Corticostriatal ensemble dynamics across heroin self-administration to reinstatement.

Corticostriatal projection neurons from prelimbic medial prefrontal cortex to the nucleus accumbens core critically regulate drug-seeking behaviors, yet the underlying encoding dynamics whereby these neurons contribute to drug seeking remain elusive. Here we use two-photon calcium imaging to visualize the activity of corticostriatal neurons in mice from the onset of heroin use to relapse. We find that the activity of these neurons is highly heterogeneous during heroin self-administration and seeking, with at least 8 distinct neuronal ensembles that display both excitatory and inhibitory encoding dynamics. These neuronal ensembles are particularly apparent during relapse, where excitatory responses are amplified compared to heroin self-administration. Moreover, we find that optogenetic inhibition of corticostriatal projection neurons attenuates heroin seeking regardless of the relapse trigger. Our results reveal the precise corticostriatal activity dynamics underlying drug-seeking behaviors and support a key role for this circuit in mediating relapse to drug seeking.

Restoration of a paraventricular thalamo-accumbal behavioral suppression circuit prevents reinstatement of heroin seeking.

Lack of behavioral suppression typifies substance use disorders, yet the neural circuit underpinnings of drug-induced behavioral disinhibition remain unclear. Here, we employ deep-brain two-photon calcium imaging in heroin self-administering mice, longitudinally tracking adaptations within a paraventricular thalamus to nucleus accumbens behavioral inhibition circuit from the onset of heroin use to reinstatement. We find that select thalamo-accumbal neuronal ensembles become profoundly hypoactive across the development of heroin seeking and use. Electrophysiological experiments further reveal persistent adaptations at thalamo-accumbal parvalbumin interneuronal synapses, whereas functional rescue of these synapses prevents multiple triggers from initiating reinstatement of heroin seeking. Finally, we find an enrichment of µ-opioid receptors in output- and cell-type-specific paraventricular thalamic neurons, which provide a mechanism for heroin-induced synaptic plasticity and behavioral disinhibition. These findings reveal key circuit adaptations that underlie behavioral disinhibition in opioid dependence and further suggest that recovery of this system would reduce relapse susceptibility.

A neuropathologic feature of brain aging: multi-lumen vascular profiles.

Cerebrovascular pathologies other than frank infarctions are commonly seen in aged brains. Here, we focus on multi-lumen vascular profiles (MVPs), which are characterized by multiple vessel lumens enclosed in a single vascular channel. Little information exists on the prevalence, risk factors, and co-pathologies of MVPs. Therefore, we used samples and data from the University of Kentucky Alzheimer's Disease Research Center (n = 91), the University of Kentucky Pathology Department (n = 31), and the University of Pittsburgh Pathology Department (n = 4) to study MVPs. Age at death was correlated with MVP density in the frontal neocortex, Brodmann Area 9 (r = 0.51; p < 0.0001). Exploratory analyses were performed to evaluate the association between conventional vascular risk factors (e.g., hypertension, diabetes), cardiovascular diseases (e.g., heart attack, arrhythmia), and cerebrovascular disease (e.g., stroke); the only nominal association with MVP density was a self-reported history of brain trauma (Prevalence Ratio = 2.1; 95 CI 1.1-3.9, before correcting for multiple comparisons). No specific associations were detected between neuropathological (e.g., brain arteriolosclerosis) or genetic (e.g., APOE) variables and MVP density. Using a tissue clearing method called SeeDB, we provide 3-dimensional images of MVPs in brain tissue. We conclude that MVPs are an age-related brain pathology and more work is required to identify their clinical-pathological correlation and associated risk factors.

An opioid-gated thalamoaccumbal circuit for the suppression of reward seeking in mice.

Suppression of dangerous or inappropriate reward-motivated behaviors is critical for survival, whereas therapeutic or recreational opioid use can unleash detrimental behavioral actions and addiction. Nevertheless, the neuronal systems that suppress maladaptive motivated behaviors remain unclear, and whether opioids disengage those systems is unknown. In a mouse model using two-photon calcium imaging in vivo, we identify paraventricular thalamostriatal neuronal ensembles that are inhibited upon sucrose self-administration and seeking, yet these neurons are tonically active when behavior is suppressed by a fear-provoking predator odor, a pharmacological stressor, or inhibitory learning. Electrophysiological, optogenetic, and chemogenetic experiments reveal that thalamostriatal neurons innervate accumbal parvalbumin interneurons through synapses enriched with calcium permeable AMPA receptors, and activity within this circuit is necessary and sufficient for the suppression of sucrose seeking regardless of the behavioral suppressor administered. Furthermore, systemic or intra-accumbal opioid injections rapidly dysregulate thalamostriatal ensemble dynamics, weaken thalamostriatal synaptic innervation of downstream neurons, and unleash reward-seeking behaviors in a manner that is reversed by genetic deletion of thalamic µ-opioid receptors. Overall, our findings reveal a thalamostriatal to parvalbumin interneuron circuit that is both required for the suppression of reward seeking and rapidly disengaged by opioids.

A Novel Assay Allowing Drug Self-Administration, Extinction, and Reinstatement Testing in Head-Restrained Mice.

Multiphoton microscopy is one of several new technologies providing unprecedented insight into the activity dynamics and function of neural circuits. Unfortunately, some of these technologies require experimentation in head-restrained animals, limiting the behavioral repertoire that can be integrated and studied. This issue is especially evident in drug addiction research, as no laboratories have coupled multiphoton microscopy with simultaneous intravenous drug self-administration, a behavioral paradigm that has predictive validity for treatment outcomes and abuse liability. Here, we describe a new experimental assay wherein head-restrained mice will press an active lever, but not inactive lever, for intravenous delivery of heroin or cocaine. Similar to freely moving animals, we find that lever pressing is suppressed through daily extinction training and subsequently reinstated through the presentation of relapse-provoking triggers (drug-associative cues, the drug itself, and stressors). Finally, we show that head-restrained mice will show similar patterns of behavior for oral delivery of a sucrose reward, a common control used for drug self-administration experiments. Overall, these data demonstrate the feasibility of combining drug self-administration experiments with technologies that require head-restraint, such as multiphoton imaging. The assay described could be replicated by interested labs with readily available materials to aid in identifying the neural underpinnings of substance use disorder.

Specialized coding patterns among dorsomedial prefrontal neuronal ensembles predict conditioned reward seeking.

Non-overlapping cell populations within dorsomedial prefrontal cortex (dmPFC), defined by gene expression or projection target, control dissociable aspects of reward seeking through unique activity patterns. However, even within these defined cell populations, considerable cell-to-cell variability is found, suggesting that greater resolution is needed to understand information processing in dmPFC. Here, we use two-photon calcium imaging in awake, behaving mice to monitor the activity of dmPFC excitatory neurons throughout Pavlovian reward conditioning. We characterize five unique neuronal ensembles that each encodes specialized information related to a sucrose reward, reward-predictive cues, and behavioral responses to those cues. The ensembles differentially emerge across daily training sessions - and stabilize after learning - in a manner that improves the predictive validity of dmPFC activity dynamics for deciphering variables related to behavioral conditioning. Our results characterize the complex dmPFC neuronal ensemble dynamics that stably predict reward availability and initiation of conditioned reward seeking following cue-reward learning.

Brain capillary pericytes exert a substantial but slow influence on blood flow.

The majority of the brain's vasculature is composed of intricate capillary networks lined by capillary pericytes. However, it remains unclear whether capillary pericytes influence blood flow. Using two-photon microscopy to observe and manipulate brain capillary pericytes in vivo, we find that their optogenetic stimulation decreases lumen diameter and blood flow, but with slower kinetics than similar stimulation of mural cells on upstream pial and precapillary arterioles. This slow vasoconstriction was inhibited by the clinically used vasodilator fasudil, a Rho-kinase inhibitor that blocks contractile machinery. Capillary pericytes were also slower to constrict back to baseline following hypercapnia-induced dilation, and slower to dilate towards baseline following optogenetically induced vasoconstriction. Optical ablation of single capillary pericytes led to sustained local dilation and a doubling of blood cell flux selectively in capillaries lacking pericyte contact. These data indicate that capillary pericytes contribute to basal blood flow resistance and slow modulation of blood flow throughout the brain.

Organizational hierarchy and structural diversity of microvascular pericytes in adult mouse cortex.

Smooth muscle cells and pericytes, together called mural cells, coordinate many distinct vascular functions. Canonically, smooth muscle cells are ring-shaped and cover arterioles with circumferential processes, whereas pericytes extend thin processes that run longitudinally along capillaries. In between these canonical mural cell types are cells with features of both smooth muscle cells and pericytes. Recent studies suggest that these transitional cells are critical for controlling blood flow to the capillary bed during health and disease, but there remains confusion on how to identify them and where they are located in the brain microvasculature. To address this issue, we measured the morphology, vascular territory, and α-smooth muscle actin content of structurally diverse mural cells in adult mouse cortex. We first imaged intact 3D vascular networks to establish the locations of major gradations in mural cell appearance as arterioles branched into capillaries. We then imaged individual mural cells occupying the regions within these gradations. This revealed two transitional cells that were often similar in appearance, but with sharply contrasting levels of α-smooth muscle actin. Our findings highlight the diversity of mural cell morphologies in brain microvasculature, and provide guidance for identification and categorization of mural cell types.

Dynamic Remodeling of Pericytes In Vivo Maintains Capillary Coverage in the Adult Mouse Brain.

Direct contact and communication between pericytes and endothelial cells is critical for maintenance of cerebrovascular stability and blood-brain barrier function. Capillary pericytes have thin processes that reach hundreds of micrometers along the capillary bed. The processes of adjacent pericytes come in close proximity but do not overlap, yielding a cellular chain with discrete territories occupied by individual pericytes. Little is known about whether this pericyte chain is structurally dynamic in the adult brain. Using in vivo two-photon imaging in adult mouse cortex, we show that while pericyte somata were immobile, the tips of their processes underwent extensions and/or retractions over days. The selective ablation of single pericytes provoked exuberant extension of processes from neighboring pericytes to contact uncovered regions of the endothelium. Uncovered capillary regions had normal barrier function but were dilated until pericyte contact was regained. Pericyte structural plasticity may be critical for cerebrovascular health and warrants detailed investigation.

Pericytes as Inducers of Rapid, Matrix Metalloproteinase-9-Dependent Capillary Damage during Ischemia.

Blood-brain barrier disruption (BBB) and release of toxic blood molecules into the brain contributes to neuronal injury during stroke and other cerebrovascular diseases. While pericytes are builders and custodians of the BBB in the normal brain, their impact on BBB integrity during ischemia remains unclear. We imaged pericyte-labeled transgenic mice with in vivo two-photon microscopy to examine the relationship between pericytes and blood plasma leakage during photothrombotic occlusion of cortical capillaries. Upon cessation of capillary flow, we observed that plasma leakage occurred with three times greater frequency in regions where pericyte somata adjoined the endothelium. Pericyte somata covered only 7% of the total capillary length in cortex, indicating that a disproportionate amount of leakage occurred from a small fraction of the capillary bed. Plasma leakage was preceded by rapid activation of matrix metalloproteinase (MMP) at pericyte somata, which was visualized at high resolution in vivo using a fluorescent probe for matrix metalloproteinase-2/9 activity, fluorescein isothiocyanate (FITC)-gelatin. Coinjection of an MMP-9 inhibitor, but not an MMP-2 inhibitor, reduced pericyte-associated FITC-gelatin fluorescence and plasma leakage. These results suggest that pericytes contribute to rapid and localized proteolytic degradation of the BBB during cerebral ischemia. SIGNIFICANCE STATEMENT: Pericytes are a key component of the neurovascular unit and are essential for normal BBB function. However, during acute ischemia, we find that pericytes are involved in creating rapid and heterogeneous BBB disruption in the capillary bed. The mechanism by which pericytes contribute to BBB damage warrants further investigation, as it may yield new therapeutic targets for acute stroke injury and other neurological diseases involving capillary flow impairment.

Pericyte structure and distribution in the cerebral cortex revealed by high-resolution imaging of transgenic mice.

Pericytes are essential for normal brain function, but many aspects of their physiology remain enigmatic due to a lack of tools to genetically target this cell population. Here, we characterize brain pericytes using two existing Cre-recombinase driver mouse lines that can serve distinct purposes in cerebrovascular research. One line expresses an inducible version of Cre under the NG2 proteoglycan promoter, which provides the sparse labeling necessary to define the morphology of single cells. These mice reveal structural differences between pericytes adjacent to arterioles versus those broadly distributed in the capillary bed that may underlie differential roles in control of vessel caliber. A second line expresses Cre constitutively under the platelet-derived growth factor receptor ß promoter and provides continuous, highly specific and near-complete labeling of pericytes and myocytes along the entire cerebrovasculature. This line provides a three-dimensional view of pericyte distribution along the cortical angioarchitecture following optical clearing of brain tissue. In combination with recent reporter lines for expression of optogenetic actuators and activity-sensitive probes, these mice may be key tools for studying pericyte biology in the intact brain.