression.

Cortical spreading depression is a slowly propagating wave of near-complete depolarization of brain cells followed by temporary suppression of neuronal activity. Accumulating evidence indicates that cortical spreading depression underlies the migraine aura and that similar waves promote tissue damage in stroke, trauma, and hemorrhage. Cortical spreading depression is characterized by neuronal swelling, profound elevation of extracellular potassium and glutamate, multiphasic blood flow changes, and drop in tissue oxygen tension. The slow speed of the cortical spreading depression wave implies that it is mediated by diffusion of a chemical substance, yet the identity of this substance and the pathway it follows are unknown. Intercellular spread between gap junction-coupled neurons or glial cells and interstitial diffusion of K(+) or glutamate have been proposed. Here we use extracellular direct current potential recordings, K(+)-sensitive microelectrodes, and 2-photon imaging with ultrasensitive Ca(2+) and glutamate fluorescent probes to elucidate the spatiotemporal dynamics of ionic shifts associated with the propagation of cortical spreading depression in the visual cortex of adult living mice. Our data argue against intercellular spread of Ca(2+) carrying the cortical spreading depression wavefront and are in favor of interstitial K(+) diffusion, rather than glutamate diffusion, as the leading event in cortical spreading depression.

Augmentation of Ca(2+) signaling in astrocytic endfeet in the latent phase of temporal lobe epilepsy.

Astrocytic endfeet are specialized cell compartments whose important homeostatic roles depend on their enrichment of water and ion channels anchored by the dystrophin associated protein complex (DAPC). This protein complex is known to disassemble in patients with mesial temporal lobe epilepsy and in the latent phase of experimental epilepsies. The mechanistic underpinning of this disassembly is an obvious target of future therapies, but remains unresolved. Here we show in a kainate model of temporal lobe epilepsy that astrocytic endfeet display an enhanced stimulation-evoked Ca(2+) signal that outlast the Ca(2+) signal in the cell bodies. While the amplitude of this Ca(2+) signal is reduced following group I/II metabotropic receptor (mGluR) blockade, the duration is sustained. Based on previous studies it has been hypothesized that the molecular disassembly in astrocytic endfeet is caused by dystrophin cleavage mediated by Ca(2+) dependent proteases. Using a newly developed genetically encoded Ca(2+) sensor, the present study bolsters this hypothesis by demonstrating long-lasting, enhanced stimulation-evoked Ca(2+) signals in astrocytic endfeet.

Stimulation-evoked Ca2+ signals in astrocytic processes at hippocampal CA3-CA1 synapses of adult mice are modulated by glutamate and ATP.

To date, it has been difficult to reveal physiological Ca(2+) events occurring within the fine astrocytic processes of mature animals. The objective of the study was to explore whether neuronal activity evokes astrocytic Ca(2+) signals at glutamatergic synapses of adult mice. We stimulated the Schaffer collateral/commissural fibers in acute hippocampal slices from adult mice transduced with the genetically encoded Ca(2+) indicator GCaMP5E driven by the glial fibrillary acidic protein promoter. Two-photon imaging revealed global stimulation-evoked astrocytic Ca(2+) signals with distinct latencies, rise rates, and amplitudes in fine processes and somata. Specifically, the Ca(2+) signals in the processes were faster and of higher amplitude than those in the somata. A combination of P2 purinergic and group I/II metabotropic glutamate receptor (mGluR) antagonists reduced the amplitude of the Ca(2+) transients by 30-40% in both astrocytic compartments. Blockage of the mGluRs alone only modestly reduced the magnitude of the stimulation-evoked Ca(2+) signals in processes and failed to affect the somatic Ca(2+) response. Local application of group I or I/II mGluR agonists or adenosine triphosphate (ATP) elicited global astrocytic Ca(2+) signals that mimicked the stimulation-evoked astrocytic Ca(2+) responses. We conclude that stimulation-evoked Ca(2+) signals in astrocytic processes at CA3-CA1 synapses of adult mice (1) differ from those in astrocytic somata and (2) are modulated by glutamate and ATP.

Neural basis of benzodiazepine reward: requirement for α2 containing GABAA receptors in the nucleus accumbens.

Despite long-standing concerns regarding the abuse liability of benzodiazepines, the mechanisms underlying properties of benzodiazepines that may be relevant to abuse are still poorly understood. Earlier studies showed that compounds selective for α1-containing GABAA receptors (α1GABAARs) are abused by humans and self-administered by animals, and that these receptors may underlie a preference for benzodiazepines as well as neuroplastic changes observed in the ventral tegmental area following benzodiazepine administration. There is some evidence, however, that even L-838, 417, a compound with antagonistic properties at α1GABAARs and agonistic properties at the other three benzodiazepine-sensitive GABAA receptor subtypes, is self-administered, and that the α2GABAARs may have a role in benzodiazepine-induced reward enhancement. Using a two-bottle choice drinking paradigm to evaluate midazolam preference and an intracranial self-stimulation (ICSS) paradigm to evaluate the impact of midazolam on reward enhancement, we demonstrated that mice carrying a histidine-to-arginine point mutation in the α2 subunit which renders it insensitive to benzodiazepines (α2(H101R) mice) did not prefer midazolam and did not show midazolam-induced reward enhancement in ICSS, in contrast to wild-type controls, suggesting that α2GABAARs are necessary for the reward enhancing effects and preference for oral benzodiazepines. Through a viral-mediated knockdown of α2GABAARs in the nucleus accumbens (NAc), we demonstrated that α2 in the NAc is necessary for the preference for midazolam. Findings imply that α2GABAARs in the NAc are involved in at least some reward-related properties of benzodiazepines, which might partially underlie repeated drug-taking behavior.

Electroconvulsive therapy induces neurogenesis in frontal rat brain areas.

Electroconvulsive therapy (ECT) is an effective therapy for several psychiatric disorders, including severe major depression, mania and certain forms of schizophrenia. It had been proposed that ECT acts by modulating local plasticity via the stimulation of neurogenesis. In fact, among antidepressant therapies, ECT is the most robust enhancer of neurogenesis in the hippocampus of rodents and non-human primates. The existence of ECT-triggered neurogenesis in other brain areas, particularly in those adjacent to the other main locus of neurogenesis, the subventricular zone (SVZ), had so far remained unknown. Here we show that ECT also strongly enhances neurogenesis in frontal brain areas, especially in the rostro-medial striatum, generating specific, small-size calretinin-positive interneurons. We provide here the first evidence that ECT stimulates neurogenesis in areas outside the hippocampus. Our data may open research possibilities that focus on the plastic changes induced by ECT in frontal limbic circuitry.

Inherited and de novo SHANK2 variants associated with autism spectrum disorder impair neuronal morphogenesis and physiology.

Mutations in the postsynaptic scaffolding gene SHANK2 have recently been identified in individuals with autism spectrum disorder (ASD) and intellectual disability. However, the cellular and physiological consequences of these mutations in neurons remain unknown. We have analyzed the functional impact caused by two inherited and one de novo SHANK2 mutations from ASD individuals (L1008_P1009dup, T1127M, R462X). Although all three variants affect spine volume and have smaller SHANK2 cluster sizes, T1127M additionally fails to rescue spine volume in Shank2 knock-down neurons. R462X is not able to rescue spine volume and dendritic branching and lacks postsynaptic clustering, indicating the most severe dysfunction. To demonstrate that R462X when expressed in mouse can be linked to physiological effects, we analyzed synaptic transmission and behavior. Principal neurons of mice expressing rAAV-transduced SHANK2-R462X present a specific, long-lasting reduction in miniature postsynaptic AMPA receptor currents. This dominant negative effect translates into dose-dependent altered cognitive behavior of SHANK2-R462X-expressing mice, with an impact on the penetrance of ASD.

Optical recording of neuronal activity with a genetically-encoded calcium indicator in anesthetized and freely moving mice.

Fluorescent calcium (Ca(2+)) indicator proteins (FCIPs) are promising tools for functional imaging of cellular activity in living animals. However, they have still not reached their full potential for in vivo imaging of neuronal activity due to limitations in expression levels, dynamic range, and sensitivity for reporting action potentials. Here, we report that viral expression of the ratiometric Ca(2+) sensor yellow cameleon 3.60 (YC3.60) in pyramidal neurons of mouse barrel cortex enables in vivo measurement of neuronal activity with high dynamic range and sensitivity across multiple spatial scales. By combining juxtacellular recordings and two-photon imaging in vitro and in vivo, we demonstrate that YC3.60 can resolve single action potential (AP)-evoked Ca(2+) transients and reliably reports bursts of APs with negligible saturation. Spontaneous and whisker-evoked Ca(2+) transients were detected in individual apical dendrites and somata as well as in local neuronal populations. Moreover, bulk measurements using wide-field imaging or fiber-optics revealed sensory-evoked YC3.60 signals in large areas of the barrel field. Fiber-optic recordings in particular enabled measurements in awake, freely moving mice and revealed complex Ca(2+) dynamics, possibly reflecting different behavior-related brain states. Viral expression of YC3.60 - in combination with various optical techniques - thus opens a multitude of opportunities for functional studies of the neural basis of animal behavior, from dendrites to the levels of local and large-scale neuronal populations.