Improved green and red GRAB sensors for monitoring dopaminergic activity in vivo.

Dopamine (DA) plays multiple roles in a wide range of physiological and pathological processes via a large network of dopaminergic projections. To dissect the spatiotemporal dynamics of DA release in both dense and sparsely innervated brain regions, we developed a series of green and red fluorescent G-protein-coupled receptor activation-based DA (GRABDA) sensors using a variety of DA receptor subtypes. These sensors have high sensitivity, selectivity and signal-to-noise ratio with subsecond response kinetics and the ability to detect a wide range of DA concentrations. We then used these sensors in mice to measure both optogenetically evoked and behaviorally relevant DA release while measuring neurochemical signaling in the nucleus accumbens, amygdala and cortex. Using these sensors, we also detected spatially resolved heterogeneous cortical DA release in mice performing various behaviors. These next-generation GRABDA sensors provide a robust set of tools for imaging dopaminergic activity under a variety of physiological and pathological conditions.

MultipleXed Population Selection and Enrichment single nucleus RNA sequencing (XPoSE-seq) enables sample identity retention during transcriptional profiling of rare populations.

Single nucleus RNA-sequencing is critical in deciphering tissue heterogeneity and identifying rare populations. However, current high throughput techniques are not optimized for rare target populations and require tradeoffs in design due to feasibility. We provide a novel snRNA pipeline, MulipleXed Population Selection and Enrichment snRNA-sequencing (XPoSE-seq), to enable targeted snRNA-seq experiments and in-depth transcriptomic characterization of rare target populations while retaining individual sample identity.

SMART: An Open-Source Extension of WholeBrain for Intact Mouse Brain Registration and Segmentation.

Mapping immediate early gene (IEG) expression across intact mouse brains allows for unbiased identification of brain-wide activity patterns underlying complex behaviors. Accurate registration of sample brains to a common anatomic reference is critical for precise assignment of IEG-positive ("active") neurons to known brain regions of interest (ROIs). While existing automated voxel-based registration methods provide a high-throughput solution, they require substantial computing power, can be difficult to implement and fail when brains are damaged or only partially imaged. Additionally, it is challenging to cross-validate these approaches or compare them to any preexisting literature based on serial coronal sectioning. Here, we present the open-source R package SMART (Semi-Manual Alignment to Reference Templates) that extends the WholeBrain R package framework to automated segmentation and semi-automated registration of intact mouse brain light-sheet fluorescence microscopy (LSFM) datasets. The SMART package was created for novice programmers and introduces a streamlined pipeline for aligning, registering, and segmenting LSFM volumetric datasets across the anterior-posterior (AP) axis, using a simple "choice game" and interactive menus. SMART provides the flexibility to register whole brains, partial brains or discrete user-chosen images, and is fully compatible with traditional sectioned coronal slice-based analyses. We demonstrate SMART's core functions using example datasets and provide step-by-step video tutorials for installation and implementation of the package. We also present a modified iDISCO+ tissue clearing procedure for uniform immunohistochemical labeling of the activity marker Fos across intact mouse brains. The SMART pipeline, in conjunction with the modified iDISCO+ Fos procedure, is ideally suited for examination and orthogonal cross-validation of brain-wide neuronal activation datasets.

Incubation of palatable food craving is associated with brain-wide neuronal activation in mice.

Studies using rodent models have shown that relapse to drug or food seeking increases progressively during abstinence, a behavioral phenomenon termed "incubation of craving." Mechanistic studies of incubation of craving have focused on specific neurobiological targets within preselected brain areas. Recent methodological advances in whole-brain immunohistochemistry, clearing, and imaging now allow unbiased brain-wide cellular resolution mapping of regions and circuits engaged during learned behaviors. However, these whole-brain imaging approaches were developed for mouse brains, while incubation of drug craving has primarily been studied in rats, and incubation of food craving has not been demonstrated in mice. Here, we established a mouse model of incubation of palatable food craving and examined food reward seeking after 1, 15, and 60 abstinence days. We then used the neuronal activity marker Fos with intact-brain mapping procedures to identify corresponding patterns of brain-wide activation. Relapse to food seeking was significantly higher after 60 abstinence days than after 1 or 15 days. Using unbiased ClearMap analysis, we identified increased activation of multiple brain regions, particularly corticostriatal structures, following 60 but not 1 or 15 abstinence days. We used orthogonal SMART2 analysis to confirm these findings within corticostriatal and thalamocortical subvolumes and applied expert-guided registration to investigate subdivision and layer-specific activation patterns. Overall, we 1) identified brain-wide activity patterns during incubation of food seeking using complementary analytical approaches and 2) provide a single-cell resolution whole-brain atlas that can be used to identify functional networks and global architecture underlying the incubation of food craving.

Inactivation of the infralimbic cortex decreases discriminative stimulus-controlled relapse to cocaine seeking in rats.

Persistent susceptibility to cue-induced relapse is a cardinal feature of addiction. Discriminative stimuli (DSs) are one type of drug-associated cue that signal drug availability (DS+) or unavailability (DS-) and control drug seeking prior to relapse. We previously established a trial-based procedure in rats to isolate DSs from context, conditioned stimuli, and other drug-associated cues during cocaine self-administration and demonstrated DS-controlled cocaine seeking up to 300 abstinence days. The behavioral and neural mechanisms underlying trial-based DS-control of drug seeking have rarely been investigated. Here we show that following discrimination training in our trial-based procedure, the DS+ and DS- independently control the expression and suppression of cocaine seeking during abstinence. Using microinjections of GABAA + GABAB receptor agonists (muscimol + baclofen) in medial prefrontal cortex, we report that infralimbic, but not prelimbic, subregion of medial prefrontal cortex is critical to persistent DS-controlled relapse to cocaine seeking after prolonged abstinence, but not DS-guided discriminated cocaine seeking or DS-controlled cocaine self-admininstration. Finally, using ex vivo whole-cell recordings from pyramidal neurons in the medial prefrontal cortex, we demonstrate that the disruption of DS-controlled cocaine seeking following infralimbic cortex microinjections of muscimol+baclofen is likely a result of suppression of synaptic transmission in the region via a presynaptic mechanism of action.

Discriminative stimuli are sufficient for incubation of cocaine craving.

In abstinent drug addicts, cues formerly associated with drug-taking experiences gain relapse-inducing potency ('incubate') over time. Animal models of incubation may help develop treatments to prevent relapse, but these models have ubiquitously focused on the role of conditioned stimuli (CSs) signaling drug delivery. Discriminative stimuli (DSs) are unique in that they exert stimulus-control over both drug taking and drug seeking behavior and are difficult to extinguish. For this reason, incubation of the excitatory effects of DSs that signal drug availability, not yet examined in preclinical studies, could be relevant to relapse prevention. We trained rats to self-administer cocaine (or palatable food) under DS control, then investigated DS-controlled incubation of craving, in the absence of drug-paired CSs. DS-controlled cocaine (but not palatable food) seeking incubated over 60 days of abstinence and persisted up to 300 days. Understanding the neural mechanisms of this DS-controlled incubation holds promise for drug relapse treatments.

Trial-based Discrimination Procedure for Studying Drug Relapse in Rats.

In abstinent drug addicts, cues formerly associated with drug-taking experiences gain relapse-inducing potency ('incubate') over time. Animal models of incubation may help in developing treatments for relapse prevention. However, these models have primarily focused on the role of conditioned stimuli (CSs) signaling drug delivery and not on discriminative stimuli (DSs), which signal drug availability and are also known to play a major role in drug relapse. We recently showed that DS-controlled cocaine seeking in rats also incubates during abstinence and persists up to 300 days. We used a trial-based procedure to train male and female rats to discriminate between two light cues: one light cue (DS+) signaled the availability of cocaine reward and the second light cue (DS-) signaled the absence of reward. Rats learned to press a central retractable lever during trials in which the DS+ cue was presented and to suppress responding when the DS- cue was presented. Here, we provide a detailed protocol for the behavioral procedure used in our study. The trial-based design of this behavior lends itself well to time-locked in vivo recording and manipulation approaches that can be used to identify neurobiological mechanisms underlying the contributions of DSs to drug relapse.

Maladaptive Decision Making in Adults with a History of Adolescent Alcohol use, in a Preclinical Model, Is Attributable to the Compromised Assignment of Incentive Value during Stimulus-Reward Learning.

According to recent WHO reports, alcohol remains the number one substance used and abused by adolescents, despite public health efforts to curb its use. Adolescence is a critical period of biological maturation where brain development, particularly the mesocorticolimbic dopamine system, undergoes substantial remodeling. These circuits are implicated in complex decision making, incentive learning and reinforcement during substance use and abuse. An appealing theoretical approach has been to suggest that alcohol alters the normal development of these processes to promote deficits in reinforcement learning and decision making, which together make individuals vulnerable to developing substance use disorders in adulthood. Previously we have used a preclinical model of voluntary alcohol intake in rats to show that use in adolescence promotes risky decision making in adulthood that is mirrored by selective perturbations in dopamine network dynamics. Further, we have demonstrated that incentive learning processes in adulthood are also altered by adolescent alcohol use, again mirrored by changes in cue-evoked dopamine signaling. Indeed, we have proposed that these two processes, risk-based decision making and incentive learning, are fundamentally linked through dysfunction of midbrain circuitry where inputs to the dopamine system are disrupted by adolescent alcohol use. Here, we test the behavioral predictions of this model in rats and present the findings in the context of the prevailing literature with reference to the long-term consequences of early-life substance use on the vulnerability to develop substance use disorders. We utilize an impulsive choice task to assess the selectivity of alcohol's effect on decision-making profiles and conditioned reinforcement to parse out the effect of incentive value attribution, one mechanism of incentive learning. Finally, we use the differential reinforcement of low rates of responding (DRL) task to examine the degree to which behavioral disinhibition may contribute to an overall decision-making profile. The findings presented here support the proposition that early life alcohol use selectively alters risk-based choice behavior through modulation of incentive learning processes, both of which may be inexorably linked through perturbations in mesolimbic circuitry and may serve as fundamental vulnerabilities to the development of substance use disorders.

Platelet-rich fibrin matrix in the management of arthroscopic repair of the rotator cuff: a prospective, randomized, double-blinded study.

BACKGROUND: Arthroscopic rotator cuff repair has a high rate of patient satisfaction. However, multiple studies have shown significant rates of anatomic failure. Biological augmentation would seem to be a reasonable technique to improve clinical outcomes and healing rates. PURPOSE: To represent a prospective, double-blinded, randomized study to assess the use of platelet-rich fibrin matrix (PRFM) in rotator cuff surgery. STUDY DESIGN: Randomized controlled trial; level of evidence, 1. METHODS: Prestudy power analysis demonstrated that a sample size of 30 patients in each group (PRFM vs control) would allow recognition of a 20% difference in perioperative pain scores. Sixty consecutive patients were randomized to either receive a commercially available PRFM product or not. Preoperative and postoperative range of motion (ROM), University of California-Los Angeles (UCLA), and simple shoulder test (SST) scores were recorded. Surgery was performed using an arthroscopic single-row technique. Visual analog scale (VAS) pain scores were obtained upon arrival to the recovery room and 1 hour postoperatively, and narcotic consumption was recorded and converted to standard narcotic equivalents. The SST and ROM measurements were taken at 3, 6, 9, and 12 weeks postoperatively, and final (1 year) American shoulder and elbow surgeons (ASES) shoulder and UCLA shoulder scores were assessed. RESULTS: There were no complications. Randomization created comparable groups except that the PRFM group was younger than the control group (mean ± SD, 59.67 ± 8.16 y vs 64.50 ± 8.59 y, respectively; P < .05). Mean surgery time was longer for the PRFM group than for the control group (83.28 ± 17.13 min vs 73.28 ± 17.18 min, respectively; P < .02). There was no significant difference in VAS scores or narcotic use between groups and no statistically significant differences in recovery of motion, SST, or ASES scores. Mean ASES scores were 82.48 ± 8.77 (PRFM group) and 82.52 ± 12.45 (controls) (F(1,56) = 0.00, P > .98). Mean UCLA shoulder scores were 27.94 ± 4.98 for the PRFM group versus 29.59 ± 1.68 for the controls (P < .046). Structural results correlated with age and size of the tear and did not differ between the groups. CONCLUSION: Platelet-rich fibrin matrix was not shown to significantly improve perioperative morbidity, clinical outcomes, or structural integrity. While longer term follow-up or different platelet-rich plasma formulations may show differences, early follow-up does not show significant improvement in perioperative morbidity, structural integrity, or clinical outcome.