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Optogenetics and calcium imaging can be combined to simultaneously stimulate and record neural activity in vivo. However, this usually requires two-photon microscopes, which are not portable nor affordable. Here we report the design and implementation of a miniaturized one-photon endoscope for performing simultaneous optogenetic stimulation and calcium imaging. By integrating digital micromirrors, the endoscope makes it possible to activate any neuron of choice within the field of view, and to apply arbitrary spatiotemporal patterns of photostimulation while imaging calcium activity. We used the endoscope to image striatal neurons from either the direct pathway or the indirect pathway in freely moving mice while activating any chosen neuron in the field of view. The endoscope also allows for the selection of neurons based on their relationship with specific animal behaviour, and to recreate the behaviour by mimicking the natural neural activity with photostimulation. The miniaturized endoscope may facilitate the study of how neural activity gives rise to behaviour in freely moving animals.

Animals can learn to repeat behaviors to earn desired rewards, a process commonly known as reinforcement learning. While previous work has implicated the ascending dopaminergic projections to the basal ganglia in reinforcement learning, little is known about the role of the hippocampus. Here we report that a specific population of hippocampal neurons and their dopaminergic innervation contribute to operant self-stimulation. These neurons are located in the dentate gyrus, receive dopaminergic projections from the locus coeruleus, and express D1 dopamine receptors. Activation of D1+ dentate neurons is sufficient for self-stimulation: mice will press a lever to earn optogenetic activation of these neurons. A similar effect is also observed with selective activation of the locus coeruleus projections to the dentate gyrus, and blocked by D1 receptor antagonism. Calcium imaging of D1+ dentate neurons revealed significant activity at the time of action selection, but not during passive reward delivery. These results reveal the role of dopaminergic innervation of the hippocampus in supporting operant reinforcement.

Gene rv3722c of Mycobacterium tuberculosis is essential for in vitro growth, and encodes a putative pyridoxal phosphate-binding protein of unknown function. Here we use metabolomic, genetic and structural approaches to show that Rv3722c is the primary aspartate aminotransferase of M. tuberculosis, and mediates an essential but underrecognized role in metabolism: nitrogen distribution. Rv3722c deficiency leads to virulence attenuation in macrophages and mice. Our results identify aspartate biosynthesis and nitrogen distribution as potential species-selective drug targets in M. tuberculosis., Gene rv3722c is essential for in vitro growth of Mycobacterium tuberculosis, but its function is unclear. Here, Jansen et al. show that Rv3722c is the primary aspartate aminotransferase of this pathogen, mediates nitrogen distribution, and is important for virulence during infection of macrophages and mice.

While in vivo calcium imaging makes it possible to record activity in defined neuronal populations with cellular resolution, optogenetics allows selective manipulation of neural activity. Recently, these two tools have been combined to stimulate and record neural activity at the same time, but current approaches often rely on two-photon microscopes that are difficult to use in freely moving animals, or one-photon fiberscopes with benchtop-based digital micromirror devices that limit system portability. To address these limitations, we have developed a new integrated system combining a one-photon endoscope and a digital micromirror device for simultaneous calcium imaging and precise optogenetic photo-stimulation (Miniscope with All-optical Patterned Stimulation and Imaging, MAPSI). Using this system, we were able to successfully image striatal neurons from either the direct pathway or the indirect pathway while simultaneously activating any neuron of choice within the field of view or synthesizing arbitrary spatio-temporal patterns of photo-stimulation in freely moving mice. We could also select neurons based on their relationship with behavior and recreate the behavior by mimicking the natural neural activity with photo-stimulation. MAPSI thus provides a powerful tool for in vivo interrogation of neural circuit function.

Most adaptive behaviors require precise tracking of targets in space. In pursuit behavior with a moving target, mice use distance to target to guide their own movement continuously. Here, we show that in the sensorimotor striatum, parvalbumin-positive fast-spiking interneurons (FSIs) can represent the distance between self and target during pursuit behavior, while striatal projection neurons (SPNs), which receive FSI projections, can represent self-velocity. FSIs are shown to regulate velocity-related SPN activity during pursuit, so that movement velocity is continuously modulated by distance to target. Moreover, bidirectional manipulation of FSI activity can selectively disrupt performance by increasing or decreasing the self-target distance. Our results reveal a key role of the FSI-SPN interneuron circuit in pursuit behavior and elucidate how this circuit implements distance to velocity transformation required for the critical underlying computation., Many natural behaviours involve tracking of a target in space. Here, the authors describe a task to assess this behaviour in mice and use in vivo electrophysiology, calcium imaging, optogenetics, and chemogenetics to investigate the role of the striatum in target pursuit.

In recent years, there have been concerns about research practices in basic and preclinical biomedical research. There have been problems with non-replicable results, and experimental designs lacking internal validity or external or translational validity. The Experimental Design Accelerator (XDA) is Internet-based, interactive software designed to help those trying to design, conduct and document rigorous, replicable and relevant experiments. It leads the investigator step-by-step through a series of decisions that will define the experimental design. It provides background regarding the significance of each decision and the advantages and disadvantages of each possible choice. For example, it leads the researcher to address issues such as choosing a research model, developing testable hypotheses, identifying extraneous variables, dealing with random and systematic error, picking appropriate sample size and picking appropriate statistical analyses. There are also sections to help conduct the experiment consistent with its design and to document the study to facilitate accurate replication. Helpful features include access to an online statistics book and provisions for rapid contact with consulting experts. A number of potential uses for such novel interactive software tools will be discussed.

Choice behavior is characterized by temporal discounting, i.e., preference for immediate rewards given a choice between immediate and delayed rewards. Agouti-related peptide (AgRP)-expressing neurons located in the arcuate nucleus of the hypothalamus (ARC) regulate food intake and energy homeostasis, yet whether AgRP neurons influence choice behavior and temporal discounting is unknown. Here, we demonstrate that motivational state potently modulates temporal discounting. Hungry mice (both male and female) strongly preferred immediate food rewards, yet sated mice were largely indifferent to reward delay. More importantly, selective optogenetic activation of AgRP-expressing neurons or their axon terminals within the posterior bed nucleus of stria terminalis (BNST) produced temporal discounting in sated mice. Furthermore, activation of neuropeptide Y (NPY) type 1 receptors (Y1Rs) within the BNST is sufficient to produce temporal discounting. These results demonstrate a profound influence of hypothalamic signaling on temporal discounting for food rewards and reveal a novel circuit that determine choice behavior.SIGNIFICANCE STATEMENTTemporal discounting is a universal phenomenon found in many species, yet the underlying neurocircuit mechanisms are still poorly understood. Our results revealed a novel neural pathway from agouti-related peptide (AgRP) neurons in the hypothalamus to the bed nucleus of stria terminalis (BNST) that regulates temporal discounting in decision-making.

Selective activation of Pf-STN pathway initiates actions in normal mice and restores movement in a mouse model of PD., The parafascicular nucleus (Pf) of the thalamus provides major projections to the basal ganglia, a set of subcortical nuclei involved in action initiation. Here, we show that Pf projections to the subthalamic nucleus (STN), but not to the striatum, are responsible for movement initiation. Because the STN is a major target of deep brain stimulation treatments for Parkinson’s disease, we tested the effect of selective stimulation of Pf-STN projections in a mouse model of PD. Bilateral dopamine depletion with 6-OHDA created complete akinesia in mice, but Pf-STN stimulation immediately and markedly restored a variety of natural behaviors. Our results therefore revealed a functionally novel neural pathway for the initiation of movements that can be recruited to rescue movement deficits after dopamine depletion. They not only shed light on the clinical efficacy of conventional STN DBS but also suggest more selective and improved stimulation strategies for the treatment of parkinsonian symptoms.

Summary Many studies in systems neuroscience use head-fixation preparations for in vivo experimentation. While head-fixation confers several advantages, one major limitation is the lack of behavioral measures that quantify whole-body movements. Here, we detail a step-by-step protocol for using a novel head-fixation device that measures the forces exerted by head-fixed mice in multiple dimensions. We further detail how this system can be used in conjunction with in vivo electrophysiology and optogenetics to study dopamine neurons in the ventral tegmental area. For complete details on the use and execution of this protocol, please refer to Hughes et al. (2020a, 2020b), Graphical Abstract, Highlights • Protocol for using a novel head-fixation device to measure head and body forces in mice • Protocol allows recording from optogenetically tagged dopamine neurons • Device can identify start and end of movements for correlation with neuron activity, Many studies in systems neuroscience use head-fixation preparations for in vivo experimentation. While head fixation confers several advantages, one major limitation is the lack of behavioral measures that quantify whole-body movements. Here, we detail a step-by-step protocol for using a novel head-fixation device that measures the forces exerted by head-fixed mice in multiple dimensions. We further detail how this system can be used in conjunction with in vivo electrophysiology and optogenetics to study dopamine neurons in the ventral tegmental area.

Organisms are defined by their genomes, yet many distinguishing features of a given organism are encoded by genes that are functionally unannotated.Mycobacterium tuberculosis(Mtb), the leading cause of death due to a single microbe, co-evolved with humans as its only known natural reservoir, yet the factors mediatingMtb’spathogenicity remain incompletely defined.rv3722cis a gene of unknown function predicted to encode a pyridoxal phosphate binding protein and to be essential forin vitrogrowth ofMtb. Using metabolomic, genetic and structural approaches, we show that Rv3722c is the primary aspartate aminotransferase ofMtband mediates an essential but underrecognized role in metabolism: nitrogen distribution. Together with the attenuation of Rv3722c-deficientMtbin macrophages and mice, these results identify aspartate biosynthesis and nitrogen distribution as potential species-selective drug targets inMtb.

Summary The ventral tegmental area (VTA) is a midbrain region implicated in a variety of motivated behaviors. However, the function of VTA GABAergic (Vgat+) neurons remains poorly understood. Here, using three-dimensional motion capture, in vivo electrophysiology, calcium imaging, and optogenetics, we demonstrate a novel function of VTAVgat+ neurons. We found three distinct populations of neurons, each representing head angle about a principal axis of rotation: yaw, roll, and pitch. For each axis, opponent cell groups were found that increase firing when the head moves in one direction and decrease firing in the opposite direction. Selective excitation and inhibition of VTAVgat+ neurons generate opposite rotational movements. Thus, VTAVgat+ neurons serve a critical role in the control of rotational kinematics while pursuing a moving target. This general-purpose steering function can guide animals toward desired spatial targets in any motivated behavior.

SummaryThe Ventral Tegmental Area (VTA) is a midbrain region implicated in a variety of motivated behaviors. However, the function of VTA GABAergic (Vgat+) neurons remains poorly understood. Here, using 3D motion capture, in vivo electrophysiology and calcium imaging, and optogenetics, we demonstrate a novel function of VTAVgat+ neurons. We found three distinct populations of neurons, each representing head angle about a principal axis of rotation: pitch, roll, and yaw. For each axis, opponent cell groups were found that increase firing when the head moves in one direction, and decrease firing in the opposite direction. Selective excitation and inhibition of VTAVgat+ neurons generate opposite rotational movements. The relationship between these neurons and head angle is degraded only at the time of reward consumption, at which point all head-angle related neuronal subpopulations show indistinguishable reward-related responses. Thus, VTAVgat+ neurons serve a critical role in the control of rotational kinematics while pursuing a moving target. This general-purpose steering function can guide animals toward desired spatial targets in any motivated behavior.

The SecA2 protein export system is critical for the virulence of Mycobacterium tuberculosis. However, the mechanism of this export pathway remains unclear. Through a screen for suppressors of a secA2 mutant, we identified a new player in the mycobacterial SecA2 pathway that we named SatS for SecA2 (two) Suppressor. In M. tuberculosis, SatS is required for the export of a subset of SecA2 substrates and for growth in macrophages. We further identify a role for SatS as a protein export chaperone. SatS exhibits multiple properties of a chaperone, including the ability to bind to and protect substrates from aggregation. Our structural studies of SatS reveal a distinct combination of a new fold and hydrophobic grooves resembling preprotein-binding sites of the SecB chaperone. These results are significant in better defining a molecular pathway for M. tuberculosis pathogenesis and in expanding our appreciation of the diversity among chaperones and protein export systems.

The parafascicular nucleus (Pf) of the thalamus sends major projections to the basal ganglia, a group of subcortical nuclei involved in action initiation and selection. Here, we used optogenetics, 3D motion capture, in vivo electrophysiology, and viral-based neuroanatomical tracing to examine the contribution of the Pf to volitional movements in mice. We discovered that Pf neurons are highly correlated with movement velocity. Stimulation of glutamatergic (Vglut2+) neurons in the Pf generates turning and orienting movements. This effect was not due to Pf projections to the striatum, but to its projections to the subthalamic nucleus (STN), a major target of deep brain stimulation (DBS) for Parkinson9s disease (PD) patients. Moreover, selective excitation of either Pf Vglut2+ terminals in the STN or Pf cell bodies that send glutamatergic projections to the STN can restore natural movements in a bilateral mouse model of PD with complete akinesia. Our results reveal a novel thalamo-subthalamic pathway regulating movement initiation, and demonstrate a circuit mechanism that could explain the clinical efficacy of DBS for relief of PD motor symptoms.