Consumption of food and water is regulated by interactions between neural circuits that govern motivational drive states, arousal, motor activity, and reward signaling, among other factors. Neurons in the lateral hypothalamus that express the neuropeptide neurotensin (LH-Nts neurons) are known to influence many of these elements, but their precise role in regulating specific aspects of ingestive behavior remains unclear. Utilizing a tightly controlled head-fixed task, we find that LH-Nts neurons strongly encode the rate of licking for water and sweet solutions, with weaker modulation of activity by solution identity and restriction state. Silencing LH-Nts neurons reduces water intake but has little direct effect on hunger or satiety. Instead, we find that these neurons impact multiple underlying behavioral components necessary for food consumption, including arousal and engagement with a novel food source, and also influence thermoregulation and metabolism. Together, these data establish tonic activity of LH-Nts neurons as a critical signal supporting exploration and volitional movement, while also delineating a role for these neurons in driving active consumption, particularly of water. Furthermore, quantitative projection mapping revealed widespread innervation of structures linked to thirst, arousal, reward, metabolism, and facial motor control, suggesting that these neurons play an important role coordinating neural circuits governing multiple different aspects of ingestion.

The periaqueductal gray (PAG) is a midbrain structure known to influence responses to both threat and reward. The PAG sends projections to the ventral tegmental area (VTA), a region critical for regulating motivated behavior via dopamine release. We previously identified a population of VTA-projecting PAG neurons that express the peptide neurotensin (Nts), a potent dopamine neuron activator. Here we find that PAG-Nts neurons co-release glutamate and Nts in the VTA to drive dopamine neuron activation. These neurons are activated by threats and threat-predictive cues and are inhibited by entry into a shelter and during reward consumption. Optogenetic stimulation elicits a robust threat response, including freezing and tail rattle, but remarkably can also drive intracranial self-stimulation. This operant reinforcement behavior is dopamine dependent while the threat response is not. Together, these results identify a dual-output circuit that engages the dopamine system, likely to increase the salience of environmental stimuli, while simultaneously driving specific threat response behaviors.

Drug-associated stimuli (cues) can usurp potent control of behavior in individuals with substance use disorders; and these effects are often attributed to altered dopamine transmission. However, there is much debate over the way in which dopamine signaling changes over the course of chronic drug use. Here, we carried out longitudinal recording and manipulation of cue-evoked dopamine release in the core of the nucleus accumbens across phases of substance use in male rats. We show that, in a subset of individuals that exhibit increased cue reactivity and escalated drug consumption, this signaling undergoes diametrically opposed changes in amplitude, determined by the context in which the cue was presented. Dopamine evoked by non-contingent cue presentation (independent of the animal's actions) increases over drug use, producing greater cue reactivity; whereas dopamine evoked by contingent cue presentation (dependent on the animal's actions) decreases over drug use, producing escalation of drug consumption. Therefore, despite being in opposite directions, these dopamine trajectories each promote cardinal features of substance use disorders.

The ventral tegmental area (VTA) is a critical node in circuits governing motivated behavior and is home to diverse populations of neurons that release dopamine, gamma-aminobutyric acid (GABA), glutamate, or combinations of these neurotransmitters. The VTA receives inputs from many brain regions, but a comprehensive understanding of input-specific activation of VTA neuronal subpopulations is lacking. To address this, we combined optogenetic stimulation of select VTA inputs with single-nucleus RNA sequencing (snRNA-seq) and highly multiplexed in situ hybridization to identify distinct neuronal clusters and characterize their spatial distribution and activation patterns. Quantification of immediate-early gene (IEG) expression revealed that different inputs activated select VTA subpopulations, which demonstrated cell-type-specific transcriptional programs. Within dopaminergic subpopulations, IEG induction levels correlated with differential expression of ion channel genes. This new transcriptomics-guided circuit analysis reveals the diversity of VTA activation driven by distinct inputs and provides a resource for future analysis of VTA cell types.

ABSTRACT Increases in drug consumption over time, also known as escalation, is a key behavioral component of substance use disorder (SUD) that is related to potential harm to users, such as overdose. Studying escalation also allows researchers to investigate the transition from casual drug use to more SUD-like drug use. Understanding the neurobiological systems that drive this transition will inform therapeutic treatments in the aim to prevent increases in drug use and the development of SUD. The kappa opioid receptor (KOR) system is typically known for its role in negative affect, which is commonly found in SUD as well. Furthermore, the KOR system has also been implicated in drug use and importantly, modulating the negative effects of drug use. However, the specific neuronal subpopulation expressing KOR involved has not been identified. Here, we first demonstrated that pharmacologically inhibiting KOR in the nucleus accumbens core (NAcC), as a whole, blocks cocaine escalation under long-access self-administration conditions. We then demonstrated that KOR expressed on ventral tegmental area (VTA) neurons but not NAcC neurons is sufficient for blocking cocaine escalation by utilizing a novel virally-mediated CRISPR-SaCas9 knock-out of the oprk1 gene. Together, this suggests that activation of KOR on VTA terminals in the NAcC drives the transition to the SUD-like phenotype of escalation of cocaine consumption.

SummaryFast-acting neurotransmitters and slow, modulatory neuropeptides are commonly co-released from neurons in the central nervous system (CNS), albeit from distinct synaptic vesicles1. The mechanisms of how co-released neurotransmitters and neuropeptides that have opposing actions, e.g., stimulatory versus inhibitory, work together to exert control of neural circuit output remain unclear. This question has been difficult to resolve due to the inability to selectively isolate these signaling pathways in a cell- and circuit-specific manner. To overcome these barriers, we developed a genetic-based anatomical disconnect procedure that utilizes distinct DNA recombinases to independently facilitate conditional in vivo CRISPR/Cas9 mutagenesis2 of neurotransmitter- and neuropeptide-related genes in distinct cell types in two different brain regions simultaneously. With this approach we demonstrate that the stimulatory neuropeptide neurotensin (Nts) and the inhibitory neurotransmitter γ-aminobutyric acid (GABA), which are co-released from neurons in the lateral hypothalamus (LH), work coordinately to activate dopamine neurons of the ventral tegmental area (VTA-DA). We show that GABA release from LH-Nts neurons acts on GABA neurons within the VTA to rapidly disinhibit VTA-DA neurons, while Nts signals through the Nts receptor 1 (Ntsr1) on VTA-DA neurons to promote a slow depolarization of these cells. Thus, these two signals act on distinct time scales through different cell types to enhance mesolimbic dopamine neuron activation, which optimizes behavioral reinforcement. These data demonstrate a circuit-based mechanism for the coordinated action of a neurotransmitter and neuropeptide with opposing effects on cell physiology.

The axonal guidance cue netrin-1 serves a critical role in neural circuit development by promoting growth cone motility, axonal branching, and synaptogenesis. Within the adult mouse brain, expression of the gene encoding (Ntn1) is highly enriched in the ventral midbrain where it is expressed in both GABAergic and dopaminergic neurons, but its function in these cell types in the adult system remains largely unknown. To address this, we performed viral-mediated, cell-type specific CRISPR-Cas9 mutagenesis of Ntn1 in the ventral tegmental area (VTA) of adult mice. Ntn1 loss-of-function in either cell type resulted in a significant reduction in excitatory postsynaptic connectivity. In dopamine neurons, the reduced excitatory tone had a minimal phenotypic behavioral outcome; however, reduced glutamatergic tone on VTA GABA neurons induced behaviors associated with a hyperdopaminergic phenotype. Simultaneous loss of Ntn1 function in both cell types largely rescued the phenotype observed in the GABA-only mutagenesis. These findings demonstrate an important role for Ntn1 in maintaining excitatory connectivity in the adult midbrain and that a balance in this connectivity within two of the major cell types of the VTA is critical for the proper functioning of the mesolimbic system.

Dopamine is broadly implicated in reinforcement learning, but how patterns of dopamine activity are generated is poorly resolved. Here, we demonstrate that two ion channels, Kv4.3 and BKCa1.1, regulate the pattern of dopamine neuron firing and dopamine release on different time scales to influence separate phases of reinforced behavior in mice. Inactivation of Kv4.3 in VTA dopamine neurons increases ex vivo pacemaker activity and excitability that is associated with increased in vivo firing rate and ramping dynamics before lever press in a learned instrumental paradigm. Loss of Kv4.3 enhances performance of the learned response and facilitates extinction. In contrast, loss of BKCa1.1 increases burst firing and phasic dopamine release that enhances learning of an instrumental response and enhances extinction burst lever pressing in early extinction that is associated with a greater change in activity between reinforced and unreinforced actions. These data demonstrate that disruption of intrinsic regulators of neuronal activity differentially affects dopamine dynamics during reinforcement and extinction learning.

Despite the widely known role of dopamine in reinforcement learning, how the patterns of dopamine release that are critical to the acquisition, performance, and extinction of conditioned responses are generated is poorly resolved. Here, we demonstrate that the coordinated actions of two ion channels, Kv4.3 and BKCa1.1, control the pattern of dopamine neuron firing and dopamine release on different time scales to regulate separate phases of reinforced behavior in mice. Inactivation of Kv4.3 in VTA dopamine neurons increases ex vivo pacemaker activity and excitability that is associated with increased in vivo ramping dynamics prior to lever press in a learned instrumental response paradigm. Loss of Kv4.3 enhances performance of the learned response and facilitates extinction. In contrast, loss of BKCa1.1 increases burst firing and phasic dopamine release that enhances learning of an instrumental response. Inactivation of BKCa1.1 enhances extinction burst lever pressing in early extinction training that is associated with increased reward prediction error signals. These data demonstrate that temporally distinct patterns of dopamine release are governed by the intrinsic regulators of the cell to shape behavior. Teaser We show that ion channels in midbrain dopamine neurons are critical for patterning action potential firing at the cell body and governing neurotransmitter release to regulate reinforcement learning.

In some individuals, drug-associated cues subsume potent control of behavior, such as the elicitation of drug craving1-3 and automatized drug use4. The intensity of this cue reactivity is highly predictive of relapse and other clinical outcomes in substance use disorders5,6. It has been postulated that this cue reactivity is driven by augmentation of dopamine release over the course of chronic drug use7. Here we carried out longitudinal recording and manipulation of cue-evoked dopamine signaling across phases of substance-use related behavior in rats. We observed a subset of individuals that exhibited increased cue reactivity and escalated drug consumption, two cardinal features of substance use disorders. In these individuals, cue-evoked phasic dopamine release underwent diametrically opposed changes in amplitude, determined by the context in which the cue is presented. Dopamine evoked by non-contingent cue presentation increased over drug use, producing greater cue reactivity; whereas dopamine evoked by contingent cue presentation decreased over drug use, producing escalation of drug consumption. Therefore, despite being in opposite directions, these dopamine trajectories each promote core symptoms of substance use disorders.

Neuropeptides play an important role in modulating mesolimbic system function. However, while synaptic inputs to the ventral tegmental area (VTA) have been extensively mapped, the sources of many neuropeptides are not well resolved. Here, we mapped the anatomical locations of three neuropeptide inputs to the VTA: neurotensin (NTS), corticotrophin releasing factor (CRF), and neurokinin B (NkB). Among numerous labeled inputs we identified the bed nucleus of the stria terminalis (BNST) as a major source of all three peptides, containing similar numbers of NTS, CRF, and NkB VTA projection neurons. Approximately 50% of BNST to VTA inputs co-expressed two or more of the peptides examined. Consistent with this expression pattern, analysis of calcium dynamics in the terminals of these inputs in the VTA revealed both common and distinct patterns of activation during appetitive and aversive conditioning. These data demonstrate additional diversification of the mesolimbic dopamine system through partially overlapping neuropeptidergic inputs.

Ion channel complexes typically consist of both pore-forming subunits and auxiliary subunits that do not directly conduct current but can regulate trafficking or alter channel properties. Isolating the role of these auxiliary subunits in neurons has proved difficult due to a lack of specific pharmacological agents and the potential for developmental compensation in constitutive knockout models. Here, we use cell-type-specific viral-mediated CRISPR/Cas9 mutagenesis to target the potassium channel auxiliary subunit Kvβ2 (Kcnab2) in dopamine neurons in the adult mouse brain. We find that mutagenesis of Kcnab2 reduces surface expression of Kv1.2, the primary Kv1 pore-forming subunit expressed in dopamine neurons, and shifts the voltage dependence of inactivation of potassium channel currents toward more hyperpolarized potentials. Loss of Kcnab2 broadens the action potential waveform in spontaneously firing dopamine neurons recorded in slice, reduces the afterhyperpolarization amplitude, and increases spike timing irregularity and excitability, all of which is consistent with a reduction in potassium channel current. Similar effects were observed with mutagenesis of the pore-forming subunit Kv1.2 (Kcna2). These results identify Kv1 currents as important contributors to dopamine neuron firing and demonstrate a role for Kvβ2 subunits in regulating the trafficking and gating properties of these ion channels. Furthermore, they demonstrate the utility of CRISPR-mediated mutagenesis in the study of previously difficult to isolate ion channel subunits.NEW & NOTEWORTHY Here, we utilize CRISPR/Cas9-mediated mutagenesis in dopamine neurons in mice to target the gene encoding Kvβ2, an auxiliary subunit that forms a part of Kv1 channel complexes. We find that the absence of Kvβ2 alters action potential properties by reducing surface expression of pore-forming subunits and shifting the voltage dependence of channel inactivation. This work establishes a new function for Kvβ2 subunits and Kv1 complexes in regulating dopamine neuron activity.

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Methods Data availability References Decision letter Author response Article and author information Metrics Abstract The axonal guidance cue netrin-1 serves a critical role in neural circuit development by promoting growth cone motility, axonal branching, and synaptogenesis. Within the adult mouse brain, expression of the gene encoding (Ntn1) is highly enriched in the ventral midbrain where it is expressed in both GABAergic and dopaminergic neurons, but its function in these cell types in the adult system remains largely unknown. To address this, we performed viral-mediated, cell-type specific CRISPR-Cas9 mutagenesis of Ntn1 in the ventral tegmental area (VTA) of adult mice. Ntn1 loss-of-function in either cell type resulted in a significant reduction in excitatory postsynaptic connectivity. In dopamine neurons, the reduced excitatory tone had a minimal phenotypic behavioral outcome; however, reduced glutamatergic tone on VTA GABA neurons induced behaviors associated with a hyperdopaminergic phenotype. Simultaneous loss of Ntn1 function in both cell types largely rescued the phenotype observed in the GABA-only mutagenesis. These findings demonstrate an important role for Ntn1 in maintaining excitatory connectivity in the adult midbrain and that a balance in this connectivity within two of the major cell types of the VTA is critical for the proper functioning of the mesolimbic system. Editor's evaluation This manuscript reports an important, previously unappreciated, non-developmental role for the guidance cue netrin-1 in midbrain physiology and related behavior in adult animals. Using multiple experimental tools in adult mice, the study convincingly shows that netrin-1 within midbrain dopamine and GABA neurons is necessary to maintain dopamine excitatory tone and plays a role in motivated and anxiety-like behavior. This paper will be of interest to neuroscientists studying dopamine function and/or motivated behavior and those interested in ways that neurodevelopmental genes can continue to play a role in neuronal function and behavior into adulthood. https://doi.org/10.7554/eLife.83760.sa0 Decision letter eLife's review process Introduction Proper regulation of the midbrain dopamine system is essential for numerous brain functions and behavior (Bissonette and Roesch, 2016). Disruption in the balance of midbrain dopamine neuron activity has been linked to several neurological and psychiatric conditions, including autism (Pavăl, 2017), schizophrenia (Hietala and Syvälahti, 1996), and substance use disorders (Ostroumov and Dani, 2018). Within the VTA, the activity of dopamine neurons is regulated in part by inhibitory (GABAergic) and excitatory (glutamatergic) synaptic input. The molecular mechanisms that maintain the balance of inhibitory and excitatory connectivity in the adult midbrain, however, remain poorly resolved. Genome-wide association studies and analysis of de novo mutations have strongly implicated genes regulating neuronal axon guidance in neurodevelopmental disorders (Gilman et al., 2012; Gulsuner et al., 2013). Although the impact of mutations in these genes early in development is likely critical for their role in neurodevelopmental disorders, many of the genes maintain high levels of expression in the adult brain, and their functions in this context are less understood. We previously demonstrated that the axonal guidance receptor Robo2 is necessary for the maintenance of inhibitory synaptic connectivity in the adult VTA (Gore et al., 2017), suggesting that axonal guidance proteins have a critical function in maintaining synaptic connectivity in the adult midbrain. Netrin-1 is predominately recognized for its role in neurodevelopmental processes (Gore et al., 2017; Manitt et al., 2010; Winberg et al., 1998; Glasgow et al., 2018; Yetnikoff et al., 2010). During development, the gene encoding netrin-1 (Ntn1) is highly expressed throughout the central nervous system (CNS). Following this critical period global expression decreases (Manitt et al., 2010), but expression within the limbic system, particularly in the ventral midbrain, persists. Consistent with the continued function of Ntn1 following early development, genetic inactivation of either Ntn1 (Winberg et al., 1998) or its receptor Dcc Glasgow et al., 2018 from forebrain glutamatergic neurons in late postnatal development results in significantly impaired spatial memory in adult mice that corresponds to a loss of hippocampal plasticity. Within the VTA, Dcc expression levels in adult mice are significantly upregulated following amphetamine exposure (Yetnikoff et al., 2010), and Dcc haploinsufficient mice display blunted locomotor response to amphetamine (Flores et al., 2005), consistent with increased excitatory synaptic strength in the VTA following amphetamine treatment (Saal et al., 2003). These results suggest a potential role for Ntn1 signaling through Dcc in regulating excitatory tone in the adult dopamine system. To determine whether Ntn1 regulates excitatory synaptic connectivity in the VTA of adult mice, we used viral-mediated, Cre-inducible CRISPR/Cas9 (Hunker et al., 2020) to selectively mutate Ntn1 in midbrain dopamine and GABA neurons. We find that Ntn1 loss of function significantly reduces postsynaptic glutamate receptor-mediated currents in a cell-autonomous manner similar to what has been reported previously in the adult hippocampus (Glasgow et al., 2018). We further show that Ntn1 loss of function in VTA GABA neurons has a more profound effect on behavior than the loss of function in VTA dopamine neurons. Intriguingly, the simultaneous loss of function of Ntn1 in both cell types of the VTA largely rescues the behavioral phenotypes observed following mutagenesis in VTA GABA neurons alone. These data support a model in which the balance of excitatory synaptic connectivity between dopamine and GABA neurons within the VTA is maintained by the persistent expression of the developmental gene Ntn1. This continued function of Ntn1 in adulthood sustains the excitatory/inhibitory equilibrium onto dopamine neurons that is critical to the function of the mesolimbic dopamine system. Results Ntn1 expression and mutagenesis in the VTA In situ hybridization analysis of Ntn1 from the Allen Institute mouse brain expression atlas (Lein et al., 2007) shows diffuse and low levels of expression throughout the adult mouse brain, with moderate expression levels in the cerebellum and hippocampus (Figure 1A), and the highest level of expression in the ventral midbrain (substantia nigra and ventral tegmental area). The VTA is comprised of multiple cell types Morales and Margolis, 2017; to determine the cell type-specific expression of Ntn1 within the heterogeneous VTA, we performed RNAscope in situ hybridization on midbrain slices from adult wild-type mice (>8 weeks of age) and probed for Ntn1, Th (tyrosine hydroxylase, a marker of dopamine neurons), and Slc32a1 (vesicular GABA transporter [Vgat], a marker of GABA neurons). We found Ntn1 expression to be present throughout the VTA, largely localized to Th-positive neurons but also present in GABA neurons (Figure 1C–F). Of the identified Ntn1 positive cells, Ntn1 expression co-localized with Th expression (dopamine producing neurons; 72.2% co-localization) and Slc32a1-expressing GABA neurons (18.1% co-localization) (Figure 1E). The remaining Ntn1 expressing cells that do not co-localize with Th or Slc32a1 are likely glutamatergic neurons (Morales and Margolis, 2017), or possibly glial cells (Phillips et al., 2022). Immunohistochemistry for Ntn1 and Th (Figure 1G) confirmed the presence of Ntn1 in dopamine and non-dopamine producing (Th-negative) cells. Figure 1 Download asset Open asset Netrin-1 is present in the adult ventral tegmental area (VTA) and expressed by both dopamine and GABA neurons. 3D display of Ntn1 (A) and Slc6a3 (B, dopamine marker) from the Allen Brain Atlas. (C–D) 20 X magnification images of in situ hybridization (RNAScope) for Ntn1 (green) and Slc32a1 (GABA marker; red, C) and Th (dopamine marker; red; D). Arrows indicate co-labeling of Ntn1 with Slc32a1 (C) or Th (D). Scale bar indicates 20 μm. (E–F) Quantification of cell type expression. Of the cells expressing Ntn1, 72.2% were dopaminergic (Th+) and 18.1% were GABAergic (Slc32a1+; E). (F) Of the total of Th+ identified cells, 64.5% co-expressed Ntn1 (35.6% did not express Ntn1), and 30.4% of Slc32a1 identified cells co-expressed Ntn1 (69.5% did not express Ntn1). (G) Immunohistochemistry confirms the presence of Ntn1 protein (red) in both Th+ (cyan) and non-dopamine cells (Th- cells, indicated by yellow arrows). Figure 1—source data 1 Cell counts. https://cdn.elifesciences.org/articles/83760/elife-83760-fig1-data1-v1.xlsx Download elife-83760-fig1-data1-v1.xlsx To selectively mutate Ntn1 in specific cell types in the VTA, we designed a single guide RNA (sgRNA) targeting exon 2 in mice (sgNtn1; Figure 2A) and cloned it into an AAV packaging plasmid containing a Cre-recombinase dependent expression cassette for SaCas9 (Hunker et al., 2020). To determine the efficiency of Ntn1 mutagenesis, we injected DAT-Cre (Slc6a3Cre/+) mice (aged 8–10 weeks) bilaterally into the VTA with either AAV-FLEX-SaCas9-HA-sgNtn1 and AAV-FLEX-YFP (DAT-Cre Ntn1-cKO mice) or AAV-FLEX-SaCas9-sgRosa26 (a gene locus with no known function; control mice). Four to five weeks following injection, we performed immunohistochemistry for Ntn1 and Th. Ntn1 conditional knockout (cKO) resulted in a significant reduction in the proportion of VTA Th-positive cells co-labeled with Ntn1 in DAT-Cre Ntn1 cKO mice compared to controls (Figure 2D–E). In contrast to previous findings following Ntn1 deletion in the substantia nigra (Jasmin et al., 2021), the average number of Th + cells per slice was not statistically different in DAT-Cre Ntn1 cKO mice compared to controls (control: 178.2 ± 12.68 and Ntn1 cKO 173.3 ± 10.75). Although, this result is consistent with Ntn1 inactivation not compromising cell viability, without a complete stereological analysis of every neuron within the VTA, we cannot definitively conclude that some cell loss did not occur. Figure 2 Download asset Open asset Virally delivered CRISPR-Cas9 complex targeting the Ntn1 locus results in a significant reduction in Ntn1 antibody staining. (A–B) Schematics summarizing cell type-specific knockout procedure. (A) Adult mice were injected bilaterally into the VTA with AAV-FLEX-SaCas9-HA-sgNtn1 and AAV-FLEX-YFP. Control mice received an equivalent volume of -sgRosa26 and/or AAV-FLEX-YFP. SaCas9 is virally delivered into the genome in the inactive orientation and returned to the active orientation only in the presence of Cre recombinase, limiting Cas9 expression to target cells. (B) Schematic of the VTA (left) showing VTA GABA neurons project to and inhibit VTA dopamine neurons. By using transgenic Cre-driver mouse lines (right) viral delivery of SaCas9 results in gene disruption in specifically VTA dopamine neurons (DAT-Cre mice, top panel), or VTA GABA neurons (Vgat-Cre mice, bottom panel). (D) Example images for Th (cyan) and Ntn1 (red) immunostaining in the ventral tegmental area (VTA) of mice injected with control or sgNtn1 CRISPR virus. (E) Quantification of the percentage of Th + cells co-labled with Ntn1 (Students t-test; t=8.179, df = 10, 62.25 ± 5.796 vs 9.586 ± 2.807, ****p<0.0001). Figure 2—source data 1 Cell counts. https://cdn.elifesciences.org/articles/83760/elife-83760-fig2-data1-v1.xlsx Download elife-83760-fig2-data1-v1.xlsx Netrin-1 regulates excitatory connectivity within the adult VTA Previous research has shown that Ntn1 regulates excitatory synaptic connectivity in the adult hippocampus (Glasgow et al., 2018). To determine the impact of Ntn1 loss of function on synaptic connectivity, DAT-Cre or Vgat-Cre (Slc32a1Cre/+) mice were injected with AAV1-FLEX-SaCas9-U6-sgNtn1 and AAV1-FLEX–YFP (Figure 3A and E). After at least four weeks, miniature excitatory postsynaptic currents (mEPSCs) were recorded from fluorescently identified dopamine or GABA neurons of the VTA. Ntn1 mutagenesis in dopamine neurons resulted in significantly reduced mEPSC amplitude and frequency (Figure 3B–D). Similarly, Ntn1 mutagenesis in VTA GABA neurons also resulted in significantly reduced mEPSC amplitude and frequency (Figure 3F–H). We did not detect significant effects on miniature inhibitory postsynaptic currents (mIPSCs) in VTA dopamine or GABA neurons following Ntn1 mutagenesis in these cells (Figure 3—figure supplement 1), suggesting Ntn1 does not play a role in regulating inhibitory connectivity in these cells. Figure 3 with 3 supplements see all Download asset Open asset Loss of Ntn1 results in a significant reduction in excitatory postsynaptic current. (A) Schematic of DAT-Cre dopamine specific Ntn1cKO. (B) Sample traces from control (top panel) and DAT Ntn1 cKO mice (bottom panel). (C–D) mEPSC amplitude (C) and frequency (D) measured from fluorescently identified dopamine neurons (n=35 controls, n=33 cKO, t=3.744, df = 66, ***p<0.001 and t=5.259, df = 66, ****p<0.0001). (E) Schematic of Vgat-Cre GABA specific Ntn1cKO. (F) Sample traces from control (top panel) and Vgat Ntn1 cKO mice (bottom panel). (G–H) mEPSC amplitude (G) and frequency (H) measured from fluorescently identified GABA neurons (n=30 controls, n=32 cKO, t=2.048, df = 60, *p<0.05, and t=3.966, df = 60, ***p<0.001). (I) Schematic of stimulating electrode placement in horizontal midbrain slice and example EPSCs. (J–K) Paired pulse ratio in dopamine (J, n=18 controls, n=21 cKO, t=1.271, df = 37, p>0.05), or GABA neurons (K, n=14 controls, n=21 cKO, t=1.105, df = 33, p>0.05). Figure 3—source data 1 EPSCs and IPSCs from targeted cells. https://cdn.elifesciences.org/articles/83760/elife-83760-fig3-data1-v1.xlsx Download elife-83760-fig3-data1-v1.xlsx Figure 3—source data 2 Additional EPSC and IPSC data from non-targeted cells. https://cdn.elifesciences.org/articles/83760/elife-83760-fig3-data2-v1.xlsx Download elife-83760-fig3-data2-v1.xlsx Because Ntn1 is a secreted protein, it is also possible that Ntn1 loss of function in one cell type could affect synaptic connectivity in adjacent neurons in which the gene was not inactivated, inducing a non-cell autonomous effect. To address this, we recorded mEPSCs from non-YFP-expressing (presumptively non-dopamine) neurons in DAT-Cre mice injected with Ntn1 CRISPR or control virus, and from non-YFP-expressing (presumptively non-GABA) neurons in Vgat-Cre injected mice. We did not observe significant non-cell autonomous effects on mEPSCs from non-targeted cells (Figure 3—figure supplement 2). Similarly, we also did not observe non-cell autonomous effects on mIPSCs from non-targeted cells (Figure 3—figure supplement 2). The observed reduction in mEPSC frequency suggests that loss of Ntn1 function could act presynaptically, potentially through postsynaptic Ntn1 secretion (Glasgow et al., 2018). To test potential presynaptic changes in vesicle release probability, we analyzed the paired-pulse ratio (PPR) of electrically evoked EPSCs delivered 50 ms apart. Ntn1 mutagenesis in either dopamine or GABA neurons did not result in a significant change in PPR compared to controls, suggesting no measurable change in presynaptic release (Figure 3J–K). To further resolve this question, we analyzed potential changes in quantal size by performing a 1/CV2 analysis of the coefficient of variation in the mEPSC amplitude. We did not detect a statistically significant change in 1/CV2 associated with Ntn1 loss in either dopamine or GABA cells, further suggesting netrin manipulation is altering either the number of or the function of postsynaptic AMPA receptors (Figure 3—figure supplement 3A, B). Our data suggest that the observed changes in mEPSCs are likely a reflection of reduced AMPA-type or NMDA-type glutamate receptor levels in postsynaptic cells. To address this, fluorescently identified dopamine neurons from DAT-Cre Ntn1 cKO mice held at –60 mV, and AMPA-evoked current was measured following bath application of 1 uM AMPA. For NMDA currents, neurons were held at +40 mV and NMDA-evoked current was measured following bath application of 50 μM NMDA. Ntn1 mutagenesis in DAT-Cre mice resulted in significantly reduced AMPA-evoked current compared to controls (Figure 3—figure supplement 3C, D). In contrast, NMDA-evoked responses were similar between the groups (Figure 3—figure supplement 3E, F). These results suggest that Ntn1 regulates AMPA receptor availability in adult VTA. Ntn1 loss of function in VTA-dopamine neurons has little effect on behavior Dopamine producing neurons of the VTA regulate multiple aspects of locomotor activity, motivated behavior, and psychomotor activation. To determine whether conditional mutagenesis of Ntn1 in dopamine neurons, and subsequent reduction in excitatory synaptic connectivity impacts these behaviors, we injected DAT-Cre mice with AAV1-FLEX-SaCas9-sgNtn1 or AAV1-FLEX-SaCas9-sgRosa26 (control) and assayed them in multiple behavioral paradigms. First, we monitored day-night locomotion in control and AAV1-FLEX-SaCas9-sgNtn1 injected DAT-Cre mice. No significant differences were detected (Figure 4B and Figure 4—figure supplement 1). Figure 4 with 1 supplement see all Download asset Open asset Ntn1 cKO in DA neurons results in little behavioral alteration. (A) Schematic summarizing cell type-specific knockout procedure. (B) Distance traveled in 15 min bins over the course of three nights and two days (n=21 control; n=15 cKO, Two-way ANOVA, Group F(1, 34)=1.169, p=0.2872, Time F(18.25, 620.6)=21.97 p<0.0001, Interaction F(251, 8534)=1.063 p=0.2380). (C) Earned reinforcers during three days of FR1 or FR5 operant conditioning (n=19 control; n=15, FR1; Group F(1, 96)=0.9761 p=0.3257, Time F(2, 96)=9.999 p=0.0001, Interaction F(2, 96)=0.006622 p=0.9934; FR5 Group F(1, 32)=0.6140, p=0.9808, Time F (2, 96)=2.786 p=0.0667, Interaction F(2, 96)=0.008669 p=0.9914). (D) Breakpoint (maximum presses per reinforcer) on a progressive ratio task (t=0.9434, df = 32, p=0.3525). (E) Lever presses per session during five days of extinction training (Group F(1, 32)=1.336, p=0.2562, Time F(4, 128)=87.55 p<0.0001, Interaction F(4, 128)=2.017 p=0.0959). (F) Acoustic startle response to varying intensity white noise stimuli (Group F(1, 31)=3.176 p=0.0845, Intensity F(1.737, 53.83)=37.74 p<0.0001, Interaction F(6, 186)=2.124 p=0.0525) (G) Percent inhibition of startle response following pre-pulse at indicated intensities (Group F(1, 96)=0.05032 p=0.8230, Intensity F(2, 96)=5.638 p=0.0048, Interaction F(2, 96)=0.2402 p=0.7870). (H) Time on edge or in center of an open field arena during a 10 min test session (Edge: t=2.897, df = 32, **p<0.01, Center: t=2.750, df = 32, **p<0.01). Figure 4—source data 1 Behavioral data for Figure 4. https://cdn.elifesciences.org/articles/83760/elife-83760-fig4-data1-v1.xlsx Download elife-83760-fig4-data1-v1.xlsx To determine whether appetitive conditioning behaviors are disrupted by the loss of Ntn1 function in VTA dopamine neurons, we assayed mice in a simple instrumental conditioning paradigm using a fixed-ratio 1 (FR1) followed by a fixed ratio 5 (FR5) schedule of reinforcement in which one or five lever presses are required to obtain a food reward, respectively. We did not observe significant differences in either of these behavioral tasks (Figure 4C). Next, we monitored motivated behavior using a progressive ratio schedule of reinforcement in which the number of lever presses required for reinforcement increases non-arithmetically (1, 2, 4, 7, 13, 19, 25, 34, 43, 52, 61, 73…), and again did not observe significant differences between control and experimental mice (Figure 4D). Following PR, we reinstated FR1 responding for three days followed by extinction training, and again did not detect any differences between the two groups (Figure 4E and Figure 4—figure supplement 1), indicating Ntn1 loss of function in VTA dopamine neurons did not alter appetitive conditioning behaviors. Although appetitive conditioning was not affected by Ntn1 loss of function in dopamine neurons, we did observe a slight but significant reduction in body weight in these mice relative to controls prior to calorie restriction (Figure 4—figure supplement 1). To determine whether sensory-motor gating is altered in mice with loss of Ntn1 function in VTA dopamine neurons, we assayed them in acoustic startle and pre-pulse inhibition (PPI) paradigms. Although acoustic startle responses were reduced in AAV1-FLEX-SaCas9-sgNtn1 injected mice, this did not reach significance (Figure 4F). Moreover, we did not observe differences in PPI percentage inhibition (Figure 4G). These results indicate that loss of Ntn1 function in VTA dopamine neurons does not appear to affect psychomotor activation. In addition to reinforcement and motivation, dopamine regulates other dimensions of affective behavior. To test whether anxiety-related behavior is affected in experimental mice relative to control mice, we assayed them in an open-field test. AAV1-FLEX-SaCas9-sgNtn1 injected DAT-Cre mice spent significantly more time on the edge of the open field arena and significantly less time in the center of the arena, consistent with an elevation in anxiety-like behavior (Figure 4H). There were no significant locomotor differences associated with the loss of Ntn1 function in the open field arena (Figure 4—figure supplement 1). Ntn1 loss of function in VTA-GABA neurons affects multiple behaviors To determine whether reducing excitatory synaptic connectivity onto VTA GABA neurons through the loss of Ntn1 function in these cells impacts behavior, we injected Vgat-Cre mice with AAV1-FLEX-SaCas9-sgNtn1 or AAV1-FLEX-SaCas9-sgRosa26 (control) into the VTA as described previously and tested these mice using the same behavioral paradigms described above. In contrast to Ntn1 mutagenesis in dopamine neurons, this manipulation in VTA GABA neurons resulted in a significant increase in locomotor activity (Figure 5B and Figure 5—figure supplement 1). Figure 5 with 1 supplement see all Download asset Open asset Ntn1 cKO in GABA ventral tegmental area (VTA) neurons resulted in significant behavioral alterations. (A) Schematic summarizing cell type-specific knockout procedure. (B) Distance traveled in 15 min bins over the course of three nights and two days (n=26 controls, n=23 cKO, Two-way ANOVA Group F(1, 11797)=527.4, ****p<0.0001, Time F(250, 11797)=14.61 p<0.0001. Interaction F(250, 11797)=1.342, p=0.0003). (C) Earned reinforcers during three days of FR1 or FR5 operant conditioning (n=18 control; n=15 cKO; FR1: Group F(1, 31)=0.08647 p=0.7707, Time F(2, 62)=30.46 p<0.0001, Interaction F(2, 62)=3.186 p=0.0482; FR5: Group F(1, 31)=4.261, *p<0.05, Time F (1.992, 61.74)=0.3131 p=0.7314, Interaction F(2, 62)=1.448 p=0.2428). (D) Breakpoint (maximum presses per reinforcer) on a progressive ratio task (t=2.577, df = 31, *p<0.05). (E) Lever presses per session during five days of extinction training (Group F(1, 31)=10.23, **p<0.01, Time F(1.491, 46.23)=83.84 p<0.0001, Interaction F(4, 124)=3.546 p=0.0089). (F) Acoustic startle response to varying intensity white noise stimuli (Group F(1, 31)=7.891, **p<0.0085, Intensity F(1.790, 55.49)=24.94 p<0.0001, Interaction F(6, 186)=2.186, p=0.0462). (G) Percent inhibition of startle response following pre-pulse at indicated intensities (Group F(1, 93)=9.181, **p<0.01, Intensity F(2, 93)=5.101 p=0.0079, Interaction F(2, 93)=0.002227 p=0.9978). (H) Time on edge or in center of open field arena during a 10 min test session (edge: t=2.248, df = 31, *p<0.05, center t=1.366, df = 33, p>0.05). Figure 5—source data 1 Behavioral data for Figure 5. https://cdn.elifesciences.org/articles/83760/elife-83760-fig5-data1-v1.xlsx Download elife-83760-fig5-data1-v1.xlsx In the FR1 schedule of reinforcement, we did not observe a significant difference between the groups; however, we observed an increase in the number of earned reinforcements in the FR5 schedule in mice with Ntn1 loss of function in VTA GABA neurons (Figure 5C). We also observed an increase in the PR schedule of reinforcement in these mice relative to controls (Figure 5D). In contrast to DAT-Cre Ntn1 cKO mice, pre-calorie restriction body weights in Vgat-Cre Ntn1 cKO mice did not differ from controls (Figure 5—figure supplement 1). Reinstatement of FR1 responding in Vgat-Cre Ntn1 cKO following PR was not different than controls (Figure 5—figure supplement 1). However, during extinction training, Vgat-Cre Ntn1 cKO mice displayed high extinction bursts (elevated pressing following reward omission) compared to controls that remained elevated on the second day of extinction training (Figure 5E). While these data likely reflect an altered motivational state with loss of Ntn1, it is also possible that the hyperactivity observed in Vgat-Cre Ntn1 cKO mice contributes to the elevated lever press rates during FR5, PR, and extinction. Analysis of sensory-motor gating in these mice revealed that Vgat-Cre mice injected with AAV1-FLEX-SaCas9-sgNtn1 had a significant reduction in the acoustic startle relative to control mice (Figure 5F) that was accompanied by a reduction in PPI (Figure 5G). Similar to mutagenesis of Ntn1 in dopamine neurons, this manipulation in GABA neurons resulted in an increase in anxiety-like behavior as demonstrated by an increased time on edge; though we only observed a trend towards a reduction in time spent in the center of the open field arena (Figure 5H). The lack of observed significance in the time in center in the context of increased edge time may reflect the hyperactivity observed following Ntn1 mutagenesis in VTA GABA neurons, consistent with this possibility, we did observe increased distance traveled during the open field test in these mice relative to controls (Figure 5—figure supplement 1). Loss of netrin-1 in dopamine neurons largely reverses the effects of Ntn1 mutagenesis in GABA neurons A loss of Ntn1 in VTA-dopamine neurons resulted in decreased excitatory synaptic input to those cells (theoretically reducing dopamine activity) (Figure 6A), and loss of Ntn1 in VTA-GABA neurons resulted in decreased excitatory tone onto GABA neurons, which would be predicted to increase dopamine activity through disinhibition (Tan et al., 2012; Figure 6A). Based on these observations, we asked whether a loss of Ntn1 in both cell types would restore the balance of activity in the midbrain, or whether there is a hierarchical effect of Ntn1 loss of function in GABA neurons. To address this, we crossed DAT-Cre with Vgat-Cre mice to develop a DAT-Cre::Vgat-Cre transgenic line, injected these mice with AAV1-FLEX-SaCas9-sgNtn1 or AAV1-FLEX-SaCas9-sgRosa26 (control) (Figure 6B), and assayed them using the previous behavioral battery. Figure 6 with 1 supplement see all Download asset Open asset Ntn1 cKO in DATIRES::Vgat-Cre mice partially rescues behavioral phenotype. (A) Model of Ntn1 loss of function in the ventral tegmental area (VTA) on excitatory and inhibitory balance. (B) Schematic of GABA and Dopamine Ntn1 cKO. (C) Distance traveled in 15 min bins over the course of three nights and two days (Two-Way ANOVA Group F(1, 45)=0.004273, p>0.05, Time F(17.16, 772.1)=23.36, p<0.0001, Interaction F (247, 11115)=1.492, p<0.0001). (D) Earned reinforcers during three days of FR1 or FR5 operant conditioning (n=21 controls, n=20 Ntn1 cKO, FR1: Group F(1, 40)=0.04247 p=0.8378, Time F(2, 80)=25.70 p<0.0001, Interaction F(2, 80)=1.402 p=0.2522; FR5: Group F(1, 40)=0.2244 p=0.6383, Time F(1.499, 59.95)=2.226 Pp0.1295, Interaction F(2, 80)=0.1385 p=0.8708) (E) Breakpoint (maximum presses per reinforcer) on a progressive ratio task (t=2.502, df = 39, *p<0.05) (F) Lever presses per session during five days of extinction training (Group F(1, 39)=6.990, *p=0.0117, Time F(2.381, 92.87)=42.95 p<0.0001, Interaction F(4, 156)=0.1470 p=0.9641). (G) Acoustic startle response to varying intensity white noise stimuli (Group F(1, 40)=0.1207 p=0.7301, Intensity F(1.775, 70.99)=36.77 p<0.0001, Interaction F(6, 240)=0.6127 p=0.7201). (G) Percent inhibition of startle response following pre-pulse at indicated intensities (Group F(1, 120)=0.9661 p=0.3276, Intensity F(2, 120)=7.067 p=0.0013, Interaction F(2, 120)=0.8861 p=0.4150). (H) Time on edge or in center of open field arena during a 10 min test session (edge: t=0.3584, df = 45 p>0,05, center: t=0.4233, df = 45, p>0.05). Figure 6—source data 1 Behavioral data for Figure 6. https://cdn.elifesciences.org/articles/83760/elife-83760-fig6-data1-v1.xlsx Download elife-83760-fig6-data1-v1.xlsx Simultaneous Ntn1 loss of function in VTA GABA and dopamine neurons largely reversed the hyperlocomotor phenotype (Figure 6C) observed with Ntn1 mutagenesis in VTA GABA neurons alone, though a modest, increase in daytime locomotion remained (Figure 6—figure supplement 1). Similarly, loss of Ntn1 in both VTA GABA and dopamine neurons resulted in operant responding during FR1 and FR5 that was similar to controls (Figure 6D) and pre-calorie restriction body weights did not differ between the groups. Motivation, as measured in the PR task, was elevated in the double transgenic Cre line following Ntn1 mutagenesis (Figure 6E) and extinction was impaired (Figure 6F), though these phenotypes were less robust than those observed in the VTA GABA-only mice. Further analysis of extinction training days four and five revealed significant differences in both the number of lever presses and the rate of lever presses between

ABSTRACT The axonal guidance cue netrin-1 serves a critical role in neural circuit development by promoting growth cone motility, axonal branching, and synaptogenesis. Within the adult mouse brain, expression of the gene encoding netrin-1 ( Ntn1) is highly enriched in the ventral midbrain where it is expressed in both GABAergic and dopaminergic neurons, but its function in these cell types in the adult system remains largely unknown. To address this, we performed viral-mediated, cell-type specific CRISPR-Cas9 mutagenesis of Ntn1 in the ventral tegmental area (VTA) of adult mice. Ntn1 loss-of-function in either cell type resulted in a significant reduction in excitatory postsynaptic connectivity. In dopamine neurons, reduced excitatory tone had a minimal phenotypic behavioral outcome; however, reduced glutamatergic tone on VTA GABA neurons induced behaviors associated with a hyperdopaminergic phenotype. Loss of Ntn1 function in both cell types simultaneously largely rescued the phenotype observed in the GABA-only mutagenesis. These findings demonstrate an important role for netrin-1 in maintaining excitatory connectivity in the adult midbrain and that a balance in this connectivity within two of the major cell types of the VTA is critical for the proper functioning of the mesolimbic system.

Discrimination between predictive and non-predictive threat stimuli decreases as threat intensity increases. The central mechanisms that mediate the transition from discriminatory to generalized threat responding remain poorly resolved. Here, we identify the stress- and dysphoria-associated kappa opioid receptor (KOR) and its ligand dynorphin (Dyn), acting in the ventral tegmental area (VTA), as a key substrate for regulating threat generalization. We identify several dynorphinergic inputs to the VTA and demonstrate that projections from the bed nucleus of the stria terminalis (BNST) and dorsal raphe nucleus (DRN) both contribute to anxiety-like behavior but differentially affect threat generalization. These data demonstrate that conditioned threat discrimination has an inverted “U” relationship with threat intensity and establish a role for KOR/Dyn signaling in the midbrain for promoting threat generalization.

The ventral tegmental area (VTA) is important for reward processing and motivation. The anatomic organization of neurotransmitter-specific inputs to the VTA remains poorly resolved. In the present study, we mapped the major neurotransmitter projections to the VTA through cell-type-specific retrograde and anterograde tracing. We found that glutamatergic inputs arose from a variety of sources and displayed some connectivity biases toward specific VTA cell types. The sources of GABAergic projections were more widespread, displayed a high degree of differential innervation of subregions in the VTA and were largely biased toward synaptic contact with local GABA neurons. Inactivation of GABA release from the two major sources, locally derived versus distally derived, revealed distinct roles for these projections in behavioral regulation. Optogenetic manipulation of individual distal GABAergic inputs also revealed differential behavioral effects. These results demonstrate that GABAergic projections to the VTA are a major contributor to the regulation and diversification of the structure. Soden et al. use cell-type-specific retrograde tracing to identify neurotransmitter-specific inputs to the ventral tegmental area, uncovering an underappreciated number of GABAergic inputs with diverse innervation patterns and behavioral functions.

Mice engineered for conditional, cell type-specific gene inactivation have dominated the field of mouse genetics because of the high efficiency of Cre-loxP-mediated recombination. Recent advances in CRISPR/Cas9 technologies have provided alternatives for rapid gene mutagenesis for loss-of-function (LOF) analysis. Whether these strategies can be streamlined for rapid genetic analysis with the efficiencies comparable with those of conventional genetic approaches has yet to be established. We show that a single adeno-associated viral (AAV) vector containing a recombinase-dependent Staphylococcus aureus Cas9 (SaCas9) and a single guide RNA (sgRNA) are as efficient as conventional conditional gene knockout and can be adapted for use in either Cre- or Flp-driver mouse lines. The efficacy of this approach is demonstrated for the analysis of GABAergic, glutamatergic, and monoaminergic neurotransmission. Using this strategy, we reveal insight into the role of GABAergic regulation of midbrain GABA-producing neurons in psychomotor activation.

Dopamine neurons of the ventral tegmental area (VTA) regulate reward association and motivation. It remains unclear whether there are distinct dopamine populations to mediate these functions. Using mouse genetics, we isolated two populations of dopamine-producing VTA neurons with divergent projections to the nucleus accumbens (NAc) core and shell. Inhibition of VTA-core-projecting neurons disrupted Pavlovian reward learning, and activation of these cells promoted the acquisition of an instrumental response. VTA-shell-projecting neurons did not regulate Pavlovian reward learning and could not facilitate acquisition of an instrumental response, but their activation could drive robust responding in a previously learned instrumental task. Both populations are activated simultaneously by cues, actions, and rewards, and this co-activation is required for robust reinforcement of behavior. Thus, there are functionally distinct dopamine populations in the VTA for promoting motivation and reward association, which operate on the same timescale to optimize behavioral reinforcement.

BACKGROUND:Studies in humans and nonhuman primates have identified a region of the dentate nucleus of the cerebellum, or the lateral cerebellar nucleus (LCN) in rodents, activated during performance of cognitive tasks involving complex spatial and sequential planning. Whether such a subdivision exists in rodents is not known. Dopamine and its receptors, which are implicated in cognitive function, are present in the cerebellar nuclei, but their function is unknown.METHODS:Using viral and genetic strategies in mice, we examined cellular phenotypes of dopamine D1 receptor-positive (D1R+) cells in the LCN with whole-cell patch clamp recordings, messenger RNA profiling, and immunohistochemistry to examine D1R expression in mouse LCN and human dentate nucleus of the cerebellum. We used chemogenetics to inhibit D1R+ neurons and examined behaviors including spatial navigation, social recognition memory, prepulse inhibition of the acoustic startle reflex, response inhibition, and working memory to test the necessity of these neurons in these behaviors.RESULTS:We identified a population of D1R+ neurons that are localized to an anatomically distinct region of the LCN. We also observed D1R+ neurons in human dentate nucleus of the cerebellum, which suggests an evolutionarily conserved population of dopamine-receptive neurons in this region. The genetic, electrophysiological, and anatomical profile of mouse D1R neurons is consistent with a heterogeneous population of gamma-aminobutyric acidergic, and to a lesser extent glutamatergic, cell types. Selective inhibition of D1R+ LCN neurons impairs spatial navigation memory, response inhibition, working memory, and prepulse inhibition of the acoustic startle reflex.CONCLUSIONS:Collectively, these data demonstrate a functional link between genetically distinct neurons in the LCN and cognitive behaviors.