DART.2: bidirectional synaptic pharmacology with thousandfold cellular specificity.

Precision pharmacology aims to manipulate specific cellular interactions within complex tissues. In this pursuit, we introduce DART.2 (drug acutely restricted by tethering), a second-generation cell-specific pharmacology technology. The core advance is optimized cellular specificity-up to 3,000-fold in 15 min-enabling the targeted delivery of even epileptogenic drugs without off-target effects. Additionally, we introduce brain-wide dosing methods as an alternative to local cannulation and tracer reagents for brain-wide dose quantification. We describe four pharmaceuticals-two that antagonize excitatory and inhibitory postsynaptic receptors, and two that allosterically potentiate these receptors. Their versatility is showcased across multiple mouse-brain regions, including cerebellum, striatum, visual cortex and retina. Finally, in the ventral tegmental area, we find that blocking inhibitory inputs to dopamine neurons accelerates locomotion, contrasting with previous optogenetic and pharmacological findings. Beyond enabling the bidirectional perturbation of chemical synapses, these reagents offer intersectional precision-between genetically defined postsynaptic cells and neurotransmitter-defined presynaptic partners.

Mechanism of local and global Ca2+ sensing by calmodulin in complex with a Ca2+ channel.

Calmodulin (CaM) in complex with Ca(2+) channels constitutes a prototype for Ca(2+) sensors that are intimately colocalized with Ca(2+) sources. The C-lobe of CaM senses local, large Ca(2+) oscillations due to Ca(2+) influx from the host channel, and the N-lobe senses global, albeit diminutive Ca(2+) changes arising from distant sources. Though biologically essential, the mechanism underlying global Ca(2+) sensing has remained unknown. Here, we advance a theory of how global selectivity arises, and we experimentally validate this proposal with methodologies enabling millisecond control of Ca(2+) oscillations seen by the CaM/channel complex. We find that global selectivity arises from rapid Ca(2+) release from CaM combined with greater affinity of the channel for Ca(2+)-free versus Ca(2+)-bound CaM. The emergence of complex decoding properties from the juxtaposition of common elements, and the techniques developed herein, promise generalization to numerous molecules residing near Ca(2+) sources.

Noradrenergic Signaling Disengages Feedforward Transmission in the Nucleus Accumbens Shell.

The nucleus accumbens shell (NAcSh) receives extensive monoaminergic input from multiple midbrain structures. However, little is known how norepinephrine (NE) modulates NAc circuit dynamics. Using a dynamic electrophysiological approach with optogenetics, pharmacology, and drugs acutely restricted by tethering (DART), we explored microcircuit-specific neuromodulatory mechanisms recruited by NE signaling in the NAcSh of parvalbumin (PV)-specific reporter mice. Surprisingly, NE had little direct effect on modulation of synaptic input at medium spiny projection neurons (MSNs). In contrast, we report that NE transmission selectively modulates glutamatergic synapses onto PV-expressing fast-spiking interneurons (PV-INs) by recruiting postsynaptically-localized α2-adrenergic receptors (ARs). The synaptic effects of α2-AR activity decrease PV-IN-dependent feedforward inhibition onto MSNs evoked via optogenetic stimulation of cortical afferents to the NAcSh. These findings provide insight into a new circuit motif in which NE has a privileged line of communication to tune feedforward inhibition in the NAcSh.SIGNIFICANCE STATEMENT The nucleus accumbens (NAc) directs reward-related motivational output by integrating glutamatergic input with diverse neuromodulatory input from monoamine centers. The present study reveals a synapse-specific regulatory mechanism recruited by norepinephrine (NE) signaling within parvalbumin-expressing interneuron (PV-IN) feedforward inhibitory microcircuits. PV-IN-mediated feedforward inhibition in the NAc is instrumental in coordinating NAc output by synchronizing the activity of medium spiny projection neurons (MSNs). By negatively regulating glutamatergic transmission onto PV-INs via α2-adrenergic receptors (ARs), NE diminishes feedforward inhibition onto MSNs to promote NAc output. These findings elucidate previously unknown microcircuit mechanisms recruited by the historically overlooked NE system in the NAc.

Movement and structure of mitochondria in oligodendrocytes and their myelin sheaths.

Mitochondria play several crucial roles in the life of oligodendrocytes. During development of the myelin sheath they are essential providers of carbon skeletons and energy for lipid synthesis. During normal brain function their consumption of pyruvate will be a key determinant of how much lactate is available for oligodendrocytes to export to power axonal function. Finally, during calcium-overload induced pathology, as occurs in ischemia, mitochondria may buffer calcium or induce apoptosis. Despite their important functions, very little is known of the properties of oligodendrocyte mitochondria, and mitochondria have never been observed in the myelin sheaths. We have now used targeted expression of fluorescent mitochondrial markers to characterize the location and movement of mitochondria within oligodendrocytes. We show for the first time that mitochondria are able to enter and move within the myelin sheath. Within the myelin sheath the highest number of mitochondria was in the cytoplasmic ridges along the sheath. Mitochondria moved more slowly than in neurons and, in contrast to their behavior in neurons and astrocytes, their movement was increased rather than inhibited by glutamate activating NMDA receptors. By electron microscopy we show that myelin sheath mitochondria have a low surface area of cristae, which suggests a low ATP production. These data specify fundamental properties of the oxidative phosphorylation system in oligodendrocytes, the glial cells that enhance cognition by speeding action potential propagation and provide metabolic support to axons.

Ca2+ channel nanodomains boost local Ca2+ amplitude.

Local Ca(2+) signals through voltage-gated Ca(2+) channels (CaVs) drive synaptic transmission, neural plasticity, and cardiac contraction. Despite the importance of these events, the fundamental relationship between flux through a single CaV channel and the Ca(2+) signaling concentration within nanometers of its pore has resisted empirical determination, owing to limitations in the spatial resolution and specificity of fluorescence-based Ca(2+) measurements. Here, we exploited Ca(2+)-dependent inactivation of CaV channels as a nanometer-range Ca(2+) indicator specific to active channels. We observed an unexpected and dramatic boost in nanodomain Ca(2+) amplitude, ten-fold higher than predicted on theoretical grounds. Our results uncover a striking feature of CaV nanodomains, as diffusion-restricted environments that amplify small Ca(2+) fluxes into enormous local Ca(2+) concentrations. This Ca(2+) tuning by the physical composition of the nanodomain may represent an energy-efficient means of local amplification that maximizes information signaling capacity, while minimizing global Ca(2+) load.

A modular switch for spatial Ca2+ selectivity in the calmodulin regulation of CaV channels.

Ca2+/calmodulin-dependent regulation of voltage-gated CaV1-2 Ca2+ channels shows extraordinary modes of spatial Ca2+ decoding and channel modulation, vital for many biological functions. A single calmodulin (CaM) molecule associates constitutively with the channel's carboxy-terminal tail, and Ca2+ binding to the C-terminal and N-terminal lobes of CaM can each induce distinct channel regulations. As expected from close channel proximity, the C-lobe responds to the roughly 100-microM Ca2+ pulses driven by the associated channel, a behaviour defined as 'local Ca2+ selectivity'. Conversely, all previous observations have indicated that the N-lobe somehow senses the far weaker signals from distant Ca2+ sources. This 'global Ca2+ selectivity' satisfies a general signalling requirement, enabling a resident molecule to remotely sense cellular Ca2+ activity, which would otherwise be overshadowed by Ca2+ entry through the host channel. Here we show that the spatial Ca2+ selectivity of N-lobe CaM regulation is not invariably global but can be switched by a novel Ca2+/CaM-binding site within the amino terminus of channels (NSCaTE, for N-terminal spatial Ca2+ transforming element). Native CaV2.2 channels lack this element and show N-lobe regulation with a global selectivity. On the introduction of NSCaTE into these channels, spatial Ca2+ selectivity transforms from a global to local profile. Given this effect, we examined CaV1.2/CaV1.3 channels, which naturally contain NSCaTE, and found that their N-lobe selectivity is indeed local. Disruption of this element produces a global selectivity, confirming the native function of NSCaTE. Thus, differences in spatial selectivity between advanced CaV1 and CaV2 channel isoforms are explained by the presence or absence of NSCaTE. Beyond functional effects, the position of NSCaTE on the channel's amino terminus indicates that CaM can bridge the amino terminus and carboxy terminus of channels. Finally, the modularity of NSCaTE offers practical means for understanding the basis of global Ca2+ selectivity.

Mouse model of Timothy syndrome recapitulates triad of autistic traits.

Autism and autism spectrum disorder (ASD) typically arise from a mixture of environmental influences and multiple genetic alterations. In some rare cases, such as Timothy syndrome (TS), a specific mutation in a single gene can be sufficient to generate autism or ASD in most patients, potentially offering insights into the etiology of autism in general. Both variants of TS (the milder TS1 and the more severe TS2) arise from missense mutations in alternatively spliced exons that cause the same G406R replacement in the Ca(V)1.2 L-type calcium channel. We generated a TS2-like mouse but found that heterozygous (and homozygous) animals were not viable. However, heterozygous TS2 mice that were allowed to keep an inverted neomycin cassette (TS2-neo) survived through adulthood. We attribute the survival to lowering of expression of the G406R L-type channel via transcriptional interference, blunting deleterious effects of mutant L-type channel overactivity, and addressed potential effects of altered gene dosage by studying Ca(V)1.2 knockout heterozygotes. Here we present a thorough behavioral phenotyping of the TS2-neo mouse, capitalizing on this unique opportunity to use the TS mutation to model ASD in mice. Along with normal general health, activity, and anxiety level, TS2-neo mice showed markedly restricted, repetitive, and perseverative behavior, altered social behavior, altered ultrasonic vocalization, and enhanced tone-cued and contextual memory following fear conditioning. Our results suggest that when TS mutant channels are expressed at levels low enough to avoid fatality, they are sufficient to cause multiple, distinct behavioral abnormalities, in line with the core aspects of ASD.

Local synaptic inhibition mediates cerebellar granule cell pattern separation and enables learned sensorimotor associations.

The cerebellar cortex has a key role in generating predictive sensorimotor associations. To do so, the granule cell layer is thought to establish unique sensorimotor representations for learning. However, how this is achieved and how granule cell population responses contribute to behavior have remained unclear. To address these questions, we have used in vivo calcium imaging and granule cell-specific pharmacological manipulation of synaptic inhibition in awake, behaving mice. These experiments indicate that inhibition sparsens and thresholds sensory responses, limiting overlap between sensory ensembles and preventing spiking in many granule cells that receive excitatory input. Moreover, inhibition can be recruited in a stimulus-specific manner to powerfully decorrelate multisensory ensembles. Consistent with these results, granule cell inhibition is required for accurate cerebellum-dependent sensorimotor behavior. These data thus reveal key mechanisms for granule cell layer pattern separation beyond those envisioned by classical models.

Natural phasic inhibition of dopamine neurons signals cognitive rigidity.

When animals unexpectedly fail, their dopamine neurons undergo phasic inhibition that canonically drives extinction learning, a cognitive-flexibility mechanism for discarding outdated strategies. However, the existing evidence equates natural and artificial phasic inhibition, despite their spatiotemporal differences. Addressing this gap, we targeted a GABAA-receptor antagonist precisely to dopamine neurons, yielding three unexpected findings. First, this intervention blocked natural phasic inhibition selectively, leaving tonic activity unaffected. Second, blocking natural phasic inhibition accelerated extinction learning, opposite to canonical mechanisms. Third, our approach selectively benefitted perseverative mice, restoring rapid extinction without affecting new reward learning. Our findings reveal that extinction learning is rapid by default and slowed by natural phasic inhibition, challenging foundational learning theories, while delineating a synaptic mechanism and therapeutic target for cognitive rigidity.

An open-source head-fixation and implant-protection system for mice.

Mice are widely used in neuroscience experiments, which often require head-fixation and attachment of skull-mounted hardware. For many experiments, these components must remain intact over weeks to months, ideally with animals group housed. Many labs have designed ad-hoc head-fixation systems, which is an inefficient process. For example, when reinventing these solutions in our lab, we faced challenges with group housing, wherein mice would chew and damage implanted cannulas and electrodes of their cage mates. We performed several non-trivial design iterations to solve this problem, and present the most successful designs as an open-source collection. The designs include a standard mounting headbar compatible with most skull-mounted hardware, a snap-on protective mouse hat (headhat) to prevent mice from chewing the hardware, and a head-fixation station to facilitate common experimental procedures. We provide 3D-printing files, detail vendors and software used to make the components of the system, and provide editable design files for maximum flexibility to individual lab requirements.

Ketamine rescues anhedonia by cell-type and input specific adaptations in the Nucleus Accumbens.

Ketamine's role in providing a rapid and sustained antidepressant response, particularly for patients unresponsive to conventional treatments, is increasingly recognized. A core symptom of depression, anhedonia, or the loss of enjoyment or interest in previously pleasurable activities, is known to be significantly alleviated by ketamine. While several hypotheses have been proposed regarding the mechanisms by which ketamine alleviates anhedonia, the specific circuits and synaptic changes responsible for its sustained therapeutic effects are not yet understood. Here, we show that the nucleus accumbens (NAc), a major hub of the reward circuitry, is essential for ketamine's effect in rescuing anhedonia in mice subjected to chronic stress, a critical risk factor in the genesis of depression in humans. Specifically, a single exposure to ketamine rescues stress-induced decreased strength of excitatory synapses on NAc D1 dopamine receptor-expressing medium spiny neurons (D1-MSNs). By using a novel cell-specific pharmacology method, we demonstrate that this cell-type specific neuroadaptation is necessary for the sustained therapeutic effects of ketamine. To test for causal sufficiency, we artificially mimicked ketamine-induced increase in excitatory strength on D1-MSNs and found that this recapitulates the behavioral amelioration induced by ketamine. Finally, to determine the presynaptic origin of the relevant glutamatergic inputs for ketamine-elicited synaptic and behavioral effects, we used a combination of opto- and chemogenetics. We found that ketamine rescues stress-induced reduction in excitatory strength at medial prefrontal cortex and ventral hippocampus inputs to NAc D1-MSNs. Chemogenetically preventing ketamine-evoked plasticity at those unique inputs to the NAc reveals a ketamine-operated input-specific control of hedonic behavior. These results establish that ketamine rescues stress-induced anhedonia via cell-type-specific adaptations as well as information integration in the NAc via discrete excitatory synapses.

An open-source transparent microelectrode array.

Objective.The micro-electrode array (MEA) is a cell-culture surface with integrated electrodes used for assays of electrically excitable cells and tissues. MEAs have been a workhorse in the study of neurons and myocytes, owing to the scalability and millisecond temporal resolution of the technology. However, traditional MEAs are opaque, precluding inverted microscope access to modern genetically encoded optical sensors and effectors.Approach. To address this gap, transparent MEAs have been developed. However, for many labs, transparent MEAs remain out of reach due to the cost of commercially available products, and the complexity of custom fabrication. Here, we describe an open-source transparent MEA based on the OpenEphys platform (Siegleet al2017J. Neural Eng.14045003).Main results.We demonstrate the performance of this transparent MEA in a multiplexed electrical and optogenetic assay of primary rat hippocampal neurons.Significance.This open-source transparent MEA and recording platform is designed to be accessible, requiring minimal microelectrode fabrication or circuit design experience. We include low-noise connectors for seamless integration with the Intan Technologies headstage, and a mechanically stable adaptor conforming to the 24-well plate footprint for compatibility with most inverted microscopes.

Acute restraint stress redirects prefrontal cortex circuit function through mGlu5 receptor plasticity on somatostatin-expressing interneurons.

Inhibitory interneurons orchestrate prefrontal cortex (PFC) activity, but we have a limited understanding of the molecular and experience-dependent mechanisms that regulate synaptic plasticity across PFC microcircuits. We discovered that mGlu5 receptor activation facilitates long-term potentiation at synapses from the basolateral amygdala (BLA) onto somatostatin-expressing interneurons (SST-INs) in mice. This plasticity appeared to be recruited during acute restraint stress, which induced intracellular calcium mobilization within SST-INs and rapidly potentiated postsynaptic strength onto SST-INs. Restraint stress and mGlu5 receptor activation each augmented BLA recruitment of SST-IN phasic feedforward inhibition, shunting information from other excitatory inputs, including the mediodorsal thalamus. Finally, studies using cell-type-specific mGlu5 receptor knockout mice revealed that mGlu5 receptor function in SST-expressing cells is necessary for restraint stress-induced changes to PFC physiology and related behaviors. These findings provide new insights into interneuron-specific synaptic plasticity mechanisms and suggest that SST-IN microcircuits may be promising targets for treating stress-induced psychiatric diseases.

Cell-Specific Chemical Delivery Using a Selective Nitroreductase-Nitroaryl Pair.

The utility of small molecules to probe or perturb biological systems is limited by the lack of cell-specificity. "Masking" the activity of small molecules using a general chemical modification and "unmasking" it only within target cells overcomes this limitation. To this end, we have developed a selective enzyme-substrate pair consisting of engineered variants of E. coli nitroreductase (NTR) and a 2-nitro- N-methylimidazolyl (NM) masking group. To discover and optimize this NTR-NM system, we synthesized a series of fluorogenic substrates containing different nitroaromatic masking groups, confirmed their stability in cells, and identified the best substrate for NTR. We then engineered the enzyme for improved activity in mammalian cells, ultimately yielding an enzyme variant (enhanced NTR, or eNTR) that possesses up to 100-fold increased activity over wild-type NTR. These improved NTR enzymes combined with the optimal NM masking group enable rapid, selective unmasking of dyes, indicators, and drugs to genetically defined populations of cells.

Deconstructing behavioral neuropharmacology with cellular specificity.

Behavior has molecular, cellular, and circuit determinants. However, because many proteins are broadly expressed, their acute manipulation within defined cells has been difficult. Here, we combined the speed and molecular specificity of pharmacology with the cell type specificity of genetic tools. DART (drugs acutely restricted by tethering) is a technique that rapidly localizes drugs to the surface of defined cells, without prior modification of the native target. We first developed an AMPAR antagonist DART, with validation in cultured neuronal assays, in slices of mouse dorsal striatum, and in behaving mice. In parkinsonian animals, motor deficits were causally attributed to AMPARs in indirect spiny projection neurons (iSPNs) and to excess phasic firing of tonically active interneurons (TANs). Together, iSPNs and TANs (i.e., D2 cells) drove akinesia, whereas movement execution deficits reflected the ratio of AMPARs in D2 versus D1 cells. Finally, we designed a muscarinic antagonist DART in one iteration, demonstrating applicability of the method to diverse targets.

Sequential ionic and conformational signaling by calcium channels drives neuronal gene expression.

Voltage-gated CaV1.2 channels (L-type calcium channel α1C subunits) are critical mediators of transcription-dependent neural plasticity. Whether these channels signal via the influx of calcium ion (Ca(2+)), voltage-dependent conformational change (VΔC), or a combination of the two has thus far been equivocal. We fused CaV1.2 to a ligand-gated Ca(2+)-permeable channel, enabling independent control of localized Ca(2+) and VΔC signals. This revealed an unexpected dual requirement: Ca(2+) must first mobilize actin-bound Ca(2+)/calmodulin-dependent protein kinase II, freeing it for subsequent VΔC-mediated accumulation. Neither signal alone sufficed to activate transcription. Signal order was crucial: Efficiency peaked when Ca(2+) preceded VΔC by 10 to 20 seconds. CaV1.2 VΔC synergistically augmented signaling by N-methyl-d-aspartate receptors. Furthermore, VΔC mistuning correlated with autistic symptoms in Timothy syndrome. Thus, nonionic VΔC signaling is vital to the function of CaV1.2 in synaptic and neuropsychiatric processes.

Neural circuits. Labeling of active neural circuits in vivo with designed calcium integrators.

The identification of active neurons and circuits in vivo is a fundamental challenge in understanding the neural basis of behavior. Genetically encoded calcium (Ca(2+)) indicators (GECIs) enable quantitative monitoring of cellular-resolution activity during behavior. However, such indicators require online monitoring within a limited field of view. Alternatively, post hoc staining of immediate early genes (IEGs) indicates highly active cells within the entire brain, albeit with poor temporal resolution. We designed a fluorescent sensor, CaMPARI, that combines the genetic targetability and quantitative link to neural activity of GECIs with the permanent, large-scale labeling of IEGs, allowing a temporally precise "activity snapshot" of a large tissue volume. CaMPARI undergoes efficient and irreversible green-to-red conversion only when elevated intracellular Ca(2+) and experimenter-controlled illumination coincide. We demonstrate the utility of CaMPARI in freely moving larvae of zebrafish and flies, and in head-fixed mice and adult flies.

Systematic mapping of the state dependence of voltage- and Ca2+-dependent inactivation using simple open-channel measurements.

The state from which channel inactivation occurs is both biologically and mechanistically critical. For example, preferential closed-state inactivation is potentiated in certain Ca(2+) channel splice variants, yielding an enhancement of inactivation during action potential trains, which has important consequences for short-term synaptic plasticity. Mechanistically, the structural substrates of inactivation are now being resolved, yielding a growing library of molecular snapshots, ripe for functional interpretation. For these reasons, there is an increasing need for experimentally direct and systematic means of determining the states from which inactivation proceeds. Although many approaches have been devised, most rely upon numerical models that require detailed knowledge of channel-state topology and gating parameters. Moreover, prior strategies have only addressed voltage-dependent forms of inactivation (VDI), and have not been readily applicable to Ca(2+)-dependent inactivation (CDI), a vital form of regulation in numerous contexts. Here, we devise a simple yet systematic approach, applicable to both VDI and CDI, for semiquantitative mapping of the states from which inactivation occurs, based only on open-channel measurements. The method is relatively insensitive to the specifics of channel gating and does not require detailed knowledge of state topology or gating parameters. Rather than numerical models, we derive analytic equations that permit determination of the states from which inactivation occurs, based on direct manipulation of data. We apply this methodology to both VDI and CDI of Ca(V)1.3 Ca(2+) channels. VDI is found to proceed almost exclusively from the open state. CDI proceeds equally from the open and nearby closed states, but is disfavored from deep closed states distant from the open conformation. In all, these outcomes substantiate and enrich conclusions of our companion paper in this issue (Tadross et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910308) that deduces endpoint mechanisms of VDI and CDI in Ca(V)1.3. More broadly, the methods introduced herein can be readily generalized for the analysis of other channel types.

Molecular endpoints of Ca2+/calmodulin- and voltage-dependent inactivation of Ca(v)1.3 channels.

Ca(2+)/calmodulin- and voltage-dependent inactivation (CDI and VDI) comprise vital prototypes of Ca(2+) channel modulation, rich with biological consequences. Although the events initiating CDI and VDI are known, their downstream mechanisms have eluded consensus. Competing proposals include hinged-lid occlusion of channels, selectivity filter collapse, and allosteric inhibition of the activation gate. Here, novel theory predicts that perturbations of channel activation should alter inactivation in distinctive ways, depending on which hypothesis holds true. Thus, we systematically mutate the activation gate, formed by all S6 segments within Ca(V)1.3. These channels feature robust baseline CDI, and the resulting mutant library exhibits significant diversity of activation, CDI, and VDI. For CDI, a clear and previously unreported pattern emerges: activation-enhancing mutations proportionately weaken inactivation. This outcome substantiates an allosteric CDI mechanism. For VDI, the data implicate a "hinged lid-shield" mechanism, similar to a hinged-lid process, with a previously unrecognized feature. Namely, we detect a "shield" in Ca(V)1.3 channels that is specialized to repel lid closure. These findings reveal long-sought downstream mechanisms of inactivation and may furnish a framework for the understanding of Ca(2+) channelopathies involving S6 mutations.