Electrochemical Determination of Fentanyl Using Carbon Nanofiber-Modified Electrodes.

In this work, we report the direct electrochemical oxidation of fentanyl using commercial screen-printed carbon electrodes (SPCEs) modified with carboxyl-functionalized carbon nanofibers (fCNFs). CNFs have surface chemistry and reactivity similar to carbon nanotubes (CNTs), yet they are easier to produce and are of a lower cost than CNTs. By monitoring the current produced during the electrochemical oxidation of fentanyl, variables such as fCNF loading, fentanyl accumulation time, electrolyte pH, and differential pulse voltammetry parameters were optimized. Under an optimized set of conditions, the fCNF/SPCEs responded linearly to fentanyl in the concentration range of 0.125-10 µM, with a limit of detection of 75 nM. The fCNF/SPCEs also demonstrated excellent selectivity against common cutting agents found in illicit drugs (e.g., glucose, sucrose, caffeine, acetaminophen, and theophylline) and interferents found in biological samples (e.g., ascorbic acid, NaCl, urea, creatinine, and uric acid). The performance of the sensor was also successfully tested using fentanyl spiked into an artificial urine sample. The straightforward electrode assembly process, low cost, ease of use, and rapid response make the fCNF/SPCEs prime candidates for the detection of fentanyl in both physiological samples and street drugs.

A spatially-resolved transcriptional atlas of the murine dorsal pons at single-cell resolution.

The "dorsal pons", or "dorsal pontine tegmentum" (dPnTg), is part of the brainstem. It is a complex, densely packed region whose nuclei are involved in regulating many vital functions. Notable among them are the parabrachial nucleus, the Kölliker Fuse, the Barrington nucleus, the locus coeruleus, and the dorsal, laterodorsal, and ventral tegmental nuclei. In this study, we applied single-nucleus RNA-seq (snRNA-seq) to resolve neuronal subtypes based on their unique transcriptional profiles and then used multiplexed error robust fluorescence in situ hybridization (MERFISH) to map them spatially. We sampled ~1 million cells across the dPnTg and defined the spatial distribution of over 120 neuronal subtypes. Our analysis identified an unpredicted high transcriptional diversity in this region and pinpointed the unique marker genes of many neuronal subtypes. We also demonstrated that many neuronal subtypes are transcriptionally similar between humans and mice, enhancing this study's translational value. Finally, we developed a freely accessible, GPU and CPU-powered dashboard ( http://harvard.heavy.ai:6273/ ) that combines interactive visual analytics and hardware-accelerated SQL into a data science framework to allow the scientific community to query and gain insights into the data.

Structure Determination of Biogenic Crystals Directly from 3D Electron Diffraction Data.

Highly reflective assemblies of purine, pteridine, and flavin crystals are used in the coloration and visual systems of many different animals. However, structure determination of biogenic crystals by single-crystal XRD is challenging due to the submicrometer size and beam sensitivity of the crystals, and powder XRD is inhibited due to the small volumes of powders, crystalline impurity phases, and significant preferred orientation. Consequently, the crystal structures of many biogenic materials remain unknown. Herein, we demonstrate that the 3D electron diffraction (3D ED) technique provides a powerful alternative approach, reporting the successful structure determination of biogenic guanine crystals (from spider integument, fish scales, and scallop eyes) from 3D ED data confirmed by analysis of powder XRD data. The results show that all biogenic guanine crystals studied are the previously known ß-polymorph. This study highlights the considerable potential of 3D ED for elucidating the structures of biogenic molecular crystals in the nanometer-to-micrometer size range. This opens up an important opportunity in the development of organic biomineralization, for which structural knowledge is critical for understanding the optical functions of biogenic materials and their possible applications as sustainable, biocompatible optical materials.

A spatially-resolved transcriptional atlas of the murine dorsal pons at single-cell resolution.

The "dorsal pons", or "dorsal pontine tegmentum" (dPnTg), is part of the brainstem. It is a complex, densely packed region whose nuclei are involved in regulating many vital functions. Notable among them are the parabrachial nucleus, the Kölliker Fuse, the Barrington nucleus, the locus coeruleus, and the dorsal, laterodorsal, and ventral tegmental nuclei. In this study, we applied single-nucleus RNA-seq (snRNA-seq) to resolve neuronal subtypes based on their unique transcriptional profiles and then used multiplexed error robust fluorescence in situ hybridization (MERFISH) to map them spatially. We sampled ~1 million cells across the dPnTg and defined the spatial distribution of over 120 neuronal subtypes. Our analysis identified an unpredicted high transcriptional diversity in this region and pinpointed many neuronal subtypes' unique marker genes. We also demonstrated that many neuronal subtypes are transcriptionally similar between humans and mice, enhancing this study's translational value. Finally, we developed a freely accessible, GPU and CPU-powered dashboard (http://harvard.heavy.ai:6273/) that combines interactive visual analytics and hardware-accelerated SQL into a data science framework to allow the scientific community to query and gain insights into the data.

Exploiting solid-state dynamic nuclear polarization NMR spectroscopy to establish the spatial distribution of polymorphic phases in a solid material.

Solid-state DNP NMR can enhance the ability to detect minor amounts of solid phases within heterogenous materials. Here we demonstrate that NMR contrast based on the transport of DNP-enhanced polarization can be exploited in the challenging case of early detection of a small amount of a minor polymorphic phase within a major polymorph, and we show that this approach can yield quantitative information on the spatial distribution of the two polymorphs. We focus on the detection of a minor amount (<4%) of polymorph III of m-aminobenzoic acid within a powder sample of polymorph I at natural isotopic abundance. Based on proposed models of the spatial distribution of the two polymorphs, simulations of 1H spin diffusion allow NMR data to be calculated for each model as a function of particle size and the relative amounts of the polymorphs. A comparison between simulated and experimental NMR data allows the model(s) best representing the spatial distribution of the polymorphs in the system to be established.

Mouse frontal cortex mediates additive multisensory decisions.

The brain can combine auditory and visual information to localize objects. However, the cortical substrates underlying audiovisual integration remain uncertain. Here, we show that mouse frontal cortex combines auditory and visual evidence; that this combination is additive, mirroring behavior; and that it evolves with learning. We trained mice in an audiovisual localization task. Inactivating frontal cortex impaired responses to either sensory modality, while inactivating visual or parietal cortex affected only visual stimuli. Recordings from >14,000 neurons indicated that after task learning, activity in the anterior part of frontal area MOs (secondary motor cortex) additively encodes visual and auditory signals, consistent with the mice's behavioral strategy. An accumulator model applied to these sensory representations reproduced the observed choices and reaction times. These results suggest that frontal cortex adapts through learning to combine evidence across sensory cortices, providing a signal that is transformed into a binary decision by a downstream accumulator.

Unraveling the Complex Solid-State Phase Transition Behavior of 1-Iodoadamantane, a Material for Which Ostensibly Identical Crystals Undergo Different Transformation Pathways.

Phase transitions in crystalline molecular solids have important implications in the fundamental understanding of materials properties and in the development of materials applications. Herein, we report the solid-state phase transition behavior of 1-iodoadamantane (1-IA) investigated using a multi-technique strategy [synchrotron powder X-ray diffraction (XRD), single-crystal XRD, solid-state NMR, and differential scanning calorimetry (DSC)], which reveals complex phase transition behavior on cooling from ambient temperature to ca. 123 K and on subsequent heating to the melting temperature (348 K). Starting from the known phase of 1-IA at ambient temperature (phase A), three low-temperature phases are identified (phases B, C, and D); the crystal structures of phases B and C are reported, together with a re-determination of the structure of phase A. Remarkably, single-crystal XRD shows that some individual crystals of phase A transform to phase B, while other crystals of phase A transform instead to phase C. Results (from powder XRD and DSC) on cooling a powder sample of phase A are fully consistent with this behavior while also revealing an additional transformation pathway from phase A to phase D. Thus, on cooling, a powder sample of phase A transforms partially to phase C (at 229 K), partially to phase D (at 226 K) and partially to phase B (at 211 K). During the cooling process, each of the phases B, C, and D is formed directly from phase A, and no transformations are observed between phases B, C, and D. On heating the resulting triphasic powder sample of phases B, C, and D from 123 K, phase B transforms to phase D (at 211 K), followed by the transformation of phase D to phase C (at 255 K), and finally, phase C transforms to phase A (at 284 K). From these observations, it is apparent that different crystals of phase A, which are ostensibly identical at the level of information revealed by XRD, must actually differ in other aspects that significantly influence their low-temperature phase transition pathways. This unusual behavior will stimulate future studies to gain deeper insights into the specific properties that control the phase transition pathways in individual crystals of this material.

A modular architecture for organizing, processing and sharing neurophysiology data.

We describe an architecture for organizing, integrating and sharing neurophysiology data within a single laboratory or across a group of collaborators. It comprises a database linking data files to metadata and electronic laboratory notes; a module collecting data from multiple laboratories into one location; a protocol for searching and sharing data and a module for automatic analyses that populates a website. These modules can be used together or individually, by single laboratories or worldwide collaborations.

Behavioral origin of sound-evoked activity in mouse visual cortex.

Sensory cortices can be affected by stimuli of multiple modalities and are thus increasingly thought to be multisensory. For instance, primary visual cortex (V1) is influenced not only by images but also by sounds. Here we show that the activity evoked by sounds in V1, measured with Neuropixels probes, is stereotyped across neurons and even across mice. It is independent of projections from auditory cortex and resembles activity evoked in the hippocampal formation, which receives little direct auditory input. Its low-dimensional nature starkly contrasts the high-dimensional code that V1 uses to represent images. Furthermore, this sound-evoked activity can be precisely predicted by small body movements that are elicited by each sound and are stereotyped across trials and mice. Thus, neural activity that is apparently multisensory may simply arise from low-dimensional signals associated with internal state and behavior.

Boron Nitride-Doped Polyphenylenic Organogels.

Herein, we describe the synthesis of the first boron nitride-doped polyphenylenic material obtained through a [4 + 2] cycloaddition reaction between a triethynyl borazine unit and a biscyclopentadienone derivative, which undergoes organogel formation in chlorinated solvents (the critical jellification concentration is 4% w/w in CHCl3). The polymer has been characterized extensively by Fourier-transform infrared spectroscopy, solid-state 13C NMR, solid-state 11B NMR, and by comparison with the isolated monomeric unit. Furthermore, the polymer gels formed in chlorinated solvents have been thoroughly characterized and studied, showing rheological properties comparable to those of polyacrylamide gels with a low crosslinker percentage. Given the thermal and chemical stability, the material was studied as a potential support for solid-state electrolytes. showing properties comparable to those of polyethylene glycol-based electrolytes, thus presenting great potential for the application of this new class of material in lithium-ion batteries.

Visuomotor learning promotes visually evoked activity in the medial prefrontal cortex.

The medial prefrontal cortex (mPFC) is necessary for executing many learned associations between stimuli and movement. It is unclear, however, how activity in the mPFC evolves across learning, and how this activity correlates with sensory stimuli and the learned movements they evoke. To address these questions, we record cortical activity with widefield calcium imaging while mice learned to associate a visual stimulus with a forelimb movement. After learning, the mPFC shows stimulus-evoked activity both during task performance and during passive viewing, when the stimulus evokes no action. This stimulus-evoked activity closely tracks behavioral performance across training, with both exhibiting a marked increase between days when mice first learn the task, followed by a steady increase with further training. Electrophysiological recordings localized this activity to the secondary motor and anterior cingulate cortex. We conclude that learning a visuomotor task promotes a route for visual information to reach the prefrontal cortex.

Task specificity in mouse parietal cortex.

Parietal cortex is implicated in a variety of behavioral processes, but it is unknown whether and how its individual neurons participate in multiple tasks. We trained head-fixed mice to perform two visual decision tasks involving a steering wheel or a virtual T-maze and recorded from the same parietal neurons during these two tasks. Neurons that were active during the T-maze task were typically inactive during the steering-wheel task and vice versa. Recording from the same neurons in the same apparatus without task stimuli yielded the same specificity as in the task, suggesting that task specificity depends on physical context. To confirm this, we trained some mice in a third task combining the steering wheel context with the visual environment of the T-maze. This hybrid task engaged the same neurons as those engaged in the steering-wheel task. Thus, participation by neurons in mouse parietal cortex is task specific, and this specificity is determined by physical context.

A transcriptomic axis predicts state modulation of cortical interneurons.

Transcriptomics has revealed that cortical inhibitory neurons exhibit a great diversity of fine molecular subtypes1-6, but it is not known whether these subtypes have correspondingly diverse patterns of activity in the living brain. Here we show that inhibitory subtypes in primary visual cortex (V1) have diverse correlates with brain state, which are organized by a single factor: position along the main axis of transcriptomic variation. We combined in vivo two-photon calcium imaging of mouse V1 with a transcriptomic method to identify mRNA for 72 selected genes in ex vivo slices. We classified inhibitory neurons imaged in layers 1-3 into a three-level hierarchy of 5 subclasses, 11 types and 35 subtypes using previously defined transcriptomic clusters3. Responses to visual stimuli differed significantly only between subclasses, with cells in the Sncg subclass uniformly suppressed, and cells in the other subclasses predominantly excited. Modulation by brain state differed at all hierarchical levels but could be largely predicted from the first transcriptomic principal component, which also predicted correlations with simultaneously recorded cells. Inhibitory subtypes that fired more in resting, oscillatory brain states had a smaller fraction of their axonal projections in layer 1, narrower spikes, lower input resistance and weaker adaptation as determined in vitro7, and expressed more inhibitory cholinergic receptors. Subtypes that fired more during arousal had the opposite properties. Thus, a simple principle may largely explain how diverse inhibitory V1 subtypes shape state-dependent cortical processing.

A structure determination protocol based on combined analysis of 3D-ED data, powder XRD data, solid-state NMR data and DFT-D calculations reveals the structure of a new polymorph of l-tyrosine.

We report the crystal structure of a new polymorph of l-tyrosine (denoted the ß polymorph), prepared by crystallization from the gas phase following vacuum sublimation. Structure determination was carried out by combined analysis of three-dimensional electron diffraction (3D-ED) data and powder X-ray diffraction (XRD) data. Specifically, 3D-ED data were required for reliable unit cell determination and space group assignment, with structure solution carried out independently from both 3D-ED data and powder XRD data, using the direct-space strategy for structure solution implemented using a genetic algorithm. Structure refinement was carried out both from powder XRD data, using the Rietveld profile refinement technique, and from 3D-ED data. The final refined structure was validated both by periodic DFT-D calculations, which confirm that the structure corresponds to an energy minimum on the energy landscape, and by the fact that the values of isotropic 13C NMR chemical shifts calculated for the crystal structure using DFT-D methodology are in good agreement with the experimental high-resolution solid-state 13C NMR spectrum. Based on DFT-D calculations using the PBE0-MBD method, the ß polymorph is meta-stable with respect to the previously reported crystal structure of l-tyrosine (now denoted the α polymorph). Crystal structure prediction calculations using the AIRSS approach suggest that there are three other plausible crystalline polymorphs of l-tyrosine, with higher energy than the α and ß polymorphs.

Neural correlates of blood flow measured by ultrasound.

Functional ultrasound imaging (fUSI) is an appealing method for measuring blood flow and thus infer brain activity, but it relies on the physiology of neurovascular coupling and requires extensive signal processing. To establish to what degree fUSI trial-by-trial signals reflect neural activity, we performed simultaneous fUSI and neural recordings with Neuropixels probes in awake mice. fUSI signals strongly correlated with the slow (<0.3 Hz) fluctuations in the local firing rate and were closely predicted by the smoothed firing rate of local neurons, particularly putative inhibitory neurons. The optimal smoothing filter had a width of ∼3 s, matched the hemodynamic response function of awake mice, was invariant across mice and stimulus conditions, and was similar in the cortex and hippocampus. fUSI signals also matched neural firing spatially: firing rates were as highly correlated across hemispheres as fUSI signals. Thus, blood flow measured by ultrasound bears a simple and accurate relationship to neuronal firing.

Biogenic Guanine Crystals Are Solid Solutions of Guanine and Other Purine Metabolites.

Highly reflective crystals of the nucleotide base guanine are widely distributed in animal coloration and visual systems. Organisms precisely control the morphology and organization of the crystals to optimize different optical effects, but little is known about how this is achieved. Here we examine a fundamental question that has remained unanswered after over 100 years of research on guanine: what are the crystals made of? Using solution-state and solid-state chemical techniques coupled with structural analysis by powder XRD and solid-state NMR, we compare the purine compositions and the structures of seven biogenic guanine crystals with different crystal morphologies, testing the hypothesis that intracrystalline dopants influence the crystal shape. We find that biogenic "guanine" crystals are not pure crystals but molecular alloys (aka solid solutions and mixed crystals) of guanine, hypoxanthine, and sometimes xanthine. Guanine host crystals occlude homogeneous mixtures of other purines, sometimes in remarkably large amounts (up to 20% of hypoxanthine), without significantly altering the crystal structure of the guanine host. We find no correlation between the biogenic crystal morphology and dopant content and conclude that dopants do not dictate the crystal morphology of the guanine host. The ability of guanine crystals to host other molecules enables animals to build physiologically "cheaper" crystals from mixtures of metabolically available purines, without impeding optical functionality. The exceptional levels of doping in biogenic guanine offer inspiration for the design of mixed molecular crystals that incorporate multiple functionalities in a single material.

Solid-State Structural Properties of Alloxazine Determined from Powder XRD Data in Conjunction with DFT-D Calculations and Solid-State NMR Spectroscopy: Unraveling the Tautomeric Identity and Pathways for Tautomeric Interconversion.

We report the solid-state structural properties of alloxazine, a tricyclic ring system found in many biologically important molecules, with structure determination carried out directly from powder X-ray diffraction (XRD) data. As the crystal structures containing the alloxazine and isoalloxazine tautomers both give a high-quality fit to the powder XRD data in Rietveld refinement, other techniques are required to establish the tautomeric form in the solid state. In particular, high-resolution solid-state 15N NMR data support the presence of the alloxazine tautomer, based on comparison between isotropic chemical shifts in the experimental 15N NMR spectrum and the corresponding values calculated for the crystal structures containing the alloxazine and isoalloxazine tautomers. Furthermore, periodic DFT-D calculations at the PBE0-MBD level indicate that the crystal structure containing the alloxazine tautomer has significantly lower energy. We also report computational investigations of the interconversion between the tautomeric forms in the crystal structure via proton transfer along two intermolecular N-H···N hydrogen bonds; DFT-D calculations at the PBE0-MBD level indicate that the tautomeric interconversion is associated with a lower energy transition state for a mechanism involving concerted (rather than sequential) proton transfer along the two hydrogen bonds. However, based on the relative energies of the crystal structures containing the alloxazine and isoalloxazine tautomers, it is estimated that under conditions of thermal equilibrium at ambient temperature, more than 99.9% of the molecules in the crystal structure will exist as the alloxazine tautomer.

Disruption of VGLUT1 in Cholinergic Medial Habenula Projections Increases Nicotine Self-Administration.

Cholinergic projections from the medial habenula (MHb) to the interpeduncular nucleus (IPN) have been studied for their complex contributions to nicotine addiction and have been implicated in nicotine reinforcement, aversion, and withdrawal. While it has been established that MHb cholinergic projections corelease glutamate, no direct evidence has demonstrated a role for this glutamate projection in nicotine consumption. In the present study, a novel floxed Slc17a7 [vesicular glutamate transporter 1 (VGLUT1)] mouse was generated and used to create conditional knock-out (cKO) mice that lack VGLUT1 in MHb cholinergic neurons. Loss of Slc17a7 expression in ventral MHb cholinergic neurons was validated using fluorescent in situ hybridization, and immunohistochemistry was used to demonstrate a corresponding reduction of VGLUT1 protein in cholinergic terminals in the IPN. We also used optogenetics-assisted electrophysiology to evoke EPSCs in IPN and observed a reduction of glutamatergic currents in the cKO, supporting the functional disruption of VGLUT1 in MHb to IPN synapses. cKO mice exhibited no gross phenotypic abnormalities and displayed normal thigmotaxis and locomotor behavior in the open-field assay. When trained to lever press for food, there was no difference between control and cKO. However, when tested in a nicotine self-administration procedure, we found that the loss of VGLUT1-mediated glutamate corelease led to increased responding for nicotine. These findings indicate that glutamate corelease from ventral MHb cholinergic neurons opposes nicotine self-administration, and provide additional support for targeting this synapse to develop potential treatments for nicotine addiction.