The brain can become transiently disconnected from the environment while maintaining vivid, internally generated experiences. This so-called 'dissociated state' can occur in pathological conditions and under the influence of psychedelics or the anesthetic ketamine (KET). The cellular and circuit mechanisms producing the dissociative state remain poorly understood. We show in mice that KET causes spontaneously active neurons to become suppressed while previously silent neurons become spontaneously activated. This switch occurs in all cortical layers and different cortical regions, is induced by both systemic and cortical application of KET and is mediated by suppression of parvalbumin and somatostatin interneuron activity and inhibition of NMDA receptors and HCN channels. Combined, our results reveal two largely non-overlapping cortical neuronal populations-one engaged in wakefulness, the other contributing to the KET-induced brain state-and may lay the foundation for understanding how the brain might become disconnected from the surrounding environment while maintaining internal subjective experiences.

Subcellular pharmacokinetic measurements have informed the study of central nervous system (CNS)-acting drug mechanisms. Recent investigations have been enhanced by the use of genetically encoded fluorescent biosensors for drugs of interest at the plasma membrane and in organelles. We describe screening and validation protocols for identifying hit pairs comprising a drug and biosensor, with each screen including 13-18 candidate biosensors and 44-84 candidate drugs. After a favorable hit pair is identified and validated via these protocols, the biosensor is then optimized, as described in other papers, for sensitivity and selectivity to the drug. We also show sample hit pair data that may lead to future intensity-based drug-sensing fluorescent reporters (iDrugSnFRs). These protocols will assist scientists to use fluorescence responses as criteria in identifying favorable fluorescent biosensor variants for CNS-acting drugs that presently have no corresponding biosensor partner. eLife (2022), DOI: 10.7554/eLife.74648 Graphical abstract.

We report the rational engineering of a remarkably stable yellow fluorescent protein (YFP), 'hyperfolder YFP' (hfYFP), that withstands chaotropic conditions that denature most biological structures within seconds, including superfolder green fluorescent protein (GFP). hfYFP contains no cysteines, is chloride insensitive and tolerates aldehyde and osmium tetroxide fixation better than common fluorescent proteins, enabling its use in expansion and electron microscopies. We solved crystal structures of hfYFP (to 1.7-Å resolution), a monomeric variant, monomeric hyperfolder YFP (1.6 Å) and an mGreenLantern mutant (1.2 Å), and then rationally engineered highly stable 405-nm-excitable GFPs, large Stokes shift (LSS) monomeric GFP (LSSmGFP) and LSSA12 from these structures. Lastly, we directly exploited the chemical stability of hfYFP and LSSmGFP by devising a fluorescence-assisted protein purification strategy enabling all steps of denaturing affinity chromatography to be visualized using ultraviolet or blue light. hfYFP and LSSmGFP represent a new generation of robustly stable fluorescent proteins developed for advanced biotechnological applications.

Selective serotonin reuptake inhibitors (SSRIs) are the most prescribed treatment for individuals experiencing major depressive disorder (MDD). The therapeutic mechanisms that take place before, during, or after SSRIs bind the serotonin transporter (SERT) are poorly understood, partially because no studies exist of the cellular and subcellular pharmacokinetic properties of SSRIs in living cells. We studied escitalopram and fluoxetine using new intensity- based drug-sensing fluorescent reporters (“iDrugSnFRs”) targeted to the plasma membrane (PM), cytoplasm, or endoplasmic reticulum (ER) of cultured neurons and mammalian cell lines. We also employed chemical detection of drug within cells and phospholipid membranes. The drugs attain equilibrium in neuronal cytoplasm and ER, at approximately the same concentration as the externally applied solution, with time constants of a few s (escitalopram) or 200-300 s (fluoxetine). Simultaneously, the drugs accumulate within lipid membranes by ≥ 18-fold (escitalopram) or 180-fold (fluoxetine), and possibly by much larger factors. Both drugs leave cytoplasm, lumen, and membranes just as quickly during washout. We synthesized membrane-impermeant quaternary amine derivatives of the two SSRIs. The quaternary derivatives are substantially excluded from membrane, cytoplasm, and ER for > 2.4 h. They inhibit SERT transport-associated currents 6- or 11-fold less potently than the SSRIs (escitalopram or fluoxetine derivative, respectively), providing useful probes for distinguishing compartmentalized SSRI effects. Although our measurements are orders of magnitude faster than the “therapeutic lag” of SSRIs, these data suggest that SSRI-SERT interactions within organelles or membranes may play roles during either the therapeutic effects or the “antidepressant discontinuation syndrome”.

Hundreds of millions of structured proteins sustain life through chemical interactions and catalytic reactions1. Though dynamic, these proteins are assumed to be built upon fixed scaffolds of secondary structure, α-helices and β-sheets. Experimentally determined structures of over >58,000 non-redundant proteins support this assumption, though it has recently been challenged by ∼100 fold-switching proteins2. These “metamorphic3” proteins, though ostensibly rare, raise the question of how many uncharacterized proteins have shapeshifting–rather than fixed–secondary structures. To address this question, we developed a comparative sequence-based approach that predicts fold-switching proteins from differences in secondary structure propensity. We applied this approach to the universally conserved NusG transcription factor family of ∼15,000 proteins, one of which has a 50-residue regulatory subunit experimentally shown to switch between α-helical and β-sheet folds4. Our approach predicted that 25% of the sequences in this family undergo similar α-helix ⇌ β-sheet transitions, a frequency two orders of magnitude larger than previously observed. Our predictions evade state-of-the-art computational methods but were confirmed experimentally by circular dichroism and nuclear magnetic resonance spectroscopy for all 10 assiduously chosen dissimilar variants. These results suggest that fold switching is a pervasive mechanism of transcriptional regulation in all kingdoms of life and imply that numerous uncharacterized proteins may also switch folds.

Mechanisms that entrain and drive rhythmic epileptiform discharges remain debated. Traditionally, this quest has been focusing on interneuronal networks driven by GABAergic connections that activate synaptic or extrasynaptic receptors. However, synchronised interneuronal discharges could also trigger a transient elevation of extracellular GABA across the tissue volume, thus raising tonic GABAA receptor conductance (Gtonic) in multiple cells. Here, we use patch-clamp GABA ‘sniffer’ and optical GABA sensor to show that periodic epileptiform discharges are preceded by region-wide, rising waves of extracellular GABA. Neural network simulations that incorporate volume-transmitted GABA signals point to mechanistic principles underpinning this relationship. We validate this hypothesis using simultaneous patch-clamp recordings from multiple nerve cells, selective optogenetic stimulation of fast-spiking interneurons. Critically, we manipulate GABA uptake to suppress extracellular GABA waves but not synaptic GABAergic transmission, which shows a clear effect on rhythm generation. Our findings thus unveil a key role of extrasynaptic, volume-transmitted GABA actions in pacing regenerative rhythmic activity in brain networks.

Synchronised rhythmic activity of the brain is thought to arise from neuronal network behaviours that rely on synaptic signalling between individual cells. This notion has been a basis to explain periodic epileptiform discharges that are driven by interneuronal networks. However, interneuronal discharges not only engage cell-cell GABAergic transmission but also control the extracellular GABA concentration ([GABA]e) and thus tonic GABAA receptor conductance (Gtonic) across the cell population. At the same time, the firing activity of interneurons shows a bell-shaped dependence on Gtonic, suggesting an innate susceptibility to self-sustained oscillations. Here, we employ patch-clamp GABA ‘sniffer’ and fast two-photon excitation imaging of GABA sensor to show that periodic epileptiform discharges are preceded by a region-wide, rising wave of extracellular GABA. Neural network simulations based on such observations reveal that it is the volume-transmitted, extrasynaptic actions of GABA targeting multiple off-target cells that drives synchronised interneuronal spiking prompting periodic epileptiform bursts. We validate this hypothesis using simultaneous patch-clamp recordings from multiple nerve cells, selective optogenetic stimulation of fast-spiking interneurons, and by revealing the role of GABA uptake. Our findings thus unveil a key role of extrasynaptic, volume-transmitted GABA actions in enabling and pacing regenerative rhythmic activity in brain networks.

The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit saturating activation kinetics and are excluded from post-synaptic densities, limiting their ability to distinguish synaptic from extrasynaptic glutamate. Using a multi-assay screen in bacteria, soluble protein, and cultured neurons, we generated novel variants with improved kinetics and signal-to-noise ratios. We also developed surface display constructs that improve iGluSnFR’s nanoscopic localization to post-synapses. The resulting indicator, iGluSnFR3, exhibits rapid non-saturating activation kinetics and reports synaptic glutamate release with improved linearity and increased specificity versus extrasynaptic signals in cultured neurons. In mouse visual cortex, imaging of iGluSnFR3 at individual boutons reported single electrophysiologically-observed action potentials with high specificity versus non-synaptic transients. In vibrissal sensory cortex Layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines.

The protein folding paradigm asserts that the three-dimensional structure of a protein is determined by its amino acid sequence. Here we show that a substantial population of proteins from the NusG superfamily of transcription factors do not adhere to this paradigm. Previous work demonstrated that one member of this superfamily has a regulatory domain that completely switches between α-helical and β-sheet folds, but the pervasiveness of this fold-switching mechanism is uncertain. To address this question, we developed a sequence-based predictor, which revealed that thousands of proteins from this superfamily switch folds. Circular dichroism and nuclear magnetic resonance spectroscopies of 10 sequence-diverse variants confirmed our predictions. By contrast, state-of-the-art methods based on the protein folding paradigm assume that related sequences adopt the same fold and thus predicted that the regulatory domains of all variants adopt only the β-sheet fold. Removal of this bias revealed that residue-residue contacts from both α-helical and β-sheet folds are conserved in a large subpopulation of fold-switching domains, poising them to assume disparate conformations. Our results suggest that fold switching is a pervasive mechanism of transcriptional regulation in all kingdoms of life and indicate that expanding the protein folding paradigm may reveal the involvement of fold-switching proteins in diverse biological processes.

Nicotinic partial agonists provide an accepted aid for smoking cessation and thus contribute to decreasing tobacco-related disease. Improved drugs constitute a continued area of study. However, there remains no reductionist method to examine the cellular and subcellular pharmacokinetic properties of these compounds in living cells. Here, we developed new intensity-based drug sensing fluorescent reporters ('iDrugSnFRs') for the nicotinic partial agonists dianicline, cytisine, and two cytisine derivatives - 10-fluorocytisine and 9-bromo-10-ethylcytisine. We report the first atomic-scale structures of liganded periplasmic binding protein-based biosensors, accelerating development of iDrugSnFRs and also explaining the activation mechanism. The nicotinic iDrugSnFRs detect their drug partners in solution, as well as at the plasma membrane (PM) and in the endoplasmic reticulum (ER) of cell lines and mouse hippocampal neurons. At the PM, the speed of solution changes limits the growth and decay rates of the fluorescence response in almost all cases. In contrast, we found that rates of membrane crossing differ among these nicotinic drugs by > 30 fold. The new nicotinic iDrugSnFRs provide insight into the real-time pharmacokinetic properties of nicotinic agonists and provide a methodology whereby iDrugSnFRs can inform both pharmaceutical neuroscience and addiction neuroscience.