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Sensory cortices, even of primary regions, are not purely unisensory. Rather, cortical neurons in sensory cortex show various forms of multisensory interactions. While some multisensory interactions naturally co-occur, the combination of others will co-occur through experience. In real life, learning and experience will result in conjunction with seemingly disparate sensory information that ultimately becomes behaviorally relevant, impacting perception, cognition, and action. Here we describe a novel auditory discrimination task in mice, designed to manipulate the expectation of upcoming trials using olfactory cues. We show that, after learning, female mice display a transient period of several days during which they exploit odor-mediated expectations for making correct decisions. Using two-photon calcium imaging of single neurons in auditory cortex (ACx) during behavior, we found that the behavioral effects of odor-mediated expectations are accompanied by an odor-induced modulation of neuronal activity. Further, we find that these effects are manifested differentially, based on the response preference of individual cells. A significant portion of effects, but not all, are consistent with a predictive coding framework. Our data show that learning novel odor-sound associations evoke changes in ACx. We suggest that behaviorally relevant multisensory environments mediate contextual effects as early as ACx. [ABSTRACT FROM AUTHOR]

Super-resolution fluorescence microscopy holds tremendous potential for discovery in neuroscience. Much of the molecular machinery and anatomic specializations that give rise to the unique and bewildering electrochemical activity of neurons are nanoscale by design, ranging somewhere between 1 nm and 1 lm. It is at this scale where most of the unknown and exciting action is and where cell biologists flock to in their dreams, but it was off limits for light microscopy until recently. While the optical principles of super-resolution microscopy are firmly established by now, the technology continues to advance rapidly in many crucial areas, enhancing its performance and reliability, and making it more accessible and user-friendly, which is sorely needed. Indeed, super-resolution microscopy techniques are nowadays widely used for visualizing immunolabeled protein distributions in fixed or living cells. However, a great potential of super-resolution microscopy for neuroscience lies in shining light on the nanoscale structures and biochemical activities in live-tissue settings, which should be developed and harnessed much more fully. In this review, we will present several vivid examples based on STED and RESOLFT super-resolution microscopy, illustrating the possibilities and challenges of nano-imaging in vivo to pique the interest of tech-developers and neurobiologists alike. We will cover recent technical progress that is facilitating in vivo applications, and share new biological insights into the nanoscale mechanisms of cellular communication between neurons and glia. [ABSTRACT FROM AUTHOR]

The superior colliculus (SC) is the midbrain center for integrating visual and multimodal sensory information. Neurons in the SC exhibit direction and orientation selectivity. Recent studies reported that neurons with similar preferences formed clusters in the mouse SC (Ahmadlou and Heimel, 2015; Feinberg and Meister, 2015; de Malmazet et al., 2018; Li et al., 2020). However, it remains controversial as to how these clusters are organized within the SC (Inayat et al., 2015; Chen et al., 2021). Here, we found that different brain states (i.e., awake or anesthetized with isoflurane) changed the selectivity of individual SC neurons and organizations of the neuronal population in both male and female mice. Using two-photon Ca2+ imaging, we examined both individual neuronal responses and the spatial patterns of their population responses. Under isoflurane anesthesia, orientation selectivity increased and a larger number of orientation-selective cells were observed when compared with the awake condition, whereas the proportions of direction-selective cells were similar in both conditions. Furthermore, direction- and orientation-selective cells located at closer positions showed more similar preferences, and cluster- like spatial patterns were enhanced. Inhibitory responses of direction-selective neurons were also reduced under isoflurane anesthesia. Thus, the changes in the spatial organization of response patterns were considered to be because of changes in the balance of excitation and inhibition, with excitation dominance, in the local circuits. These results provide new insights into the possibility that the functional organization of feature selectivity in the brain is affected by brain state. [ABSTRACT FROM AUTHOR]

The axon initial segment (AIS) is a specialized neuronal compartment in which synaptic input is converted into action potential (AP) output. This process is supported by a diverse complement of sodium, potassium, and calcium channels (CaV). Different classes of sodium and potassium channels are scaffolded at specific sites within the AIS, conferring unique functions, but how calcium channels are functionally distributed within the AIS is unclear. Here, we use conventional two-photon laser scanning and diffraction-limited, high-speed spot two-photon imaging to resolve AP-evoked calcium dynamics in the AIS with high spatiotemporal resolution. In mouse layer 5 prefrontal pyramidal neurons, calcium influx was mediated by a mix of CaV2 and CaV3 channels that differentially localized to discrete regions. CaV3 functionally localized to produce nanodomain hotspots of calcium influx that coupled to ryanodine-sensitive stores, whereas CaV2 localized to non-hotspot regions. Thus, different pools of CaVs appear to play distinct roles in AIS function. [ABSTRACT FROM AUTHOR]

There has been considerable research showing populations of neurons encoding for different aspects of space in the brain. Recently, several studies using two-photon calcium imaging and virtual navigation have identified "spatially" modulated neurons in the posterior cortex. We enquire here whether the presence of such spatial representations may be a cortex-wide phenomenon and, if so, whether these representations can be organized in the absence of the hippocampus. To this end, we imaged the dorsal cortex of mice running on a treadmill populated with tactile cues. A high percentage (40-80%) of the detected neurons exhibited sparse, spatially localized activity, with activity fields uniformly localized over the track. The development of this location specificity was impaired by hippocampal damage. Thus, there is a substantial population of neurons distributed widely over the cortex that collectively form a continuous representation of the explored environment, and hippocampal outflow is necessary to organize this phenomenon. [ABSTRACT FROM AUTHOR]

Multiphoton microscopy (MPM) has emerged as one of the most powerful and widespread technologies to monitor the activity of neuronal networks in awake, behaving animals over long periods of time. MPM development spanned across decades and crucially depended on the concurrent improvement of calcium indicators that report neuronal activity as well as surgical protocols, head fixation approaches, and innovations in optics and microscopy technology. Here we review the last decade of MPM development and highlight how in vivo imaging has matured and diversified, making it now possible to concurrently monitor thousands of neurons across connected brain areas or, alternatively, small local networks with sampling rates in the kilohertz range. This review includes different laser scanning approaches, such as multibeam technologies as well as recent developments to image deeper into neuronal tissues using new, long-wavelength laser sources. As future development will critically depend on our ability to resolve and discriminate individual neuronal spikes, we will also describe a simple framework that allows performing quantitative comparisons between the reviewed MPM instruments. Finally, we provide our own opinion on how the most recent MPM developments can be leveraged at scale to enable the next generation of discoveries in brain function. [ABSTRACT FROM AUTHOR]

Understanding seizure formation and spread remains a critical goal of epilepsy research. We used fast in vivo two-photon calcium imaging in male mouse neocortex to reconstruct, with single-cell resolution, the dynamics of acute (4-aminopyridine) focal cortical seizures as they originate within a spatially confined seizure initiation site (intrafocal region), and subsequently propagate into neighboring cortical areas (extrafocal region). We find that seizures originate as local neuronal ensembles within the initiation site. This abnormal hyperactivity engages increasingly larger areas in a saltatory fashion until it breaks into neighboring cortex, where it proceeds smoothly and is then detected electrophysiologically (LFP). Interestingly, PV inhibitory interneurons have spatially heterogeneous activity in intrafocal and extrafocal territories, ruling out a simple role of inhibition in seizure formation and spread. We propose a two-step model for the progression of focal seizures, where neuronal ensembles activate first, generating a microseizure, followed by widespread neural activation in a traveling wave through neighboring cortex during macroseizures. [ABSTRACT FROM AUTHOR]

Astrocytes can control basal synaptic strength and arteriole tone via their resting Ca2+ activity. However, whether resting astrocyte Ca2+ can adjust to a new steady-state level, with an impact on surrounding brain cells, remains unknown. Using two-photon Ca2+ imaging in male rat acute brain slices of the somatosensory neocortex, we found that theta burst neural activity produced an unexpected long-lasting reduction in astrocyte free Ca2+ in the soma and endfeet. The drop in intracellular Ca2+ was attenuated by antagonists targeting multiple ionotropic and metabotropic glutamate receptors, and intracellular cascades involved Ca2+ stores and nitric oxide. The reduction in astrocyte endfoot Ca2+ was coincident with an increase in arteriole tone, and both the Ca2+ drop and the tone change were prevented by an NMDA receptor antagonist. Astrocyte patch-clamp experiments verified that the glutamate receptors in question were located on astrocytes and that Ca2+ changes within astrocytes were responsible for the long-lasting change in arteriole diameter caused by theta burst neural activity. In astrocytes from animals that lived in an enriched environment, we measured a relatively lower resting Ca2+ level that occluded any further drop in Ca2+ in response to theta burst activity. These data suggest that electrically evoked patterns of neural activity or natural experience can adjust steady-state resting astrocyte Ca2+ and that the effect has an impact on basal arteriole diameter. [ABSTRACT FROM AUTHOR]

Discriminating between temporal features in sensory stimuli is critical to complex behavior and decision-making. However, how sensory cortical circuit mechanisms contribute to discrimination between subsecond temporal components in sensory events is unclear. To elucidate the mechanistic underpinnings of timing in primary visual cortex (V1), we recorded from V1 using two-photon calcium imaging in awake-behaving mice performing a go/no-go discrimination timing task, which was composed of patterns of subsecond audiovisual stimuli. In both conditions, activity during the early stimulus period was temporally coordinated with the preferred stimulus. However, while network activity increased in the preferred condition, network activity was increasingly suppressed in the nonpreferred condition over the stimulus period. Multiple levels of analyses suggest that discrimination between subsecond intervals that are contained in rhythmic patterns can be accomplished by local neural dynamics in V1., Competing Interests: The authors declare no competing financial interests., (Copyright © 2023 Post et al.)

Dendritic spines are a morphological feature of the majority of excitatory synapses in the mammalian neocortex and are motile structures with shapes and lifetimes that change throughout development. Proper cortical development and function, including cortical contributions to learning and memory formation, require appropriate experience-dependent dendritic spine remodeling. Dendritic spine abnormalities have been reported for many neurodevelopmental disorders, including Angelman syndrome (AS), which is caused by the loss of the maternally inherited UBE3A allele (encoding ubiquitin protein ligase E3A). Prior studies revealed that UBE3A protein loss leads to reductions in dendritic spine density and diminished excitatory synaptic transmission. However, the decrease in spine density could come from either a reduction in spine formation or an increase in spine elimination. Here, we used acute and longitudinal in vivo two-photon microscopy to investigate developmental and experience-dependent changes in the numbers, dynamics, and morphology of layer 5 pyramidal neuron apical dendritic spines in the primary visual cortex of control and AS model mice (Ube3am-/p+ mice). We found that neurons in AS model mice undergo a greater elimination of dendritic spines than wild-type mice during the end of the first postnatal month. However, when raised in darkness, spine density and dynamics were indistinguishable between control and AS model mice, which indicates that decreased spine density in AS model mice reflects impaired experience-driven spine maintenance. Our data thus demonstrate an experience-dependent anatomical substrate by which the loss of UBE3A reduces dendritic spine density and disrupts cortical circuitry. [ABSTRACT FROM AUTHOR]

In secondary epilepsy, a seizure-prone neural network evolves during the latent period between brain injury and the onset of spontaneous seizures. The nature of the evolution is largely unknown, and even its completeness at the onset of seizures has recently been challenged by measures of gradually decreasing intervals between subsequent seizures. Sequential calcium imaging of neuronal activity, in the pyramidal cell layer of mouse hippocampal in vitro preparations, during early post-traumatic epileptogenesis demonstrated rapid increases in the fraction of neurons that participate in interictal activity. This was followed by more gradual increases in the rate at which individual neurons join each developing seizure, the pairwise correlation of neuronal activities as a function of the distance separating the pair, and network-wide measures of functional connectivity. These data support the continued evolution of synaptic connectivity in epileptic networks beyond the latent period: early seizures occur when recurrent excitatory pathways are largely polysynaptic, while ongoing synaptic remodeling after the onset of epilepsy enhances intranetwork connectivity as well as the onset and spread of seizure activity. [ABSTRACT FROM AUTHOR]

Pyramidal neurons in layers 2/3 and 5 of primary somatosensory cortex (S1) exhibit somewhat modest synaptic plasticity after whisker input deprivation. Whether neurons involved at earlier steps of sensory processing show more or less plasticity has not yet been examined. Here, we used longitudinal in vivo two-photon microscopy to investigate dendritic spine dynamics in apical tufts of GFP-expressing layer 4 (L4) pyramidal neurons of the vibrissal (barrel) S1 after unilateral whisker trimming. First, we characterize the molecular, anatomical, and electrophysiological properties of identified L4 neurons in Ebf2-Cre transgenic mice. Next, we show that input deprivation results in a substantial (~50%) increase in the rate of dendritic spine loss, acutely (4-8 d) after whisker trimming. This robust synaptic plasticity in L4 suggests that primary thalamic recipient pyramidal neurons in S1 may be particularly sensitive to changes in sensory experience. Ebf2-Cre mice thus provide a useful tool for future assessment of initial steps of sensory processing in S1. [ABSTRACT FROM AUTHOR]

The membrane protein Nogo-A is known as an inhibitor of axonal outgrowth and regeneration in the CNS. However, its physiological functions in the normal adult CNS remain incompletely understood. Here, we investigated the role of Nogo-A in cortical synaptic plasticity and motor learning in the uninjured adult rodent motor cortex. Nogo-A and its receptor NgR1 are present at cortical synapses. Acute treatment of slices with function-blocking antibodies (Abs) against Nogo-A or against NgR1 increased long-term potentiation (LTP) induced by stimulation of layer 2/3 horizontal fibers. Furthermore, anti-Nogo-A Ab treatment increased LTP saturation levels, whereas long-term depression remained unchanged, thus leading to an enlarged synaptic modification range. In vivo, intrathecal application of Nogo-A-blocking Abs resulted in a higher dendritic spine density at cortical pyramidal neurons due to an increase in spine formation as revealed by in vivo two-photon microscopy. To investigate whether these changes in synaptic plasticity correlate with motor learning, we trained rats to learn a skilled forelimb-reaching task while receiving anti-Nogo-A Abs. Learning of this cortically controlled precision movement was improved upon anti-Nogo-A Ab treatment. Our results identify Nogo-A as an influential molecular modulator of synaptic plasticity and as a regulator for learning of skilled movements in the motor cortex. [ABSTRACT FROM AUTHOR]

Spiking correlations between neocortical neurons provide insight into the underlying synaptic connectivity that defines cortical microcircuitry. Here, using two-photon calcium fluorescence imaging, we observed the simultaneous dynamics of hundreds of neurons in slices of mouse primary visual cortex (V1). Consistent with a balance of excitation and inhibition, V1 dynamics were characterized by a linear scaling between firing rate and circuit size. Using lagged firing correlations between neurons, we generated functional wiring diagrams to evaluate the topological features of V1 microcircuitry. We found that circuit connectivity exhibited both cyclic graph motifs, indicating recurrent wiring, and acyclic graph motifs, indicating feedforward wiring. After overlaying the functional wiring diagrams onto the imaged field of view, we found properties consistent with Rentian scaling: wiring diagrams were topologically efficient because they minimized wiring with a modular architecture. Within single imaged fields of view, V1 contained multiple discrete circuits that were overlapping and highly interdigitated but were still distinct from one another. The majority of neurons that were shared between circuits displayed peri-event spiking activity whose timing was specific to the active circuit, whereas spike times for a smaller percentage of neurons were invariant to circuit identity. These data provide evidence that V1 microcircuitry exhibits balanced dynamics, is efficiently arranged in anatomical space, and is capable of supporting a diversity of multineuron spike firing patterns from overlapping sets of neurons. [ABSTRACT FROM AUTHOR]

Functional mapping and microstimulation studies suggest that recovery after stroke damage can be attributed to surviving brain regions taking on the functional roles of lost tissues. Although this model is well supported by data, it is not clear how activity in single neurons is altered in relation to cortical functional maps. It is conceivable that individual surviving neurons could adopt new roles at the expense of their usual function. Alternatively, neurons that contribute to recovery may take on multiple functions and exhibit a wider repertoire of neuronal processing. In vivo two-photon calcium imaging was used in adult mice within reorganized forelimb and hindlimb somatosensory functional maps to determine how the response properties of individual neurons and glia were altered during recovery from ischemic damage over a period of 2- 8 weeks. Single-cell calcium imaging revealed that the limb selectivity of individual neurons was altered during recovery from ischemia, such that neurons normally selective for a single contralateral limb processed information from multiple limbs. Altered limb selectivity was most prominent in border regions between stroke-altered forelimb and hindlimb macroscopic map representations, and peaked 1 month after the targeted insult. Two months after stroke, individual neurons near the center of reorganized functional areas became more selective for a preferred limb. These previously unreported forms of plasticity indicate that in adult animals, seemingly hardwired cortical neurons first adopt wider functional roles as they develop strategies to compensate for loss of specific sensory modalities after forms of brain damage such as stroke. [ABSTRACT FROM AUTHOR]

Astrocytes play an essential role in the maintenance and protection of the brain, which we reported was diminished with age. Here, we demonstrate that activation of a purinergic receptor (P2Y-R) signaling pathway, in astrocytes, significantly increases the resistance of astrocytes and neurons to oxidative stress. Interestingly, P2Y-R activation in old astrocytes increased their resistance to oxidative stress to levels that were comparable with stimulated young astrocytes. P2Y-R enhanced neuroprotection was blocked by oligomycin and by Xestospongin C, inhibitors of the ATP synthase and of inositol (1,4,5) triphosphate (IP3 ) binding to the IP3 receptor, respectively. Treatment of astrocytes with a membrane permeant analog of IP3 also protected astrocytes against oxidative stress. These data indicate that P2Y-R enhanced astrocyte neuroprotection is mediated by a Ca2+-dependent increase in mitochondrial metabolism. These data also reveal a signaling pathway that can rapidly respond to central energy needs throughout the aging process. [ABSTRACT FROM AUTHOR]

Spine loss represents a common hallmark of neurodegenerative diseases. However, little is known about the underlying mechanisms, especially the relationship between spine elimination and neuritic destruction. We imaged cortical dendrites throughout a neurodegenerative disease using scrapie in mice as a model. Two-photon in vivo imaging over 2 months revealed a linear decrease of spine density. Interestingly, only persistent spines (lifetime≥8 d) disappeared, whereas the density of transient spines (lifetime≤4 d) was unaffected. Before spine loss, dendritic varicosities emerged preferentially at sites where spines protrude from the dendrite. These results implicate that the location where the spine protrudes from the dendrite may be particularly vulnerable and that dendritic varicosities may actually cause spine loss. [ABSTRACT FROM AUTHOR]

Synaptic transmission initiates a cascade of signal transduction events that couple neuronal activity to local changes in blood flow and oxygenation. Although a number of vasoactive molecules and specific cell types have been implicated, the transformation of stimulusinduced activation of neuronal circuits to hemodynamic changes is still unclear. We use somatosensory stimulation and a suite of in vivo imaging tools to study neurovascular coupling in rat primary somatosensory cortex. Our stimulus evoked a central region of net neuronal depolarization surrounded by net hyperpolarization. Hemodynamic measurements revealed that predominant depolarization corresponded to an increase in oxygenation, whereas predominant hyperpolarization corresponded to a decrease in oxygenation. On the microscopic level of single surface arterioles, the response was composed of a combination of dilatory and constrictive phases. Critically, the relative strength of vasoconstriction covaried with the relative strength of oxygenation decrease and neuronal hyperpolarization. These results suggest that a neuronal inhibition and concurrent arteriolar vasoconstriction correspond to a decrease in blood oxygenation, which would be consistent with a negative blood oxygenation level-dependent functional magnetic resonance imaging signal. [ABSTRACT FROM AUTHOR]

Although plastic changes are known to occur in developing and adult cortex, it remains unclear whether these changes require remodeling of cortical circuitry whereby synapses are formed and eliminated or whether they rely on changes in the strength of existing synapses. To determine the structural stability of dendritic spines and axon terminals in vivo, we chose two approaches. First, we performed time-lapse two-photon imaging of dendritic spine motility of layer 5 pyramidal neurons in juvenile [postnatal day 28 (P28)] mice in visual, auditory, and somatosensory cortices. We found that there were differences in basal rates of dendritic spine motility of the same neuron type in different cortices, with visual cortex exhibiting the least structural dynamics. Rewiring visual input into the auditory cortex at birth, however, failed to alter dendritic spine motility, suggesting that structural plasticity rates might be intrinsic to the cortical region. Second, we investigated the persistence of both the presynaptic (axon terminals) and postsynaptic (dendritic spine) structures in young adult mice (P40-P61), using chronic in vivo two-photon imaging in different sensory areas. Both terminals and spines were relatively stable, with > 80% persisting over a 3 week period in all sensory regions. Axon terminals were more stable than dendritic spines. These data suggest that changes in network function during adult learning and memory might occur through changes in the strength and efficacy of existing synapses as well as some remodeling of connectivity through the loss and gain of synapses. [ABSTRACT FROM AUTHOR]

Ca2+ influx through synaptic NMDA receptors (NMDA-Rs) triggers a variety of adaptive cellular processes. To probe NMDA-R-mediated [Ca2+] signaling, we used two-photon glutamate uncaging to stimulate NMDA-Rs on individual dendritic spines of CA1 pyramidal neurons in rat brain slices. We measured NMDA-R currents at the soma and NMDA-R-mediated [Ca2+] transients in stimulated spines (Δ[Ca2+]). Uncaging-evoked NMDA-R current amplitudes were independent of the size of the stimulated spine, implying that smaller spines contain higher densities of functional NMDA-Rs. The ratio of Δ[Ca2+] over NMDA-R current was highly variable (factor of 10) across spines, especially for small spines. These differences were not explained by heterogeneity in spine sizes or diffusional coupling between spines and their parent dendrites. In addition, we find that small spines have NMDA-R currents that are sensitive to NMDA-R NR2B subunit-specific antagonists. With block of NR2B-containing receptors, the range of Δ[Ca2+]/NMDA-R current ratios and their average value were much reduced. Our data suggest that individual spines can regulate the subunit composition of their NMDA-Rs and the effective fractional Ca2+ current through these receptors. [ABSTRACT FROM AUTHOR]

Thalamic projections to prefrontal cortex are important for executive aspects of attention. Using two-photon imaging in prefrontal brain slices, we show that nicotine and the wakefulness neuropeptide hypocretin (orexin) excite the same identified synapses of the thalamocortical arousal pathway within the prefrontal cortex. Although it is known that attention can be improved when nicotine is infused directly into the midlayer of the prefrontal cortex in the rat, the effects of hypocretin on attention are not known. The overlap in thalamocortical synapses excited by hypocretin and nicotine and the lack of direct postsynaptic effects prompted us to compare their effects on a sustained and divided attention task in the rat. Similar to nicotine, infusions of hypocretin-2 peptide into the prefrontal cortex significantly improved accuracy under high attentional demand without effects on other performance measures. We show for the first time that hypocretin can improve attentional processes relevant to executive functions of the prefrontal cortex. [ABSTRACT FROM AUTHOR]

Action potentials backpropagate into the dendritic trees of pyramidal neurons, reporting output activity to the sites of synaptic input and provoking long-lasting changes in synaptic strength. It is unclear how this retrograde signal is modified by neural network activity. Using whole-cell recordings from somata, apical trunks, and dendritic tuft branches of layer 2/3 pyramidal neurons in vivo, we show that network-driven subthreshold membrane depolarizations ("up states") occur simultaneously throughout the apical dendritic tree. This spontaneous synaptic activity enhances action potential-evoked calcium influx into the distal apical dendrite by promoting action potential backpropagation. Hence, somatic feedback to the dendrites becomes stronger with increasing network activity. [ABSTRACT FROM AUTHOR]

Genetically encoded Ca2+ indicators (GECIs) based on fluorescent proteins (XFPs) and Ca2+-binding proteins [like calmodulin (CAM)] have great potential for the study of subcellular Ca2+ signaling and for monitoring activity in populations of neurons. However, interpreting GECI fluorescence in terms of neural activity and cytoplasmic-free Ca2+ concentration ([Ca2+]) is complicated by the nonlinear interactions between Ca2+ binding and GECI fluorescence. We have characterized GECIs in pyramidal neurons in cultured hippocampal brain slices, focusing on indicators based on circularly permuted XFPs [GCaMP (Nakai et al., 2001), Camgaroo2 (Griesbeck et al., 2001 ), and Inverse Pericam (Nagai et al., 2001)]. Measurements of fluorescence changes evoked by trains of action potentials revealed that GECIs have little sensitivity at low action potential frequencies compared with synthetic [Ca2+] indicators with similar affinities for Ca2+. The sensitivity of GECIs improved for high-frequency trains of action potentials, indicating that GECIs are supralinear indicators of neural activity. Simultaneous measurement of GECI fluorescence and [Ca2+] revealed supralinear relationships. We compared GECI fluorescence saturation with CaM Ca2+-dependent structural transitions. Our data suggest that GCaMP and Camgaroo2 report CaM structural transitions in the presence and absence of CaM-binding peptide, respectively. [ABSTRACT FROM AUTHOR]

While humans and other mammals exhibit adaptation to odorants, the neural mechanisms and brain locations involved in this process are incompletely understood. One possibility is that it primarily occurs as a result of the interactions between odorants and odorant receptors on the olfactory sensory neurons in the olfactory epithelium. In this scenario, adaptation would arise as a peripheral phenomenon transmitted to the brain. An alternative possibility is that adaptation occurs because of processing in the brain. We made an initial test of these possibilities using a two-color imaging strategy to simultaneously measure the activity of the olfactory receptor nerve terminals (input to the bulb) and mitral/tufted cell apical dendrites (output from the bulb) in anesthetized and awake mice. Repeated odor stimulation at the same concentration resulted in a decline in the bulb output, while the input remained relatively stable. Thus, the mammalian olfactory bulb appears to participate in generating the perception of olfactory adaptation under this stimulus condition. Similar experiments conducted previously showed that the bulb may also participate in the perception of concentration invariance of odorant recognition (Storace and Cohen, 2017); thus, the bulb is simultaneously carrying out more than one computation, as is true of other mammalian brain regions and perhaps is the case for all animals with sophisticated nervous systems. However, in contrast with other sensory systems (Van Essen et al., 1992), the very first processing stage in the olfactory system has an output that may directly represent perceptions., (Copyright © 2021 Storace and Cohen.)

The axon initial segment (AIS) is a specialized neuronal compartment in which synaptic input is converted into action potential (AP) output. This process is supported by a diverse complement of sodium, potassium, and calcium channels (Ca V ). Different classes of sodium and potassium channels are scaffolded at specific sites within the AIS, conferring unique functions, but how calcium channels are functionally distributed within the AIS is unclear. Here, we use conventional two-photon laser scanning and diffraction-limited, high-speed spot two-photon imaging to resolve AP-evoked calcium dynamics in the AIS with high spatiotemporal resolution. In mouse layer 5 prefrontal pyramidal neurons, calcium influx was mediated by a mix of Ca V 2 and Ca V 3 channels that differentially localized to discrete regions. Ca V 3 functionally localized to produce nanodomain hotspots of calcium influx that coupled to ryanodine-sensitive stores, whereas Ca V 2 localized to non-hotspot regions. Thus, different pools of Ca V s appear to play distinct roles in AIS function. SIGNIFICANCE STATEMENT The axon initial segment (AIS) is the site where synaptic input is transformed into action potential (AP) output. It achieves this function through a diverse complement of sodium, potassium, and calcium channels (Ca V ). While the localization and function of sodium channels and potassium channels at the AIS is well described, less is known about the functional distribution of Ca V s. We used high-speed two-photon imaging to understand activity-dependent calcium dynamics in the AIS of mouse neocortical pyramidal neurons. Surprisingly, we found that calcium influx occurred in two distinct domains: Ca V 3 generates hotspot regions of calcium influx coupled to calcium stores, whereas Ca V 2 channels underlie diffuse calcium influx between hotspots. Therefore, different Ca V classes localize to distinct AIS subdomains, possibly regulating distinct cellular processes., (Copyright © 2021 the authors.)

Membrane voltage oscillations in layer 1 (L1) of primary sensory cortices might be important indicators of cortical gain control, attentional focusing, and signal integration. However, electric field recordings are hampered by the low seal resistance of electrodes close to the brain surface. To study L1 membrane voltage oscillations, we synthesized a new voltage-sensitive dye, di1-ANNINE (anellated hemicyanine)-6plus, that can diffuse into tissue. We applied it with a new surgery, leaving the dura intact but allowing injection of large quantities of staining solution, and imaged cortical membrane potential oscillations with two-photon microscopy depth-resolved (25-100 μm below dura) in anesthetized and awake mice. We found delta (0.5-4 Hz), theta (4-10 Hz), low beta (10-20 Hz), and low gamma (30-40 Hz) oscillations. All oscillations were stronger in awake animals. While the power of delta, theta, and low beta oscillations increased with depth, the power of low gamma was more constant throughout L1. These findings identify L1 as an important coordination hub for the dynamic binding process of neurons mediated by oscillations., (Copyright © 2020 Dalphin et al.)

Two-photon imaging studies in mouse primary visual cortex (V1) consistently report that around half of the neurons respond to oriented grating stimuli. However, in cats and primates, nearly all neurons respond to such stimuli. Here we show that mouse V1 responsiveness and selectivity strongly depends on neuronal depth. Moving from superficial layer 2 down to layer 4, the percentage of visually responsive neurons nearly doubled, ultimately reaching levels similar to what is seen in other species. Over this span, the amplitude of neuronal responses also doubled. Moreover, stimulus selectivity was also modulated, not only with depth but also with response amplitude. Specifically, we found that orientation and direction selectivity were greater in stronger responding neurons, but orientation selectivity decreased with depth whereas direction selectivity increased. Importantly, these depth-dependent trends were found not just between layer 2/3 and layer 4 but at different depths within layer 2/3 itself. Thus, neuronal depth is an important factor to consider when pooling neurons for population analyses. Furthermore, the inability to drive the majority of cells in superficial layer 2/3 of mouse V1 with grating stimuli indicates that there may be fundamental differences in the micro-circuitry and role of V1 between rodents and other mammals., (Copyright © 2020 O’Herron et al.)

Odor information is transmitted from olfactory sensory neurons to principal neurons at the glomeruli of the olfactory bulb. The intraglomerular neuronal circuit also includes hundreds of interneurons referred to as juxtaglomerular (JG) cells. Stimulus selectivity is well correlated among many JG cells that are associated with the same glomerulus, consistent with their highly homogeneous sensory inputs. However, much less is known about the temporal aspects of their activity, including the temporal coordination of their odor-evoked responses. As many JG cells within a glomerular module respond to the same stimulus, the extent to which their activity is temporally aligned will affect the temporal profile of their population inhibitory inputs. Using random-access high-speed two-photon microscopy, we recorded the odor-evoked calcium transients of mouse JG cells and compared the onset latency and rise time among neurons putatively associated with the same and different glomeruli. Whereas the overall onset latencies of odor-evoked transients were distributed across a ∼150 ms time window, those from cells putatively associated with the same glomerulus were confined to a much narrower window of several tens of milliseconds. This result suggests that onset latency primarily depends on the associated glomerulus. We also observed glomerular specificity in the rise time. The glomerulus-specific temporal pattern of odor-evoked activity implies that the temporal patterns of inputs from the intraglomerular circuit are unique to individual glomerulus-odor pairs, which may contribute to efficient shaping of the temporal pattern of activity in the principal neurons.

Astrocytes can control basal synaptic strength and arteriole tone via their resting Ca 2+ activity. However, whether resting astrocyte Ca 2+ can adjust to a new steady-state level, with an impact on surrounding brain cells, remains unknown. Using two-photon Ca 2+ imaging in male rat acute brain slices of the somatosensory neocortex, we found that theta burst neural activity produced an unexpected long-lasting reduction in astrocyte free Ca 2+ in the soma and endfeet. The drop in intracellular Ca 2+ was attenuated by antagonists targeting multiple ionotropic and metabotropic glutamate receptors, and intracellular cascades involved Ca 2+ stores and nitric oxide. The reduction in astrocyte endfoot Ca 2+ was coincident with an increase in arteriole tone, and both the Ca 2+ drop and the tone change were prevented by an NMDA receptor antagonist. Astrocyte patch-clamp experiments verified that the glutamate receptors in question were located on astrocytes and that Ca 2+ changes within astrocytes were responsible for the long-lasting change in arteriole diameter caused by theta burst neural activity. In astrocytes from animals that lived in an enriched environment, we measured a relatively lower resting Ca 2+ level that occluded any further drop in Ca 2+ in response to theta burst activity. These data suggest that electrically evoked patterns of neural activity or natural experience can adjust steady-state resting astrocyte Ca 2+ and that the effect has an impact on basal arteriole diameter. SIGNIFICANCE STATEMENT The field of astrocyte-neuron and astrocyte-arteriole interactions is currently in a state of refinement. Experimental evidence ex vivo suggests that direct manipulation of astrocyte-free Ca 2+ regulates synaptic signaling and local blood flow control; however, in vivo experiments fail to link synaptically evoked astrocyte Ca 2+ transients and immediate changes to various astrocyte-mediated processes. To clarify this discrepancy, we examined a different aspect of astrocyte Ca 2+ : the resting, steady-state free Ca 2+ of astrocytes, its modulation, and its potential role in the tonic regulation of surrounding brain cells. We found that ex vivo or in vivo neural activity induced a long-lasting reduction in resting free astrocyte Ca 2+ and that this phenomenon changed arteriole tone., (Copyright © 2017 the authors 0270-6474/17/378150-16$15.00/0.)

The response properties of neurons to sensory stimuli have been used to identify their receptive fields and to functionally map sensory systems. In primary visual cortex, most neurons are selective to a particular orientation and spatial frequency of the visual stimulus. Using two-photon calcium imaging of neuronal populations from the primary visual cortex of mice, we have characterized the response properties of neurons to various orientations and spatial frequencies. Surprisingly, we found that the orientation selectivity of neurons actually depends on the spatial frequency of the stimulus. This dependence can be easily explained if one assumed spatially asymmetric Gabor-type receptive fields. We propose that receptive fields of neurons in layer 2/3 of visual cortex are indeed spatially asymmetric, and that this asymmetry could be used effectively by the visual system to encode natural scenes.

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., (Copyright © 2015 the authors 0270-6474/15/353016-06$15.00/0.)