Neuronal silence as a prosurvival factor for adult-born olfactory bulb interneurons.

Adult-born cells, arriving daily into the rodent olfactory bulb, either integrate into the neural circuitry or get eliminated. However, whether these two populations differ in their morphological or functional properties remains unclear. Using longitudinal in vivo two-photon imaging, we monitored dendritic morphogenesis, odor-evoked responsiveness, ongoing Ca2+ signaling, and survival/death of adult-born juxtaglomerular neurons (abJGNs). We found that the maturation of abJGNs is accompanied by a significant reduction in dendritic complexity, with surviving and subsequently eliminated cells showing similar degrees of dendritic remodeling. Surprisingly, ∼63% of eliminated abJGNs acquired odor responsiveness before death, with amplitudes and time courses of odor-evoked responses similar to those recorded in surviving cells. However, the subsequently eliminated cell population exhibited significantly higher ongoing Ca2+ signals, with a difference visible even 10 days before death. Quantitative supervised machine learning analysis revealed a relationship between the abJGNs' activity and survival probability, with low neuronal activity being supportive for survival.

Endogenous but not sensory-driven activity controls migration, morphogenesis and survival of adult-born juxtaglomerular neurons in the mouse olfactory bulb.

The development and survival of adult-born neurons are believed to be driven by sensory signaling. Here, in vivo analyses of motility, morphology and Ca2+ signaling, as well as transcriptome analyses of adult-born juxtaglomerular cells with reduced endogenous excitability (via cell-specific overexpression of either Kv1.2 or Kir2.1 K+ channels), revealed a pronounced impairment of migration, morphogenesis, survival, and functional integration of these cells into the mouse olfactory bulb, accompanied by a reduction in cytosolic Ca2+ fluctuations, phosphorylation of CREB and pCREB-mediated gene expression. Moreover, K+ channel overexpression strongly downregulated genes involved in neuronal migration, differentiation, and morphogenesis and upregulated apoptosis-related genes, thus locking adult-born cells in an immature and vulnerable state. Surprisingly, cells deprived of sensory-driven activity developed normally. Together, the data reveal signaling pathways connecting the endogenous intermittent neuronal activity/Ca2+ fluctuations as well as enhanced Kv1.2/Kir2.1 K+ channel function to migration, maturation, and survival of adult-born neurons.

Stable behavioral state-specific large scale activity patterns in the developing cortex of neonates.

Intrinsic neuronal activity is a hallmark of the developing brain. In rodents, a handful of such activities were described in different cortical areas but the unifying macroscopic perspective is still lacking. Here we combined large-scale in vivo Ca2+ imaging of the dorsal cortex in non-anesthetized neonatal mice with mathematical analyses to reveal unique behavioral state-specific maps of intrinsic activity. These maps were remarkably stable over time within and across experiments and used patches of correlated activity with little hemispheric symmetry as well as stationary and propagating waves as building blocks. Importantly, the maps recorded during motion and rest were almost inverse, with frontoparietal areas active during motion and posterior-lateral areas active at rest. The retrosplenial cortex engaged in both resting- and motion-related activities via functional long-range connections with respective cortical areas. The data obtained bind different region-specific activity patterns described so far into a single consistent picture and set the stage for future inactivation studies, probing the exact function of this complex activity pattern for cortical wiring in neonates.

Unique Functional Properties of Mature Adult-Born Neurons in the Mouse Olfactory Bulb.

The rodent olfactory bulb (OB) is continuously supplied with adult-born cells maturing into GABAergic neurons. Using in vivo ratiometric Ca2+ imaging to readout ongoing and sensory-driven activity, we asked whether mature adult-born cells (mABCs) in the glomerular layer of the bulb become functionally identical to resident GABAergic (ResGABA) neurons. In awake head-restrained mice the two cell populations differed significantly in terms of ongoing spontaneous activity, with 24% of mABCs contributing to a strongly active cell cluster, absent among ResGABA cells. Odor-evoked responses of mABCs were sparse, less reliable, and had smaller amplitudes compared with ResGABA cells. The opposite was seen under anesthesia, with response reliability increasing and response size of mABCs becoming larger than that of ResGABA cells. Furthermore, ongoing activity of mABCs showed increased sensitivity to ketamine/xylazine and was selectively blocked by the antagonist of serotonin receptors methysergide. These functional features of mABCs clearly distinguish them from other OB interneurons.

Spontaneous calcium transients in the immature adult-born neurons of the olfactory bulb.

Spontaneous neuronal activity and concomitant intracellular Ca2+ signaling are abundant during early perinatal development and are well known for their key role in neuronal proliferation, migration, differentiation and wiring. However, much less is known about the in vivo patterns of spontaneous Ca2+ signaling in immature adult-born cells. Here, by using two-photon Ca2+ imaging, we analyzed spontaneous in vivo Ca2+ signaling in adult-born juxtaglomerular cells of the mouse olfactory bulb over the time period of 5 weeks, from the day of their arrival in the glomerular layer till their stable integration into the preexisting neural network. We show that spontaneous Ca2+ transients are ubiquitously present in adult-born cells right after their arrival, require activation of voltage-gated Na+ channels and are little sensitive to isoflurane anesthesia. Interestingly, several parameters of this spontaneous activity, such as the area under the curve, the time spent in the active state as well as the fraction of continuously active cells show a bell-shaped dependence on cell's age, all peaking in 3-4 weeks old cells. This data firmly document the in vivo presence of spontaneous Ca2+ signaling during the layer-specific maturation of adult-born neurons in the olfactory bulb and motivate further analyses of the functional role(s) of this activity.

Intracellular Ca2+ stores control in vivo neuronal hyperactivity in a mouse model of Alzheimer's disease.

Neuronal hyperactivity is the emerging functional hallmark of Alzheimer's disease (AD) in both humans and different mouse models, mediating an impairment of memory and cognition. The mechanisms underlying neuronal hyperactivity remain, however, elusive. In vivo Ca2+ imaging of somatic, dendritic, and axonal activity patterns of cortical neurons revealed that both healthy aging and AD-related mutations augment neuronal hyperactivity. The AD-related enhancement occurred even without amyloid deposition and neuroinflammation, mainly due to presenilin-mediated dysfunction of intracellular Ca2+ stores in presynaptic boutons, likely causing more frequent activation of synaptic NMDA receptors. In mutant but not wild-type mice, store emptying reduced both the frequency and amplitude of presynaptic Ca2+ transients and, most importantly, normalized neuronal network activity. Postsynaptically, the store dysfunction was minor and largely restricted to hyperactive cells. These findings identify presynaptic Ca2+ stores as a key element controlling AD-related neuronal hyperactivity and as a target for disease-modifying treatments.

A new approach for ratiometric in vivo calcium imaging of microglia.

Microglia, resident immune cells of the brain, react to the presence of pathogens/danger signals with a large repertoire of functional responses including morphological changes, proliferation, chemotaxis, production/release of cytokines, and phagocytosis. In vitro studies suggest that many of these effector functions are Ca2+-dependent, but our knowledge about in vivo Ca2+ signalling in microglia is rudimentary. This is mostly due to technical reasons, as microglia largely resisted all attempts of in vivo labelling with Ca2+ indicators. Here, we introduce a novel approach, utilizing a microglia-specific microRNA-9-regulated viral vector, enabling the expression of a genetically-encoded ratiometric Ca2+ sensor Twitch-2B in microglia. The Twitch-2B-assisted in vivo imaging enables recording of spontaneous and evoked microglial Ca2+ signals and allows for the first time to monitor the steady state intracellular Ca2+ levels in microglia. Intact in vivo microglia show very homogenous and low steady state intracellular Ca2+ levels. However, the levels increase significantly after acute slice preparation and cell culturing along with an increase in the expression of activation markers CD68 and IL-1ß. These data identify the steady state intracellular Ca2+ level as a versatile microglial activation marker, which is highly sensitive to the cell's environment.

Long-term in vivo single-cell tracking reveals the switch of migration patterns in adult-born juxtaglomerular cells of the mouse olfactory bulb.

The behavior of adult-born cells can be easily monitored in cell culture or in lower model organisms, but longitudinal observation of individual mammalian adult-born cells in their native microenvironment still proves to be a challenge. Here we have established an approach named optical cell positioning system for long-term in vivo single-cell tracking, which integrates red-green-blue cell labeling with repeated angiography. By combining this approach with in vivo two-photon imaging technique, we characterized the in vivo migration patterns of adult-born neurons in the olfactory bulb. In contrast to the traditional view of mere radial migration of adult-born cells within the bulb, we found that juxtaglomerular cells switch from radial migration to long distance lateral migration upon arrival in their destination layer. This unique long-distance lateral migration has characteristic temporal (stop-and-go) and spatial (migratory, unidirectional or multidirectional) patterns, with a clear cell age-dependent decrease in the migration speed. The active migration of adult-born cells coincides with the time period of initial fate determination and is likely to impact on the integration sites of adult-born cells, their odor responsiveness, as well as their survival rate.

GABA depolarizes immature neurons and inhibits network activity in the neonatal neocortex in vivo.

A large body of evidence from in vitro studies suggests that GABA is depolarizing during early postnatal development. However, the mode of GABA action in the intact developing brain is unknown. Here we examine the in vivo effects of GABA in cells of the upper cortical plate using a combination of electrophysiological and Ca(2+)-imaging techniques. We report that at postnatal days (P) 3-4, GABA depolarizes the majority of immature neurons in the occipital cortex of anaesthetized mice. At the same time, GABA does not efficiently activate voltage-gated Ca(2+) channels and fails to induce action potential firing. Blocking GABA(A) receptors disinhibits spontaneous network activity, whereas allosteric activation of GABA(A) receptors has the opposite effect. In summary, our data provide evidence that in vivo GABA acts as a depolarizing neurotransmitter imposing an inhibitory control on network activity in the neonatal (P3-4) neocortex.

In vivo odourant response properties of migrating adult-born neurons in the mouse olfactory bulb.

Juxtaglomerular neurons (JGNs) of the mammalian olfactory bulb are generated throughout life. Their integration into the preexisting neural network, their differentiation and survival therein depend on sensory activity, but when and how these adult-born cells acquire responsiveness to sensory stimuli remains unknown. In vivo two-photon imaging of retrovirally labelled adult-born JGNs reveals that ~90% of the cells arrive at the glomerular layer after day post injection (DPI) 7. After arrival, adult-born JGNs are still migrating, but at DPI 9, 52% of them have odour-evoked Ca(2+) signals. Their odourant sensitivity closely resembles that of the parent glomerulus and surrounding JGNs, and their spontaneous and odour-evoked spiking is similar to that of their resident neighbours. Our data reveal a remarkably rapid functional integration of adult-born cells into the preexisting neural network. The mature pattern of odour-evoked responses of these cells strongly contrasts with their molecular phenotype, which is typical of immature, migrating neuroblasts.

Two-photon chloride imaging using MQAE in vitro and in vivo.

This protocol describes a technique for high-resolution chloride imaging of living cells using a quinoline-based chloride (Cl(-)) indicator dye, MQAE (N-[6-methoxyquinolyl] acetoethyl ester). Bath-applied to acute brain slices, MQAE provides high-quality labeling of neuronal cells and their processes. In living anesthetized mice, cortical cells are labeled using the multicell bolus loading procedure. In combination with two-photon microscopy, this procedure enables in vivo visualization of cell bodies of neurons and astrocytes as well as some astrocytic processes and allows one to monitor changes in the intracellular chloride concentration in dozens of individual cells.

Disruption of the olivo-cerebellar circuit by Purkinje neuron-specific ablation of BK channels.

The large-conductance voltage- and calcium-activated potassium (BK) channels are ubiquitously expressed in the brain and play an important role in the regulation of neuronal excitation. Previous work has shown that the total deletion of these channels causes an impaired motor behavior, consistent with a cerebellar dysfunction. Cellular analyses showed that a decrease in spike firing rate occurred in at least two types of cerebellar neurons, namely in Purkinje neurons (PNs) and in Golgi cells. To determine the relative role of PNs, we developed a cell-selective mouse mutant, which lacked functional BK channels exclusively in PNs. The behavioral analysis of these mice revealed clear symptoms of ataxia, indicating that the BK channels of PNs are of major importance for normal motor coordination. By using combined two-photon imaging and patch-clamp recordings in these mutant mice, we observed a unique type of synaptic dysfunction in vivo, namely a severe silencing of the climbing fiber-evoked complex spike activity. By performing targeted pharmacological manipulations combined with simultaneous patch-clamp recordings in PNs, we obtained direct evidence that this silencing of climbing fiber activity is due to a malfunction of the tripartite olivo-cerebellar feedback loop, consisting of the inhibitory synaptic connection of PNs to the deep cerebellar nuclei (DCN), followed by a projection of inhibitory DCN afferents to the inferior olive, the origin of climbing fibers. Taken together, our results establish an essential role of BK channels of PNs for both cerebellar motor coordination and feedback regulation in the olivo-cerebellar loop.

Sparsification of neuronal activity in the visual cortex at eye-opening.

Eye-opening represents a turning point in the function of the visual cortex. Before eye-opening, the visual cortex is largely devoid of sensory inputs and neuronal activities are generated intrinsically. After eye-opening, the cortex starts to integrate visual information. Here we used in vivo two-photon calcium imaging to explore the developmental changes of the mouse visual cortex by analyzing the ongoing spontaneous activity. We found that before eye-opening, the activity of layer 2/3 neurons consists predominantly of slow wave oscillations. These waves were first detected at postnatal day 8 (P8). Their initial very low frequency (0.01 Hz) gradually increased during development to approximately 0.5 Hz in adults. Before eye-opening, a large fraction of neurons (>75%) was active during each wave. One day after eye-opening, this dense mode of recruitment changed to a sparse mode with only 36% of active neurons per wave. This was followed by a progressive decrease during the following weeks, reaching 12% of active neurons per wave in adults. The possible role of visual experience for this process of sparsification was investigated by analyzing dark-reared mice. We found that sparsification also occurred in these mice, but that the switch from a dense to a sparse activity pattern was delayed by 3-4 days as compared with normally-reared mice. These results reveal a modulatory contribution of visual experience during the first days after eye-opening, but an overall dominating role of intrinsic factors. We propose that the transformation in network activity from dense to sparse is a prerequisite for the changed cortical function at eye-opening.

Improved calcium imaging in transgenic mice expressing a troponin C-based biosensor.

Fluorescent Ca(2+) indicator proteins (FCIPs) are attractive tools for studying Ca(2+) dynamics in live cells. Here we describe transgenic mouse lines expressing a troponin C (TnC)-based biosensor. The biosensor is widely expressed in neurons and has improved Ca(2+) sensitivity both in vitro and in vivo. This allows FCIP-based two-photon Ca(2+) imaging of distinct neurons and their dendrites in vivo, and opens a new avenue for structure-function analysis of intact neuronal circuits.

Dendritic spikes and activity-dependent synaptic plasticity.

Whereas the regenerative nature of action potential conduction in axons has been known since the late 1940s, neuronal dendrites have been considered as passive cables transferring incoming synaptic activity to the soma. The relatively recent discovery that neuronal dendrites contain active conductances has revolutionized our view of information processing in neurons. In many neuronal cell types, sodium action potentials initiated at the axon initial segment can back-propagate actively into the dendrite thereby serving, for the dendrite, as an indicator of the output activity of the neuron. In addition, the dendrites themselves can initiate action-potential-like regenerative responses, so-called dendritic spikes, that are mediated either by the activation of sodium, calcium, and/or N-methyl-D-aspartate receptor channels. Here, we review the recent experimental and theoretical evidence for a role of regenerative dendritic activity in information processing within neurons and, especially, in activity-dependent synaptic plasticity.

Properties of the new fluorescent Na+ indicator CoroNa Green: comparison with SBFI and confocal Na+ imaging.

Neuronal activity causes substantial Na+ transients in fine cellular processes such as dendrites and spines. The physiological consequences of such Na+ transients are still largely unknown. High-resolution Na+ imaging is pivotal to study these questions, and, up to now, two-photon imaging with the fluorescent Na+ indicator sodium-binding benzofuran isophthalate (SBFI) has been the primary method of choice. Recently, a new Na+ indicator dye, CoroNa Green (CoroNa), that has its absorbance maximum at 492 nm, has become available. In the present study, we have compared the properties of SBFI with those of CoroNa by performing Na+ measurements in neurons of hippocampal slices. We show that CoroNa is suitable for measurement of Na+ transients using non-confocal wide-field imaging with a CCD camera. However, substantial transmembrane dye leakage and lower Na+ sensitivity are clearly disadvantages when compared to SBFI. We also tested CoroNa for its suitability for high-resolution imaging of Na+ transients using a confocal laser scanning system. We demonstrate that CoroNa, in contrast to SBFI, can be employed for confocal imaging using a conventional argon laser and report the first Na+ measurements in dendrites using this dye. In conclusion, CoroNa may prove to be a valuable tool for confocal Na+ imaging in fine cellular processes.

From modulator to mediator: rapid effects of BDNF on ion channels.

Neurotrophins (NTs) are [?AUTHOR] a family of structurally related, secreted proteins that regulate the survival, differentiation and maintenance of function of different populations of peripheral and central neurons.1,2 Among these, BDNF (brain-derived neurotrophic factor) has drawn considerable interest because both its synthesis and secretion are increased by physiological levels of activity, indicating a unique role of this neurotrophin in coupling neuronal activity to structural and functional properties of neuronal circuits. In addition to its classical neurotrophic effects, which are evident within hours or days and which usually result from changes in cellular gene expression, BDNF exerts acute effects on synaptic transmission and is involved in the induction of long-term potentiation. Many of these rapid effects of BDNF are mediated by its modulation of ion channel properties following TrkB-mediated activation of intracellular second messenger cascades and protein phosphorylation. However, recent reports have shown that BDNF not only acts as a modulator of ion channels, but can also directly and rapidly gate a Na(+) channel, thereby assigning BDNF the properties of a classical excitatory transmitter. Thus, BDNF, in addition its role as a potent neuromodulator, emerges as an excitatory transmitter-like substance which acutely controls resting membrane potential, neuronal excitability, synaptic transmission and participates in the induction of synaptic plasticity.

Neurotrophin action on a rapid timescale.

Mechanisms underlying the fast action of neurotrophins include intracellular Ca(2+) signaling, neuronal excitation, augmentation of synaptic excitation by modulation of N-methyl-d-aspartate receptor activity and control of synaptic inhibition through the regulation of the K(+)-Cl(-) cotransporter KCC2. The fastest action of brain-derived neurotrophic factor and neurotrophin-4/5 occurs within milliseconds, and involves activation of TrkB and the opening of the Na(+) channel Na(v)1.9. Through these rapid actions, neurotrophins shape neuronal activity, modulate synaptic transmission and produce instructive signals for the induction of long-term changes in the efficacy of synaptic transmission.

Single-shock LTD by local dendritic spikes in pyramidal neurons of mouse visual cortex.

Mammalian dendrites are active structures, capable of regenerative electrical activity. Dendritic spikes can mediate synaptic plasticity and could enrich the computational properties of neurons. Besides sodium-based action potentials, which can propagate throughout the dendritic tree, neocortical pyramidal neurons also sustain dendritic spikes that are spatially restricted. The function of these 'local' dendritic spikes is unknown. We show that local spikes, which require activation of N-methyl-d-aspartate receptors (NMDARs), induce long-term synaptic depression (LTD) in layer 5 pyramidal neurons. This depression does not require somatic spiking and is input specific. Moreover, a single synaptic stimulus can evoke a dendritic spike and a brief local dendritic calcium transient, and is sufficient for the full induction of LTD.

Postsynaptic Induction of BDNF-Mediated Long-Term Potentiation.

Brain-derived neurotrophic factor (BDNF) and other neurotrophins are critically involved in long-term potentiation (LTP). Previous reports point to a presynaptic site of neurotrophin action. By imaging dentate granule cells in mouse hippocampal slices, we identified BDNF-evoked Ca2+ transients in dendrites and spines, but not at presynaptic sites. Pairing a weak burst of synaptic stimulation with a brief dendritic BDNF application caused an immediate and robust induction of LTP. LTP induction required activation of postsynaptic Ca2+ channels and N-methyl-d-aspartate receptors and was prevented by the blockage of postsynaptic Ca2+ transients. Thus, our results suggest that BDNF-mediated LTP is induced postsynaptically. Our finding that dendritic spines are the exclusive synaptic sites for rapid BDNF-evoked Ca2+ signaling supports this conclusion.