Cell specific single viral vector CRISPR/Cas9 editing and genetically encoded tool delivery in the central and peripheral nervous systems.

Gene manipulation strategies using germline knockout, conditional knockout, and more recently CRISPR/Cas9 are crucial tools for advancing our understanding of the nervous system. However, traditional gene knockout approaches can be costly and time consuming, may lack cell-type specificity, and can induce germline recombination. Viral gene editing presents and an exciting alternative to more rapidly study genes of unknown function; however, current strategies to also manipulate or visualize edited cells are challenging due to the large size of Cas9 proteins and the limited packaging capacity of adeno-associated viruses (AAVs). To overcome these constraints, we have developed an alternative gene editing strategy using a single AAV vector and mouse lines that express Cre-dependent Cas9 to achieve efficient cell-type specific editing across the nervous system. Expressing Cre-dependent Cas9 in specific cell types in transgenic mouse lines affords more space to package guide RNAs for gene editing together with Cre-dependent, genetically encoded tools to manipulate, map, or monitor neurons using a single virus. We validated this strategy with three commonly used tools in neuroscience: ChRonos, a channelrhodopsin, for manipulating synaptic transmission using optogenetics; GCaMP8f for recording Ca2+ transients using fiber photometry, and mCherry for anatomical tracing of axonal projections. We tested these tools in multiple brain regions and cell types, including GABAergic neurons in the nucleus accumbens (NAc), glutamatergic neurons projecting from the ventral pallidum (VP) to the lateral habenula (LHb), dopaminergic neurons in the ventral tegmental area (VTA), and parvalbumin (PV)-positive proprioceptive neurons in the periphery. This flexible approach should be useful to identify novel genes that affect synaptic transmission, circuit activity, or morphology with a single viral injection.

Oral oxycodone self-administration leads to features of opioid misuse in male and female mice.

Use of prescription opioids, particularly oxycodone, is an initiating factor driving the current opioid epidemic. There are several challenges with modelling oxycodone abuse. First, prescription opioids including oxycodone are orally self-administered and have different pharmacokinetics and dynamics than morphine or fentanyl, which have been more commonly used in rodent research. This oral route of administration determines the pharmacokinetic profile, which then influences the establishment of drug-reinforcement associations in animals. Moreover, the pattern of intake and the environment in which addictive drugs are self-administered are critical determinants of the levels of drug intake, of behavioural sensitization and of propensity to relapse behaviour. These are all important considerations when modelling prescription opioid use, which is characterized by continuous drug access in familiar environments. Thus, to model features of prescription opioid use and the transition to abuse, we designed an oral, homecage-based oxycodone self-administration paradigm. Mice voluntarily self-administer oxycodone in this paradigm without any taste modification such as sweeteners, and the majority exhibit preference for oxycodone, escalation of intake, physical signs of dependence and reinstatement of seeking after withdrawal. In addition, a subset of animals demonstrate drug taking that is resistant to aversive consequences. This model is therefore translationally relevant and useful for studying the neurobiological substrates of prescription opioid abuse.

Ventral arkypallidal neurons inhibit accumbal firing to promote reward consumption.

The nucleus accumbens shell (NAcSh) and the ventral pallidum (VP) are critical for reward processing, although the question of how coordinated activity within these nuclei orchestrates reward valuation and consumption remains unclear. Inhibition of NAcSh firing is necessary for reward consumption, but the source of this inhibition remains unknown. Here, we report that a subpopulation of VP neurons, the ventral arkypallidal (vArky) neurons, project back to the NAcSh, where they inhibit NAcSh neurons in vivo in mice. Consistent with this pathway driving reward consumption via inhibition of the NAcSh, calcium activity of vArky neurons scaled with reward palatability (which was dissociable from reward seeking) and predicted the subsequent drinking behavior during a free-access paradigm. Activation of the VP-NAcSh pathway increased ongoing reward consumption while amplifying hedonic reactions to reward. These results establish a pivotal role for vArky neurons in the promotion of reward consumption through modulation of NAcSh firing in a value-dependent manner.

An Open-Source, Automated Home-Cage Sipper Device for Monitoring Liquid Ingestive Behavior in Rodents.

Measuring ingestive behavior of liquids in rodents is commonly used in studies of reward, metabolism, and circadian biology. Common approaches for measuring liquid intake in real time include computer-tethered lickometers or video-based systems. Additionally, liquids can be measured or weighed to determine the amount consumed without real-time sensing. Here, we built a photobeam-based sipper device that has the following advantages over traditional methods: (1) it is battery powered and fits in vivarium caging to allow home-cage measurements; (2) it quantifies the intake of two different liquids simultaneously for preference studies; (3) it is low cost and easily constructed, enabling high-throughput experiments; and (4) it is open source so that others can modify it to fit their experimental needs. We validated the performance of this device in three experiments. First, we calibrated our device using time-lapse video-based measurements of liquid intake and correlated sipper interactions with liquid intake. Second, we used the sipper device to measure preference for water versus chocolate milk, demonstrating its utility for two-bottle choice tasks. Third, we integrated the device with fiber photometry, establishing its utility for measuring neural activity in studies of ingestive behavior. This device requires no special equipment or caging, and is small, battery powered, and wireless, allowing it to be placed directly in rodent home cages. The total cost of fabrication is less than $100, and all design files and code are open source. Together, these factors greatly increase scalability and utility for a variety of behavioral neuroscience applications.

Ventral pallidal modulation of aversion processing.

Responding to aversive and rewarding stimuli is essential to survival. The ventral pallidum (VP) is a critical node in the mesolimbic network, being the primary output of the nucleus accumbens and projecting to the lateral habenula (LHb) and ventral tegmental area (VTA). The VP is thus poised to modulate the habenula-tegmental circuitry and contribute to processing both rewarding and aversive stimuli. Here, we integrate human functional imaging, behavioral pharmacology in rodents, and recent optogenetic circuit dissection studies of the VP with a focus on the role of the neurochemically-distinct subpopulations in aversion processing. These recent results support a model in which glutamatergic VP neurons play a unique role in aversion processing, while canonical GABAergic VP neurons promote reinforcement and encode the hedonic value of reward. Genetic ablation of glutamatergic, but not GABAergic VP neurons abolishes devaluation of natural reward (sucrose) by pairing with an aversive stimulus (lithium chloride injection). Both of these populations modulate activity throughout the LHb and VTA, which is necessary for expression of adaptive behavior in response to rewarding or aversive stimuli. Future work will address how neuromodulators such as endogenous opioids or dopamine shape function and plasticity within these distinct populations of VP neurons, when these subpopulations are engaged during learning responses to rewarding and aversive stimuli, and how their activity is altered in models of reward-related disorders. Answering these questions will be necessary to understand the basis and ultimately develop targeted therapies for disorders of reward/aversion processing, such as affective, chronic pain and substance use disorders.

Reward behaviour is regulated by the strength of hippocampus-nucleus accumbens synapses.

Reward drives motivated behaviours and is essential for survival, and therefore there is strong evolutionary pressure to retain contextual information about rewarding stimuli. This drive may be abnormally strong, such as in addiction, or weak, such as in depression, in which anhedonia (loss of pleasure in response to rewarding stimuli) is a prominent symptom. Hippocampal input to the shell of the nucleus accumbens (NAc) is important for driving NAc activity1,2 and activity-dependent modulation of the strength of this input may contribute to the proper regulation of goal-directed behaviours. However, there have been few robust descriptions of the mechanisms that underlie the induction or expression of long-term potentiation (LTP) at these synapses, and there is, to our knowledge, no evidence about whether such plasticity contributes to reward-related behaviour. Here we show that high-frequency activity induces LTP at hippocampus-NAc synapses in mice via canonical, but dopamine-independent, mechanisms. The induction of LTP at this synapse in vivo drives conditioned place preference, and activity at this synapse is required for conditioned place preference in response to a natural reward. Conversely, chronic stress, which induces anhedonia, decreases the strength of this synapse and impairs LTP, whereas antidepressant treatment is accompanied by a reversal of these stress-induced changes. We conclude that hippocampus-NAc synapses show activity-dependent plasticity and suggest that their strength may be critical for contextual reward behaviour.

Glutamatergic Ventral Pallidal Neurons Modulate Activity of the Habenula-Tegmental Circuitry and Constrain Reward Seeking.

BACKGROUND: The ability to appropriately integrate and respond to rewarding and aversive stimuli is essential for survival. The ventral pallidum (VP) plays a critical role in processing both rewarding and aversive stimuli. However, the VP is a heterogeneous structure, and how VP subpopulations integrate into larger reward networks to ultimately modulate these behaviors is not known. We identify a noncanonical population of glutamatergic VP neurons that play a unique role in responding to aversive stimuli and constraining inappropriate reward seeking. METHODS: Using neurochemical, genetic, and electrophysiological approaches, we characterized glutamatergic VP neurons (n = 4-8 mice/group). We performed patch clamp and in vivo electrophysiology recordings in the lateral habenula, rostromedial tegmental nucleus, and ventral tegmental area to determine the effect of glutamatergic VP neuron activation in these target regions (n = 6-10 mice/group). Finally, we selectively optogenetically stimulated glutamatergic VP neurons in a real-time place preference task and ablated these neurons using a virally expressed caspase to determine their necessity for reward seeking. RESULTS: Glutamatergic VP neurons exhibit little overlap with cholinergic or gamma-aminobutyric acidergic markers, the canonical VP subtypes, and exhibit distinct membrane properties. Glutamatergic VP neurons innervate and increase firing activity of the lateral habenula, rostromedial tegmental nucleus, and gamma-aminobutyric acidergic ventral tegmental area neurons. While nonselective optogenetic stimulation of the VP induced a robust place preference, selective activation of glutamatergic VP neurons induced a place avoidance. Viral ablation of glutamatergic VP neurons increased reward responding and abolished taste aversion to sucrose. CONCLUSIONS: Glutamatergic VP neurons constitute a noncanonical subpopulation of VP neurons. These glutamatergic VP neurons increase activity of the lateral habenula, rostromedial tegmental nucleus, and gamma-aminobutyric acidergic ventral tegmental area neurons and adaptively constrain reward seeking.