SCAMPR, a single-cell automated multiplex pipeline for RNA quantification and spatial mapping.

Spatial gene expression, achieved classically through in situ hybridization, is a fundamental tool for topographic phenotyping of cell types in the nervous system. Newly developed techniques allow for visualization of multiple mRNAs at single-cell resolution and greatly expand the ability to link gene expression to tissue topography, yet there are challenges in efficient quantification and analysis of these high-dimensional datasets. We have therefore developed the single-cell automated multiplex pipeline for RNA (SCAMPR), facilitating rapid and accurate segmentation of neuronal cell bodies using a dual immunohistochemistry-RNAscope protocol and quantification of low- and high-abundance mRNA signals using open-source image processing and automated segmentation tools. Proof of principle using SCAMPR focused on spatial mapping of gene expression by peripheral (vagal nodose) and central (visual cortex) neurons. The analytical effectiveness of SCAMPR is demonstrated by identifying the impact of early life stress on gene expression in vagal neuron subtypes.

MET Receptor Tyrosine Kinase Regulates Lifespan Ultrasonic Vocalization and Vagal Motor Neuron Development.

The intrinsic muscles of the larynx are innervated by the vagal motor nucleus ambiguus (nAmb), which provides direct motor control over vocal production in humans and rodents. Here, we demonstrate in mice using the Phox2b Cre line, that conditional embryonic deletion of the gene encoding the MET receptor tyrosine kinase (MET) in the developing brainstem (cKO) results in highly penetrant, severe deficits in ultrasonic vocalization in early postnatal life. Major deficits and abnormal vocalization patterns persist into adulthood in more than 70% of mice, with the remaining recovering the ability to vocalize, reflecting heterogeneity in circuit restitution. We show that underlying the functional deficits, conditional deletion of Met results in a loss of approximately one-third of MET+ nAmb motor neurons, which begins as early as embryonic day 14.5. The loss of motor neurons is specific to the nAmb, as other brainstem motor and sensory nuclei are unaffected. In the recurrent laryngeal nerve, through which nAmb motor neurons project to innervate the larynx, there is a one-third loss of axons in cKO mice. Together, the data reveal a novel, heterogenous MET-dependence, for which MET differentially affects survival of a subset of nAmb motor neurons necessary for lifespan ultrasonic vocal capacity.

A role for reward valuation in the serotonergic modulation of impulsivity.

RATIONALE: Impulsive behavior is a deleterious component of a number of mental health disorders but has few targeted pharmacotherapies. One contributing factor to the difficulty in understanding the neural substrates of disordered impulsivity is the diverse presentations of impulsive behavior. Defining the behavioral and cognitive processes which contribute to different subtypes of impulsivity is important for understanding the neural underpinnings of dysregulated impulsive behavior. METHODS: Using a mouse model for disordered impulsivity, our goal was to identify behavioral and cognitive processes that are associated with increased impulsivity. Specifically, we were interested in the facets of impulsivity modulated by serotonin signaling. We used mice lacking the serotonin 1B receptor (5-HT1BR) and measured different types of impulsivity as well as goal-directed responding, extinction, habitual-like behavior, cue reactivity, and reward reactivity. RESULTS: Mice lacking expression of 5-HT1BR had increased levels of impulsive action, goal-directed responding, and motivation, with no differences seen in rate of extinction, development of habitual behavior, delay discounting, or effort-based discounting. Interestingly, mice lacking 5-HT1BR expression also showed an overall increase in the choice of higher value rewards, increased hedonic responses to sweet rewards, and responded more for cues that predict reward. We developed a novel paradigm to demonstrate that increasing anticipated reward value could directly increase impulsive action. Furthermore, we found that 5-HT1BR KO-induced impulsivity could be ameliorated by decreasing the reward value relative to controls, suggesting that the increased 5-HT1BR-associated impulsive action may be a result of increased reward valuation. CONCLUSIONS: Taken together, these data show that the effects of serotonin on impulsive action are mediated through the modulation of hedonic value, which may alter the reward representations that motivate action. Overall, this data supports a role for reward value as an important substrate in impulsive action which may drive clinically relevant increases in impulsivity.

The Behavioral Effects of the Antidepressant Tianeptine Require the Mu-Opioid Receptor.

Depression is a debilitating chronic illness that affects around 350 million people worldwide. Current treatments, such as selective serotonin reuptake inhibitors, are not ideal because only a fraction of patients achieve remission. Tianeptine is an effective antidepressant with a previously unknown mechanism of action. We recently reported that tianeptine is a full agonist at the mu opioid receptor (MOR). Here we demonstrate that the acute and chronic antidepressant-like behavioral effects of tianeptine in mice require MOR. Interestingly, while tianeptine also produces many opiate-like behavioral effects such as analgesia and reward, it does not lead to tolerance or withdrawal. Furthermore, the primary metabolite of tianeptine (MC5), which has a longer half-life, mimics the behavioral effects of tianeptine in a MOR-dependent fashion. These results point to the possibility that MOR and its downstream signaling cascades may be novel targets for antidepressant drug development.

Genetic and Modeling Approaches Reveal Distinct Components of Impulsive Behavior.

Impulsivity is an endophenotype found in many psychiatric disorders including substance use disorders, pathological gambling, and attention deficit hyperactivity disorder. Two behavioral features often considered in impulsive behavior are behavioral inhibition (impulsive action) and delayed gratification (impulsive choice). However, the extent to which these behavioral constructs represent distinct facets of behavior with discrete biological bases is unclear. To test the hypothesis that impulsive action and impulsive choice represent statistically independent behavioral constructs in mice, we collected behavioral measures of impulsivity in a single cohort of mice using well-validated operant behavioral paradigms. Mice with manipulation of serotonin 1B receptor (5-HT1BR) expression were included as a model of disordered impulsivity. A factor analysis was used to characterize correlations between the measures of impulsivity and to identify covariates. Using two approaches, we dissociated impulsive action from impulsive choice. First, the absence of 5-HT1BRs caused increased impulsive action, but not impulsive choice. Second, based on an exploratory factor analysis, a two-factor model described the data well, with measures of impulsive action and choice separating into two independent factors. A multiple-indicator multiple-causes analysis showed that 5-HT1BR expression and sex were significant covariates of impulsivity. Males displayed increased impulsivity in both dimensions, whereas 5-HT1BR expression was a predictor of increased impulsive action only. These data support the conclusion that impulsive action and impulsive choice are distinct behavioral phenotypes with dissociable biological influences that can be modeled in mice. Our work may help inform better classification, diagnosis, and treatment of psychiatric disorders, which present with disordered impulsivity.