A one-step protocol to generate impermeable fluorescent HaloTag substrates for in situ live cell application and super-resolution imaging.

Communication between cells is largely orchestrated by proteins on the cell surface, which allow information transfer across the cell membrane. Super-resolution and single-molecule visualization of these proteins can be achieved by genetically grafting HTP (HaloTag Protein) into the protein of interest followed by brief incubation of cells with a dye-HTL (dye-linked HaloTag Ligand). This approach allows for use of cutting-edge fluorophores optimized for specific optical techniques or a cell-impermeable dye-HTL to selectively label surface proteins without labeling intracellular copies. However, these two goals often conflict, as many high-performing dyes exhibit membrane permeability. Traditional methods to eliminate cell permeability face synthetic bottlenecks and risk altering photophysical properties. Here we report that dye-HTL reagents can be made cell-impermeable by inserting a charged sulfonate directly into the HTL, leaving the dye moiety unperturbed. This simple, one-step method requires no purification and is compatible with both the original HTL and second-generation HTL.2, the latter offering accelerated labeling. We validate such compounds, termed dye-SHTL ('dye shuttle') conjugates, in live cells via widefield microscopy, demonstrating exclusive membrane staining of extracellular HTP fusion proteins. In transduced primary hippocampal neurons, we label mGluR2, a neuromodulatory G protein-coupled receptor (GPCR), with dyes optimized for stimulated emission by depletion (STED) super-resolution microscopy, allowing unprecedented accuracy in distinguishing surface and receptors from those in internal compartments of the presynaptic terminal, important in neural communication. This approach offers broad utility for surface-specific protein labelling.

Behavioral state and stimulus strength regulate the role of somatostatin interneurons in stabilizing network activity.

Inhibition stabilization enables cortical circuits to encode sensory signals across diverse contexts. Somatostatin-expressing (SST) interneurons are well-suited for this role through their strong recurrent connectivity with excitatory pyramidal cells. We developed a cortical circuit model predicting that SST cells become increasingly important for stabilization as sensory input strengthens. We tested this prediction in mouse primary visual cortex by manipulating excitatory input to SST cells, a key parameter for inhibition stabilization, with a novel cell-type specific pharmacological method to selectively block glutamatergic receptors on SST cells. Consistent with our model predictions, we find antagonizing glutamatergic receptors drives a paradoxical facilitation of SST cells with increasing stimulus contrast. In addition, we find even stronger engagement of SST-dependent stabilization when the mice are aroused. Thus, we reveal that the role of SST cells in cortical processing gradually switches as a function of both input strength and behavioral state.

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.

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.

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.

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.

Publisher Correction: A repeated molecular architecture across thalamic pathways.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A repeated molecular architecture across thalamic pathways.

The thalamus is the central communication hub of the forebrain and provides the cerebral cortex with inputs from sensory organs, subcortical systems and the cortex itself. Multiple thalamic regions send convergent information to each cortical region, but the organizational logic of thalamic projections has remained elusive. Through comprehensive transcriptional analyses of retrogradely labeled thalamic neurons in adult mice, we identify three major profiles of thalamic pathways. These profiles exist along a continuum that is repeated across all major projection systems, such as those for vision, motor control and cognition. The largest component of gene expression variation in the mouse thalamus is topographically organized, with features conserved in humans. Transcriptional differences between these thalamic neuronal identities are tied to cellular features that are critical for function, such as axonal morphology and membrane properties. Molecular profiling therefore reveals covariation in the properties of thalamic pathways serving all major input modalities and output targets, thus establishing a molecular framework for understanding the thalamus.

Dissociable Structural and Functional Hippocampal Outputs via Distinct Subiculum Cell Classes.

The mammalian hippocampus, comprised of serially connected subfields, participates in diverse behavioral and cognitive functions. It has been postulated that parallel circuitry embedded within hippocampal subfields may underlie such functional diversity. We sought to identify, delineate, and manipulate this putatively parallel architecture in the dorsal subiculum, the primary output subfield of the dorsal hippocampus. Population and single-cell RNA-seq revealed that the subiculum can be divided into two spatially adjacent subregions associated with prominent differences in pyramidal cell gene expression. Pyramidal cells occupying these two regions differed in their long-range inputs, local wiring, projection targets, and electrophysiological properties. Leveraging gene-expression differences across these regions, we use genetically restricted neuronal silencing to show that these regions differentially contribute to spatial working memory. This work provides a coherent molecular-, cellular-, circuit-, and behavioral-level demonstration that the hippocampus embeds structurally and functionally dissociable streams within its serial architecture.

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.

Design and Synthesis of a Calcium-Sensitive Photocage.

Photolabile protecting groups (or "photocages") enable precise spatiotemporal control of chemical functionality and facilitate advanced biological experiments. Extant photocages exhibit a simple input-output relationship, however, where application of light elicits a photochemical reaction irrespective of the environment. Herein, we refine and extend the concept of photolabile groups, synthesizing the first Ca(2+) -sensitive photocage. This system functions as a chemical coincidence detector, releasing small molecules only in the presence of both light and elevated [Ca(2+) ]. Caging a fluorophore with this ion-sensitive moiety yields an "ion integrator" that permanently marks cells undergoing high Ca(2+) flux during an illumination-defined time period. Our general design concept demonstrates a new class of light-sensitive material for cellular imaging, sensing, and targeted molecular delivery.

Hipposeq: a comprehensive RNA-seq database of gene expression in hippocampal principal neurons.

Clarifying gene expression in narrowly defined neuronal populations can provide insight into cellular identity, computation, and functionality. Here, we used next-generation RNA sequencing (RNA-seq) to produce a quantitative, whole genome characterization of gene expression for the major excitatory neuronal classes of the hippocampus; namely, granule cells and mossy cells of the dentate gyrus, and pyramidal cells of areas CA3, CA2, and CA1. Moreover, for the canonical cell classes of the trisynaptic loop, we profiled transcriptomes at both dorsal and ventral poles, producing a cell-class- and region-specific transcriptional description for these populations. This dataset clarifies the transcriptional properties and identities of lesser-known cell classes, and moreover reveals unexpected variation in the trisynaptic loop across the dorsal-ventral axis. We have created a public resource, Hipposeq (http://hipposeq.janelia.org), which provides analysis and visualization of these data and will act as a roadmap relating molecules to cells, circuits, and computation in the hippocampus.

Spatial Gene-Expression Gradients Underlie Prominent Heterogeneity of CA1 Pyramidal Neurons.

Tissue and organ function has been conventionally understood in terms of the interactions among discrete and homogeneous cell types. This approach has proven difficult in neuroscience due to the marked diversity across different neuron classes, but it may be further hampered by prominent within-class variability. Here, we considered a well-defined canonical neuronal population­hippocampal CA1 pyramidal cells (CA1 PCs)­and systematically examined the extent and spatial rules of transcriptional heterogeneity. Using next-generation RNA sequencing, we identified striking variability in CA1 PCs, such that the differences within CA1 along the dorsal-ventral axis rivaled differences across distinct pyramidal neuron classes. This variability emerged from a spectrum of continuous gene-expression gradients, producing a transcriptional profile consistent with a multifarious continuum of cells. This work reveals an unexpected amount of variability within a canonical and narrowly defined neuronal population and suggests that continuous, within-class heterogeneity may be an important feature of neural circuits.

Thy1-GCaMP6 transgenic mice for neuronal population imaging in vivo.

Genetically-encoded calcium indicators (GECIs) facilitate imaging activity of genetically defined neuronal populations in vivo. The high intracellular GECI concentrations required for in vivo imaging are usually achieved by viral gene transfer using adeno-associated viruses. Transgenic expression of GECIs promises important advantages, including homogeneous, repeatable, and stable expression without the need for invasive virus injections. Here we present the generation and characterization of transgenic mice expressing the GECIs GCaMP6s or GCaMP6f under the Thy1 promoter. We quantified GCaMP6 expression across brain regions and neurons and compared to other transgenic mice and AAV-mediated expression. We tested three mouse lines for imaging in the visual cortex in vivo and compared their performance to mice injected with AAV expressing GCaMP6. Furthermore, we show that GCaMP6 Thy1 transgenic mice are useful for long-term, high-sensitivity imaging in behaving mice.

A neuron-based screening platform for optimizing genetically-encoded calcium indicators.

Fluorescent protein-based sensors for detecting neuronal activity have been developed largely based on non-neuronal screening systems. However, the dynamics of neuronal state variables (e.g., voltage, calcium, etc.) are typically very rapid compared to those of non-excitable cells. We developed an electrical stimulation and fluorescence imaging platform based on dissociated rat primary neuronal cultures. We describe its use in testing genetically-encoded calcium indicators (GECIs). Efficient neuronal GECI expression was achieved using lentiviruses containing a neuronal-selective gene promoter. Action potentials (APs) and thus neuronal calcium levels were quantitatively controlled by electrical field stimulation, and fluorescence images were recorded. Images were segmented to extract fluorescence signals corresponding to individual GECI-expressing neurons, which improved sensitivity over full-field measurements. We demonstrate the superiority of screening GECIs in neurons compared with solution measurements. Neuronal screening was useful for efficient identification of variants with both improved response kinetics and high signal amplitudes. This platform can be used to screen many types of sensors with cellular resolution under realistic conditions where neuronal state variables are in relevant ranges with respect to timing and amplitude.