Neural circuitry for maternal oxytocin release induced by infant cries.

Oxytocin is a neuropeptide that is important for maternal physiology and childcare, including parturition and milk ejection during nursing1-6. Suckling triggers the release of oxytocin, but other sensory cues-specifically, infant cries-can increase the levels of oxytocin in new human mothers7, which indicates that cries can activate hypothalamic oxytocin neurons. Here we describe a neural circuit that routes auditory information about infant vocalizations to mouse oxytocin neurons. We performed in vivo electrophysiological recordings and photometry from identified oxytocin neurons in awake maternal mice that were presented with pup calls. We found that oxytocin neurons responded to pup vocalizations, but not to pure tones, through input from the posterior intralaminar thalamus, and that repetitive thalamic stimulation induced lasting disinhibition of oxytocin neurons. This circuit gates central oxytocin release and maternal behaviour in response to calls, providing a mechanism for the integration of sensory cues from the offspring in maternal endocrine networks to ensure modulation of brain state for efficient parenting.

Real-time visualization of structural dynamics of synapses in live cells in vivo.

The structural plasticity of synapses is crucial for regulating brain functions. However, currently available methods for studying synapse organization based on split fluorescent proteins (FPs) have been limited in assessing synaptic dynamics in vivo due to the irreversible binding of split FPs. Here, we develop 'SynapShot', a method for visualizing the structural dynamics of intact synapses by combining dimerization-dependent FPs (ddFPs) with engineered synaptic adhesion molecules. SynapShot allows real-time monitoring of reversible and bidirectional changes of synaptic contacts under physiological stimulation. The application of green and red ddFPs in SynapShot enables simultaneous visualization of two distinct populations of synapses. Notably, the red-shifted SynapShot is highly compatible with blue light-based optogenetic techniques, allowing for visualization of synaptic dynamics while precisely controlling specific signaling pathways. Furthermore, we demonstrate that SynapShot enables real-time monitoring of structural changes in synaptic contacts in the mouse brain during both primitive and higher-order behaviors.

A single-component, light-assisted uncaging switch for endoproteolytic release.

Proteases function as pivotal molecular switches, initiating numerous biological events. Notably, potyviral protease, derived from plant viruses, has emerged as a trusted proteolytic switch in synthetic biological circuits. To harness their capabilities, we have developed a single-component photocleavable switch, termed LAUNCHER (Light-Assisted UNcaging switCH for Endoproteolytic Release), by employing a circularly permutated tobacco etch virus protease and a blue-light-gated substrate, which are connected by fine-tuned intermodular linkers. As a single-component system, LAUNCHER exhibits a superior signal-to-noise ratio compared with multi-component systems, enabling precise and user-controllable release of payloads. This characteristic renders LAUNCHER highly suitable for diverse cellular applications, including transgene expression, tailored subcellular translocation and optochemogenetics. Additionally, the plug-and-play integration of LAUNCHER into existing synthetic circuits facilitates the enhancement of circuit performance. The demonstrated efficacy of LAUNCHER in improving existing circuitry underscores its significant potential for expanding its utilization in various applications.

Temporally precise labeling and control of neuromodulatory circuits in the mammalian brain.

Few tools exist to visualize and manipulate neurons that are targets of neuromodulators. We present iTango, a light- and ligand-gated gene expression system based on a light-inducible split tobacco etch virus protease. Cells expressing the iTango system exhibit increased expression of a marker gene in the presence of dopamine and blue-light exposure, both in vitro and in vivo. We demonstrated the iTango system in a behaviorally relevant context, by inducing expression of optogenetic tools in neurons under dopaminergic control during a behavior of interest. We thereby gained optogenetic control of these behaviorally relevant neurons. We applied the iTango system to decipher the roles of two classes of dopaminergic neurons in the mouse nucleus accumbens in a sensitized locomotor response to cocaine. Thus, the iTango platform allows for control of neuromodulatory circuits in a genetically and functionally defined manner with spatial and temporal precision.

Emergence of functional subnetworks in layer 2/3 cortex induced by sequential spikes in vivo.

During cortical circuit development in the mammalian brain, groups of excitatory neurons that receive similar sensory information form microcircuits. However, cellular mechanisms underlying cortical microcircuit development remain poorly understood. Here we implemented combined two-photon imaging and photolysis in vivo to monitor and manipulate neuronal activities to study the processes underlying activity-dependent circuit changes. We found that repeated triggering of spike trains in a randomly chosen group of layer 2/3 pyramidal neurons in the somatosensory cortex triggered long-term plasticity of circuits (LTPc), resulting in the increased probability that the selected neurons would fire when action potentials of individual neurons in the group were evoked. Significant firing pattern changes were observed more frequently in the selected group of neurons than in neighboring control neurons, and the induction was dependent on the time interval between spikes, N-methyl-D-aspartate (NMDA) receptor activation, and Calcium/calmodulin-dependent protein kinase II (CaMKII) activation. In addition, LTPc was associated with an increase of activity from a portion of neighboring neurons with different probabilities. Thus, our results demonstrate that the formation of functional microcircuits requires broad network changes and that its directionality is nonrandom, which may be a general feature of cortical circuit assembly in the mammalian cortex.

Glutamate induces de novo growth of functional spines in developing cortex.

Mature cortical pyramidal neurons receive excitatory inputs onto small protrusions emanating from their dendrites called spines. Spines undergo activity-dependent remodelling, stabilization and pruning during development, and similar structural changes can be triggered by learning and changes in sensory experiences. However, the biochemical triggers and mechanisms of de novo spine formation in the developing brain and the functional significance of new spines to neuronal connectivity are largely unknown. Here we develop an approach to induce and monitor de novo spine formation in real time using combined two-photon laser-scanning microscopy and two-photon laser uncaging of glutamate. Our data demonstrate that, in mouse cortical layer 2/3 pyramidal neurons, glutamate is sufficient to trigger de novo spine growth from the dendrite shaft in a location-specific manner. We find that glutamate-induced spinogenesis requires opening of NMDARs (N-methyl-D-aspartate-type glutamate receptors) and activation of protein kinase A (PKA) but is independent of calcium-calmodulin-dependent kinase II (CaMKII) and tyrosine kinase receptor B (TrkB) receptors. Furthermore, newly formed spines express glutamate receptors and are rapidly functional such that they transduce presynaptic activity into postsynaptic signals. Together, our data demonstrate that early neural connectivity is shaped by activity in a spatially precise manner and that nascent dendrite spines are rapidly functionally incorporated into cortical circuits.

Development of Anionically Decorated Caged Neurotransmitters: In Vitro Comparison of 7-Nitroindolinyl- and 2-(p-Phenyl-o-nitrophenyl)propyl-Based Photochemical Probes.

Neurotransmitter uncaging, especially that of glutamate, has been used to study synaptic function for over 30 years. One limitation of caged glutamate probes is the blockade of γ-aminobutyric acid (GABA)-A receptor function. This problem comes to the fore when the probes are applied at the high concentrations required for effective two-photon photolysis. To mitigate such problems one could improve the photochemical properties of caging chromophores and/or remove receptor blockade. We show that addition of a dicarboxylate unit to the widely used 4-methoxy-7-nitroindolinyl-Glu (MNI-Glu) system reduced the off-target effects by about 50-70 %. When the same strategy was applied to an electron-rich 2-(p-Phenyl-o-nitrophenyl)propyl (PNPP) caging group, the pharmacological improvements were not as significant as in the MNI case. Finally, we used very extensive biological testing of the PNPP-caged Glu (more than 250 uncaging currents at single dendritic spines) to show that nitro-biphenyl caging chromophores have two-photon uncaging efficacies similar to that of MNI-Glu.

Dopamine-mediated formation of a memory module in the nucleus accumbens for goal-directed navigation.

Spatial memories guide navigation efficiently toward desired destinations. However, the neuronal and circuit mechanisms underlying the encoding of goal locations and its translation into goal-directed navigation remain unclear. Here we demonstrate that mice rapidly form a spatial memory of a shelter during shelter experiences, guiding escape behavior toward the goal location-a shelter-when under threat. Dopaminergic neurons in the ventral tegmental area and their projection to the nucleus accumbens (NAc) encode safety signals associated with the shelter. Optogenetically induced phasic dopamine signals are sufficient to create a place memory that directs escape navigation. Converging dopaminergic and hippocampal glutamatergic inputs to the NAc mediate the formation of a goal-related memory within a subpopulation of NAc neurons during shelter experiences. Artificial co-activation of this goal-related NAc ensemble with neurons in the dorsal periaqueductal gray was sufficient to trigger memory-guided, rather than random, escape behavior. These findings provide causal evidence of cognitive circuit modules linking memory with goal-directed action.

H-Ras induces exuberant de novo dendritic protrusion growth in mature neurons regardless of cell type.

Dendritic protrusions, mainly spines and filopodia, correlate with excitatory synapses in pyramidal neurons (PyNs), but this relationship may not apply universally. We found that ectopic H-Ras expression increased protrusions across various cortical cell types, including layer 2/3 PyNs, parvalbumin (PV)-, and vasoactive intestinal peptide (VIP)-positive interneurons (INs) in the primary motor cortex. The probability of detecting protrusions correlated with local H-Ras activity, indicating its role in protrusion formation. H-Ras overexpression led to high turnover rates by adding protrusions. Two-photon photolysis of glutamate induced de novo spine formation in mature H-Ras expressing neurons, suggesting H-Ras's effect is not limited to early development. In PyNs and PV-INs, but not VIP-INs, spine neck lengths shifted to filopodia-like phenotypes. H-Ras primarily induced filopodia in PyNs and spines in PV- and VIP-INs. Increased protrusions in H-Ras-transfected PyNs lacked key excitatory synaptic proteins and did not affect miniature excitatory postsynaptic currents (mEPSCs), suggesting multifaceted roles beyond excitatory synapses.

Light-gated integrator for highlighting kinase activity in living cells.

Protein kinases are key signaling nodes that regulate fundamental biological and disease processes. Illuminating kinase signaling from multiple angles can provide deeper insights into disease mechanisms and improve therapeutic targeting. While fluorescent biosensors are powerful tools for visualizing live-cell kinase activity dynamics in real time, new molecular tools are needed that enable recording of transient signaling activities for post hoc analysis and targeted manipulation. Here, we develop a light-gated kinase activity coupled transcriptional integrator (KINACT) that converts dynamic kinase signals into "permanent" fluorescent marks. KINACT enables robust monitoring of kinase activity across scales, accurately recording subcellular PKA activity, highlighting PKA activity distribution in 3D cultures, and identifying PKA activators and inhibitors in high-throughput screens. We further leverage the ability of KINACT to drive signaling effector expression to allow feedback manipulation of the balance of GαsR201C-induced PKA and ERK activation and dissect the mechanisms of oncogenic G protein signaling.

Light-gated Integrator for Highlighting Kinase Activity in Living Cells.

Protein kinases are key signaling nodes that regulate fundamental biological and disease processes. Illuminating kinase signaling from multiple angles can provide deeper insights into disease mechanisms and improve therapeutic targeting. While fluorescent biosensors are powerful tools for visualizing live-cell kinase activity dynamics in real time, new molecular tools are needed that enable recording of transient signaling activities for post hoc analysis and targeted manipulation. Here, we develop a light-gated kinase activity coupled transcriptional integrator (KINACT) that converts dynamic kinase signals into "permanent" fluorescent marks. KINACT enables robust monitoring of kinase activity across scales, accurately recording subcellular PKA activity, highlighting PKA signaling heterogeneity in 3D cultures, and identifying PKA activators and inhibitors in high-throughput screens. We further leverage the ability of KINACT to drive signaling effector expression to allow feedback manipulation of the balance of GαsR201C-induced PKA and ERK activation and dissect the mechanisms of oncogenic G protein signaling.

Optically selective two-photon uncaging of glutamate at 900 nm.

We have synthesized a 7-diethylaminocoumarin (DEAC) derivative that allows wavelength-selective two-photon uncaging at 900 nm versus 720 nm. This new caging chromophore, called DEAC450, has an extended π-electron moiety at the 3-position that shifts the absorption spectrum maximum of DEAC from 375 to 450 nm. Two-photon excitation at 900 nm was more than 60-fold greater than at 720 nm. Two-photon uncaging of DEAC450-Glu at 900 nm at spine heads on pyramidal neurons in acutely isolated brain slices generated postsynaptic responses that were similar to spontaneous postsynaptic excitatory miniature currents, whereas significantly higher energies at 720 nm evoked no currents. Since many nitroaromatic caged compounds are two-photon active at 720 nm, optically selective uncaging of DEAC450-caged biomolecules at 900 nm may allow facile two-color optical interrogation of bimodal signaling pathways in living tissue with high resolution for the first time.

Piccolo and bassoon maintain synaptic vesicle clustering without directly participating in vesicle exocytosis.

Piccolo and bassoon are highly homologous multidomain proteins of the presynaptic cytomatrix whose function is unclear. Here, we generated piccolo knockin/knockout mice that either contain wild-type levels of mutant piccolo unable to bind Ca(2+) (knockin), approximately 60% decreased levels of piccolo that is C-terminally truncated (partial knockout), or <5% levels of piccolo (knockout). All piccolo mutant mice were viable and fertile, but piccolo knockout mice exhibited increased postnatal mortality. Unexpectedly, electrophysiology and electron microscopy of piccolo-deficient synapses failed to uncover a major phenotype either in acute hippocampal slices or in cultured cortical neurons. To unmask potentially redundant functions of piccolo and bassoon, we thus acutely knocked down expression of bassoon in wild-type and piccolo knockout neurons. Despite a nearly complete loss of piccolo and bassoon, however, we still did not detect an electrophysiological phenotype in cultured piccolo- and bassoon-deficient neurons in either GABAergic or glutamatergic synaptic transmission. In contrast, electron microscopy revealed a significant reduction in synaptic vesicle clustering in double bassoon/piccolo-deficient synapses. Thus, we propose that piccolo and bassoon play a redundant role in synaptic vesicle clustering in nerve terminals without directly participating in neurotransmitter release.

Serotonin in the orbitofrontal cortex enhances cognitive flexibility.

Cognitive flexibility is a brain's ability to switch between different rules or action plans depending on the context. However, cellular level understanding of cognitive flexibility have been largely unexplored. We probed a specific serotonergic pathway from dorsal raphe nuclei (DRN) to the orbitofrontal cortex (OFC) while animals are performing reversal learning task. We found that serotonin release from DRN to the OFC promotes reversal learning. A long-range connection between these two brain regions was confirmed anatomically and functionally. We further show that spatiotemporally precise serotonergic action directly enhances the excitability of OFC neurons and offers enhanced spike probability of OFC network. Serotonergic action facilitated the induction of synaptic plasticity by enhancing Ca2+ influx at dendritic spines in the OFC. Thus, our findings suggest that a key signature of flexibility is the formation of choice specific ensembles via serotonin-dependent synaptic plasticity.

An adaptive behavioral control motif mediated by cortical axo-axonic inhibition.

Genetically defined subgroups of inhibitory interneurons are thought to play distinct roles in learning, but heterogeneity within these subgroups has limited our understanding of the scope and nature of their specific contributions. Here we reveal that the chandelier cell (ChC), an interneuron type that specializes in inhibiting the axon-initial segment (AIS) of pyramidal neurons, establishes cortical microcircuits for organizing neural coding through selective axo-axonic synaptic plasticity. We found that organized motor control is mediated by enhanced population coding of direction-tuned premotor neurons, with tuning refined through suppression of irrelevant neuronal activity. ChCs contribute to learning-dependent refinements by providing selective inhibitory control over individual pyramidal neurons rather than global suppression. Quantitative analysis of structural plasticity across axo-axonic synapses revealed that ChCs redistributed inhibitory weights to individual pyramidal neurons during learning. These results demonstrate an adaptive logic of the inhibitory circuit motif responsible for organizing distributed neural representations. Thus, ChCs permit efficient cortical computation in a targeted cell-specific manner.

Sleep and circadian rhythm disruption by NPTX2 loss of function.

Sleep and circadian rhythm disruption (SCRD) is commonly observed in aging, especially in individuals who experience progressive cognitive decline to mild cognitive impairment (MCI) and Alzheimer's disease (AD). However, precise molecular mechanisms underlying the association between SCRD and aging are not fully understood. Orexin A is a well-characterized "sleep neuropeptide" that is expressed in hypothalamic neurons and evokes wake behavior. The importance of Orexin is exemplified in narcolepsy where it is profoundly down-regulated. Interestingly, the synaptic immediate early gene NPTX2 is co-expressed in Orexin neurons and is similarly reduced in narcolepsy. NPTX2 is also down-regulated in CSF of some cognitively normal older individuals and predicts the time of transition from normal cognition to MCI. The association between Orexin and NPTX2 is further evinced here where we observe that Orexin A and NPTX2 are highly correlated in CSF of cognitively normal aged individuals and raises the question of whether SCRD that are typically attributed to Orexin A loss of function may be modified by concomitant NPTX2 down-regulation. Is NPTX2 an effector of sleep or simply a reporter of orexin-dependent SCRD? To address this question, we examined NPTX2 KO mice and found they retain Orexin expression in the brain and so provide an opportunity to examine the specific contribution of NPTX2 to SCRD. Our results reveal that NPTX2 KO mice exhibit a disrupted circadian onset time, coupled with increased activity during the sleep phase, suggesting difficulties in maintaining states. Sleep EEG indicates distinct temporal allocation shifts across vigilance states, characterized by reduced wake and increased NREM time. Evident sleep fragmentation manifests through alterations of event occurrences during Wake and NREM, notably during light transition periods, in conjunction with an increased frequency of sleep transitions in NPTX2 KO mice, particularly between Wake and NREM. EEG spectral analysis indicated significant shifts in power across various frequency bands in the wake, NREM, and REM states, suggestive of disrupted neuronal synchronicity. An intriguing observation is the diminished occurrence of sleep spindles, one of the earliest measures of human sleep disruption, in NPTX2 KO mice. These findings highlight the effector role of NPTX2 loss of function as an instigator of SCRD and a potential mediator of sleep disruption in aging.

An adaptive behavioral control motif mediated by cortical axo-axonic inhibition.

Neural circuits are reorganized with specificity during learning. Genetically-defined subgroups of inhibitory interneurons are thought to play distinct roles in learning, but heterogeneity within these subgroups has limited our understanding of the scope and nature of their specific contributions to learning. Here we reveal that the chandelier cell (ChC), an interneuron type that specializes in inhibiting the axon-initial segment (AIS) of pyramidal neurons, establishes cortical microcircuits for organizing neural coding through selective axo-axonic synaptic plasticity. We find that organized motor control is mediated by enhanced population coding of direction-tuned premotor neurons, whose tuning is refined through suppression of irrelevant neuronal activity. ChCs are required for learning-dependent refinements via providing selective inhibitory control over pyramidal neurons rather than global suppression. Quantitative analysis on structural plasticity of axo-axonic synapses revealed that ChCs redistributed inhibitory weights to individual pyramidal neurons during learning. These results demonstrate an adaptive logic of the inhibitory circuit motif responsible for organizing distributed neural representations. Thus, ChCs permit efficient cortical computation in a target cell specific manner, which highlights the significance of interneuron diversity.

Exuberant de novo dendritic spine growth in mature neurons.

Dendritic spines are structural correlates of excitatory synapses maintaining stable synaptic communications. However, this strong spine-synapse relationship was mainly characterized in excitatory pyramidal neurons (PyNs), raising a possibility that inferring synaptic density from dendritic spine number may not be universally applied to all neuronal types. Here we found that the ectopic expression of H-Ras increased dendritic spine numbers regardless of cortical cell types such as layer 2/3 pyramidal neurons (PyNs), parvalbumin (PV)- and vasoactive intestinal peptide (VIP)-positive interneurons (INs) in the primary motor cortex (M1). The probability of detecting dendritic spines was positively correlated with the magnitude of H-Ras activity, suggesting elevated local H-Ras activity is involved in the process of dendritic spine formation. H-Ras overexpression caused high spine turnover rate via adding more spines rather than eliminating them. Two-photon photolysis of glutamate triggered de novo dendritic spine formation in mature neurons, suggesting H-Ras induced spine formation is not restricted to the early development. In PyNs and PV-INs, but not VIP-INs, we observed a shift in average spine neck length towards longer filopodia-like phenotypes. The portion of dendritic spines lacking key excitatory synaptic proteins were significantly increased in H-Ras transfected neurons, suggesting that these increased spines have other distinct functions. High spine density caused by H-Ras did not result in change in the frequency or the amplitude of miniature excitatory postsynaptic currents (mEPSCs). Thus, our results propose that dendritic spines possess more multifaceted functions beyond the morphological proxy of excitatory synapse.

Long-term potentiation selectively expressed by NMDA receptors at hippocampal mossy fiber synapses.

The mossy fiber to CA3 pyramidal cell synapse (mf-CA3) provides a major source of excitation to the hippocampus. Thus far, these glutamatergic synapses are well recognized for showing a presynaptic, NMDA receptor-independent form of LTP that is expressed as a long-lasting increase of transmitter release. Here, we show that in addition to this "classical" LTP, mf-CA3 synapses can undergo a form of LTP characterized by a selective enhancement of NMDA receptor-mediated transmission. This potentiation requires coactivation of NMDA and mGlu5 receptors and a postsynaptic calcium rise. Unlike classical LTP, expression of this mossy fiber LTP is due to a PKC-dependent recruitment of NMDA receptors specifically to the mf-CA3 synapse via a SNARE-dependent process. Having two mechanistically different forms of LTP may allow mf-CA3 synapses to respond with more flexibility to the changing demands of the hippocampal network.

Current and future techniques for detecting oxytocin: Focusing on genetically-encoded GPCR sensors.

Oxytocin is a neuropituitary hormone that is involved in a wide range of psychosocial behaviors. Despite its psychophysiological importance as a neuromodulator in the CNS, effective techniques capable of monitoring oxytocin dynamics or testing related behavioral consequences are limited. Along with an explosive advancement in synthetic biology, high-performance genetically-encoded neuromodulator sensors are being developed. Here we comprehensively review the current methodologies available for detecting oxytocin in neuroscience. Their strengths and weaknesses are discussed, and a graphical summary is plotted for better comparison of techniques. We also suggest future directions for next generation oxytocin sensor development and their working principles.