Publications

Modern indirect calorimetry systems allow for high-frequency time series measurements of the factors affected by thermogenesis: energy intake and energy expenditure. These indirect calorimetry systems generate a flood of raw data recording oxygen consumption, carbon dioxide production, physical activity, and food intake among other factors. Analysis of these data requires time-consuming manual manipulation for formatting, data cleaning, quality control, and visualization. Beyond data handling, analyses of indirect calorimetry experiments require specialized statistical treatment to account for differential contributions of fat mass and lean mass to metabolic rates.Here we describe how to use the software package CalR version 1.2, to analyze indirect calorimetry data from three examples of thermogenesis, cold exposure, adrenergic agonism, and hyperthyroidism in mice, by providing standardized methods for reproducible research. CalR is a free online tool with an easy-to-use graphical user interface to import data files from the Columbus Instruments' CLAMS, Sable Systems' Promethion, and TSE Systems' PhenoMaster. Once loaded, CalR can quickly visualize experimental results and perform basic statistical analyses. We present a framework that standardizes the data structures and analyses of indirect calorimetry experiments to provide reusable and reproducible methods for the physiological data affecting body weight.

Neuron-glia interactions play a critical role in the regulation of synapse formation and circuit assembly. Here we demonstrate that canonical Sonic hedgehog (Shh) pathway signaling in cortical astrocytes acts to coordinate layer-specific synaptic connectivity. We show that the Shh receptor Ptch1 is expressed by cortical astrocytes during development and that Shh signaling is necessary and sufficient to promote the expression of genes involved in regulating synaptic development and layer-enriched astrocyte molecular identity. Loss of Shh in layer V neurons reduces astrocyte complexity and coverage by astrocytic processes in tripartite synapses; conversely, cell-autonomous activation of Shh signaling in astrocytes promotes cortical excitatory synapse formation. Furthermore, Shh-dependent genes Lrig1 and Sparc distinctively contribute to astrocyte morphology and synapse formation. Together, these results suggest that Shh secreted from deep-layer cortical neurons acts to specialize the molecular and functional features of astrocytes during development to shape circuit assembly and function.

Many mammalian neurons release multiple neurotransmitters to activate diverse classes of postsynaptic ionotropic receptors. Entopeduncular nucleus somatostatin (EP Sst+) projection neurons to the lateral habenula (LHb) release both glutamate and GABA, but it is unclear whether these are packaged into the same or segregated pools of synaptic vesicles. Here, we describe a method combining electrophysiology, spatially patterned optogenetics, and computational modeling designed to analyze the mechanism of glutamate/GABA co-release in mouse brain. We find that the properties of postsynaptic currents elicited in LHb neurons by optogenetically activating EP Sst+ terminals are only consistent with co-packaging of glutamate/GABA into individual vesicles. Furthermore, presynaptic neuromodulators that weaken EP Sst+ to LHb synapses maintain the co-packaging of glutamate/GABA while reducing vesicular release probability. Our approach is applicable to the study of multi-transmitter neurons throughout the brain, and our results constrain the mechanisms of neuromodulation and synaptic integration in LHb.

SignificanceTo obtain rewards in changing and uncertain environments, animals must adapt their behavior. We found that mouse choice and trial-to-trial switching behavior in a dynamic and probabilistic two-choice task could be modeled by equivalent theoretical, algorithmic, and descriptive models. These models capture components of evidence accumulation, choice history bias, and stochasticity in mouse behavior. Furthermore, they reveal that mice adapt their behavior in different environmental contexts by modulating their level of stickiness to their previous choice. Despite deviating from the behavior of a theoretically ideal observer, the empirical models achieve comparable levels of near-maximal reward. These results make predictions to guide interrogation of the neural mechanisms underlying flexible decision-making strategies.

The field of implantable optical neural interfaces has recently enabled the interrogation of neural circuitry with both cell-type specificity and spatial resolution in sub-cortical structures of the mouse brain. This generated the need to integrate multiple optical channels within the same implantable device, motivating the requirement of multiplexing and demultiplexing techniques. In this article, we present an orthogonalization method of the far-field space to introduce mode-division demultiplexing for collecting fluorescence from the implantable tapered optical fibers. This is achieved by exploiting the correlation between the transversal wavevector k t of the guided light and the position of the fluorescent sources along the implant, an intrinsic property of the taper waveguide. On these bases, we define a basis of orthogonal vectors in the Fourier space, each of which is associated with a depth along the taper, to simultaneously detect and demultiplex the collected signal when the probe is implanted in fixed mouse brain tissue. Our approach complements the existing multiplexing techniques used in silicon-based photonics probes with the advantage of a significant simplification of the probe itself.

Disinhibitory neurons throughout the mammalian cortex are powerful enhancers of circuit excitability and plasticity. The differential expression of neuropeptide receptors in disinhibitory, inhibitory, and excitatory neurons suggests that each circuit motif may be controlled by distinct neuropeptidergic systems. Here, we reveal that a bombesin-like neuropeptide, gastrin-releasing peptide (GRP), recruits disinhibitory cortical microcircuits through selective targeting and activation of vasoactive intestinal peptide (VIP)-expressing cells. Using a genetically encoded GRP sensor, optogenetic anterograde stimulation, and trans-synaptic tracing, we reveal that GRP regulates VIP cells most likely via extrasynaptic diffusion from several local and long-range sources. In vivo photometry and CRISPR-Cas9-mediated knockout of the GRP receptor (GRPR) in auditory cortex indicate that VIP cells are strongly recruited by novel sounds and aversive shocks, and GRP-GRPR signaling enhances auditory fear memories. Our data establish peptidergic recruitment of selective disinhibitory cortical microcircuits as a mechanism to regulate fear memories.

The biochemical state of neurons, and of cells in general, is regulated by extracellular factors, including neurotransmitters, neuromodulators, and growth hormones. Interactions of an animal with its environment trigger neuromodulator release and engage biochemical transduction cascades to modulate synapse and cell function. Although these processes are thought to enact behavioral adaption to changing environments, when and where in the brain they are induced has been mysterious because of the challenge of monitoring biochemical state in real time in defined neurons in behaving animals. Here, we describe a method allowing measurement of activity of protein kinase A (PKA), an important intracellular effector for neuromodulators, in freely moving mice. To monitor PKA activity in vivo, we use a genetically targeted sensor (FLIM-AKAR) and fluorescence lifetime photometry (FLiP). This article describes how to set up a FLiP system and obtain robust recordings of net PKA phosphorylation state in vivo. The methods should be generally useful to monitor other pathways for which fluorescence lifetime reporters exist. \copyright 2021 Wiley Periodicals LLC. Basic Protocol 1: Building a FLiP system Basic Protocol 2: FLIM-AKAR viral injection and fiber implantation for FLiP measurement Basic Protocol 3: Performing measurements using FLiP.

The ability to suppress actions that lead to a negative outcome and explore alternative actions is necessary for optimal decision making. Although the basal ganglia have been implicated in these processes1-5, the circuit mechanisms underlying action selection and exploration remain unclear. Here, using a simple lateralized licking task, we show that indirect striatal projection neurons (iSPN) in the basal ganglia contribute to these processes through modulation of the superior colliculus (SC). Optogenetic activation of iSPNs suppresses contraversive licking and promotes ipsiversive licking. Activity in lateral superior colliculus (lSC), a region downstream of the basal ganglia, is necessary for task performance and predicts lick direction. Furthermore, iSPN activation suppresses ipsilateral lSC, but surprisingly excites contralateral lSC, explaining the emergence of ipsiversive licking. Optogenetic inactivation reveals inter-collicular competition whereby each hemisphere of the superior colliculus inhibits the other, thus allowing the indirect pathway to disinhibit the contralateral lSC and trigger licking. Finally, inactivating iSPNs impairs suppression of devalued but previously rewarded licking and reduces exploratory licking. Our results reveal that iSPNs engage the competitive interaction between lSC hemispheres to trigger a motor action and suggest a general circuit mechanism for exploration during action selection.

As the scientific community seeks efficient optical neural interfaces with sub-cortical structures of the mouse brain, a wide set of technologies and methods is being developed to monitor cellular events through fluorescence signals generated by genetically encoded molecules. Among these technologies, tapered optical fibers (TFs) take advantage of the modal properties of narrowing waveguides to enable both depth-resolved and wide-volume light collection from scattering tissue, with minimized invasiveness with respect to standard flat fiber stubs (FFs). However, light guided in patch cords as well as in FFs and TFs can result in autofluorescence (AF) signal, which can act as a source of time-variable noise and limit their application to probe fluorescence lifetime in vivo. In this work, we compare the AF signal of FFs and TFs, highlighting the influence of the cladding composition on AF generation. We show that the autofluorescence signal generated in TFs has a peculiar coupling pattern with guided modes, and that far-field detection can be exploited to separate functional fluorescence from AF. On these bases, we provide evidence that TFs can be employed to implement depth-resolved fluorescence lifetime photometry, potentially enabling the extraction of a new set of information from deep brain regions, as time-correlating single photon counting starts to be applied in freely-moving animals to monitor the intracellular biochemical state of neurons.

G-protein-coupled-receptor (GPCR) signaling is exquisitely controlled to achieve spatial and temporal specificity. The endogenous protein kinase inhibitor peptide (PKI) confines the spatial and temporal spread of the activity of protein kinase A (PKA), which integrates inputs from three major types of GPCRs. Despite its wide usage as a pharmaceutical inhibitor of PKA, it was unclear whether PKI only inhibits PKA activity. Here, the effects of PKI on 55 mouse kinases were tested in in vitro assays. We found that in addition to inhibiting PKA activity, both PKI (6-22) amide and full-length PKIα facilitated the activation of multiple isoforms of protein kinase C (PKC), albeit at much higher concentrations than necessary to inhibit PKA. Thus, our results call for appropriate interpretation of experimental results using PKI as a pharmaceutical agent. Furthermore, our study lays the foundation to explore the potential functions of PKI in regulating PKC activity and in coordinating PKC and PKA activities.

The release of urine, or micturition, serves a fundamental physiological function and, in many species, is critical for social communication. In mice, the pattern of urine release is modulated by external and internal factors and transmitted to the spinal cord via the pontine micturition center (PMC). Here, we exploited a behavioral paradigm in which mice, depending on strain, social experience, and sensory context, either vigorously cover an arena with small urine spots or deposit urine in a few isolated large spots. We refer to these micturition modes as, respectively, high and low territory-covering micturition (TCM) and find that the presence of a urine stimulus robustly induces high TCM in socially isolated mice. Comparison of the brain networks activated by social isolation and by urine stimuli to those upstream of the PMC identified the lateral hypothalamic area as a potential modulator of micturition modes. Indeed, chemogenetic manipulations of the lateral hypothalamus can switch micturition behavior between high and low TCM, overriding the influence of social experience and sensory context. Our results suggest that both inhibitory and excitatory signals arising from a network upstream of the PMC are integrated to determine context- and social-experience-dependent micturition patterns.

Reinforcement learning models postulate that neurons that release dopamine encode information about action and action outcome, and provide a teaching signal to striatal spiny projection neurons in the form of dopamine release(1). Dopamine is thought to guide learning via dynamic and differential modulation of protein kinase A (PKA) in each class of spiny projection neuron(2). However, the real-time relationship between dopamine and PKA in spiny projection neurons remains untested in behaving animals. Here we monitor the activity of dopamine-releasing neurons, extracellular levels of dopamine and net PKA activity in spiny projection neurons in the nucleus accumbens of mice during learning. We find positive and negative modulation of dopamine that evolves across training and is both necessary and sufficient to explain concurrent fluctuations in the PKA activity of spiny projection neurons. Modulations of PKA in spiny projection neurons that express type-1 and type-2 dopamine receptors are dichotomous, such that these neurons are selectively sensitive to increases and decreases, respectively, in dopamine that occur at different phases of learning. Thus, PKA-dependent pathways in each class of spiny projection neuron are asynchronously engaged by positive or negative dopamine signals during learning.

We propose a feedback-assisted direct laser writing method to perform laser ablation of fiber optic devices in which their light-collection signal is used to optimize their properties. A femtosecond-pulsed laser beam is used to ablate a metal coating deposited around a tapered optical fiber, employed to show the suitability of the approach to pattern devices with a small radius of curvature. During processing, the same pulses generate two-photon fluorescence in the surrounding environment and the signal is monitored to identify different patterning regimes over time through spectral analysis. The employed fs beam mostly interacts with the metal coating, leaving almost intact the underlying silica and enabling fluorescence to couple with a specific subset of guided modes, as verified by far-field analysis. Although the method is described here for tapered optical fibers used to obtain efficient light collection in the field of optical neural interfaces, it can be easily extended to other waveguide-based devices and represents a general approach to support the implementation of a closed-loop laser ablation system of fiber optics.

In the basal ganglia (BG), anatomically segregated and topographically organized feedforward circuits are thought to modulate multiple behaviors in parallel. Although topographically arranged BG circuits have been described, the extent to which these relationships are maintained across the BG output nuclei and in downstream targets is unclear. Here, using focal trans-synaptic anterograde tracing, we show that the motor-action-related topographical organization of the striatum is preserved in all BG output nuclei. The topography is also maintained downstream of the BG and in multiple parallel closed loops that provide striatal input. Furthermore, focal activation of two distinct striatal regions induces either licking or turning, consistent with their respective anatomical targets of projection outside of the BG. Our results confirm the parallel model of BG function and suggest that the integration and competition of information relating to different behavior occur largely outside of the BG.

Proper brain function depends on neurovascular coupling: neural activity rapidly increases local blood flow to meet moment-to-moment changes in regional brain energy demand(1). Neurovascular coupling is the basis for functional brain imaging(2), and impaired neurovascular coupling is implicated in neurodegeneration(1). The underlying molecular and cellular mechanisms of neurovascular coupling remain poorly understood. The conventional view is that neurons or astrocytes release vasodilatory factors that act directly on smooth muscle cells (SMCs) to induce arterial dilation and increase local blood flow(1). Here, using two-photon microscopy to image neural activity and vascular dynamics simultaneously in the barrel cortex of awake mice under whisker stimulation, we found that arteriolar endothelial cells (aECs) have an active role in mediating neurovascular coupling. We found that aECs, unlike other vascular segments of endothelial cells in the central nervous system, have abundant caveolae. Acute genetic perturbations that eliminated caveolae in aECs, but not in neighbouring SMCs, impaired neurovascular coupling. Notably, caveolae function in aECs is independent of the endothelial NO synthase (eNOS)-mediated NO pathway. Ablation of both caveolae and eNOS completely abolished neurovascular coupling, whereas the single mutants exhibited partial impairment, revealing that the caveolae-mediated pathway in aECs is a major contributor to neurovascular coupling. Our findings indicate that vasodilation is largely mediated by endothelial cells that actively relay signals from the central nervous system to SMCs via a caveolae-dependent pathway.

The mouse cerebral cortex contains neurons that express choline acetyltransferase (ChAT) and are a potential local source of acetylcholine. However, the neurotransmitters released by cortical ChAT(+) neurons and their synaptic connectivity are unknown. We show that the nearly all cortical ChAT(+) neurons in mice are specialized VIP(+) interneurons that release GABA strongly onto other inhibitory interneurons and acetylcholine sparsely onto layer 1 interneurons and other VIP(+)/ChAT(+) interneurons. This differential transmission of ACh and GABA based on the postsynaptic target neuron is reflected in VIP(+)/ChAT(+) interneuron pre-synaptic terminals, as quantitative molecular analysis shows that only a subset of these are specialized to release acetylcholine. In addition, we identify a separate, sparse population of non-VIP ChAT(+) neurons in the medial prefrontal cortex with a distinct developmental origin that robustly release acetylcholine in layer 1. These results demonstrate both cortex-region heterogeneity in cortical ChAT(+) interneurons and target-specific co-release of acetylcholine and GABA.

The actions of neuromodulation are thought to mediate the ability of the mammalian brain to dynamically adjust its functional state in response to changes in the environment. Altered neurotransmitter (NT) and neuromodulator (NM) signaling is central to the pathogenesis or treatment of many human neurological and psychiatric disorders, including Parkinson's disease, schizophrenia, depression, and addiction. To reveal the precise mechanisms by which these neurochemicals regulate healthy and diseased neural circuitry, one needs to measure their spatiotemporal dynamics in the living brain with great precision. Here, we discuss recent development, optimization, and applications of optical approaches to measure the spatial and temporal profiles of NT and NM release in the brain using genetically encoded sensors for in vivo studies.

Neurons communicate by the activity-dependent release of small-molecule neurotransmitters packaged into synaptic vesicles (SVs). Although many molecules have been identified as neurotransmitters, technical limitations have precluded a full metabolomic analysis of SV content. Here, we present a workflow to rapidly isolate SVs and to interrogate their metabolic contents at high-resolution using mass spectrometry. We validated the enrichment of glutamate in SVs of primary cortical neurons using targeted polar metabolomics. Unbiased and extensive global profiling of SVs isolated from these neurons revealed that the only detectable polar metabolites they contain are the established neurotransmitters glutamate and GABA. In addition, we adapted the approach to enable quick capture of SVs directly from brain tissue and determined the neurotransmitter profiles of diverse brain regions in a cell-type-specific manner. The speed, robustness, and precision of this method to interrogate SV contents will facilitate novel insights into the chemical basis of neurotransmission.

Viral vectors are essential tools for the study of neural circuits, with glycoprotein-deleted rabies viruses being widely used for monosynaptic retrograde tracing to map connectivity between specific cell types in the nervous system. However, the use of rabies virus is limited by the cytotoxicity and the inflammatory responses these viruses trigger. While components of the rabies virus genome contribute to its cytotoxic effects, the function of other neuronal and non-neuronal cells within the vicinity of the infected host neurons in either effecting or mitigating virally-induced tissue damage are still being elucidated. Here, we analyzed 60,212 single-cell RNA profiles to assess both global and cell-type-specific transcriptional responses in the mouse dorsal raphe nucleus (DRN) following intracranial injection of glycoprotein-deleted rabies viruses and axonal infection of dorsal raphe serotonergic neurons. Gene pathway analyses revealed a down-regulation of genes involved in metabolic processes and neurotransmission following infection. We also identified several transcriptionally diverse leukocyte populations that infiltrate the brain and are distinct from resident immune cells. Cell type-specific patterns of cytokine expression showed that antiviral responses were likely orchestrated by Type I and Type II interferon signaling from microglia and infiltrating CD4(+) T cells, respectively. Additionally, we uncovered transcriptionally distinct states of microglia along an activation trajectory that may serve different functions, which range from surveillance to antigen presentation and cytokine secretion. Intercellular interactions inferred from transcriptional data suggest that CD4(+) T cells facilitate microglial state transitions during the inflammatory response. Our study uncovers the heterogeneity of immune cells mediating neuroinflammatory responses and provides a critical evaluation of the compatibility between rabies-mediated connectivity mapping and single-cell transcriptional profiling. These findings provide additional insights into the distinct contributions of various cell types in mediating different facets of antiviral responses in the brain and will facilitate the design of strategies to circumvent immune responses to improve the efficacy of viral gene delivery.

Recent success in identifying gene-regulatory elements in the context of recombinant adeno-associated virus vectors has enabled cell-type-restricted gene expression. However, within the cerebral cortex these tools are largely limited to broad classes of neurons. To overcome this limitation, we developed a strategy that led to the identification of multiple new enhancers to target functionally distinct neuronal subtypes. By investigating the regulatory landscape of the disease gene Scn1a, we discovered enhancers selective for parvalbumin (PV) and vasoactive intestinal peptide-expressing interneurons. Demonstrating the functional utility of these elements, we show that the PV-specific enhancer allowed for the selective targeting and manipulation of these neurons across vertebrate species, including humans. Finally, we demonstrate that our selection method is generalizable and characterizes additional PV-specific enhancers with exquisite specificity within distinct brain regions. Altogether, these viral tools can be used for cell-type-specific circuit manipulation and hold considerable promise for use in therapeutic interventions.