Elucidating an Affective Pain Circuit that Creates a Threat Memory.

Animals learn to avoid harmful situations by associating a neutral stimulus with a painful one, resulting in a stable threat memory. In mammals, this form of learning requires the amygdala. Although pain is the main driver of aversive learning, the mechanism that transmits pain signals to the amygdala is not well resolved. Here, we show that neurons expressing calcitonin gene-related peptide (CGRP) in the parabrachial nucleus are critical for relaying pain signals to the central nucleus of amygdala and that this pathway may transduce the affective motivational aspects of pain. Genetic silencing of CGRP neurons blocks pain responses and memory formation, whereas their optogenetic stimulation produces defensive responses and a threat memory. The pain-recipient neurons in the central amygdala expressing CGRP receptors are also critical for establishing a threat memory. The identification of the neural circuit conveying affective pain signals may be pertinent for treating pain conditions with psychiatric comorbidities.

Circuit coordination of opposing neuropeptide and neurotransmitter signals.

Fast-acting neurotransmitters and slow, modulatory neuropeptides are co-released from neurons in the central nervous system, albeit from distinct synaptic vesicles1. The mechanisms of how co-released neurotransmitters and neuropeptides that have opposing actions-for example, stimulatory versus inhibitory-work together to exert control of neural circuit output remain unclear. This has been difficult to resolve owing to the inability to selectively isolate these signalling pathways in a cell- and circuit-specific manner. Here we developed a genetic-based anatomical disconnect procedure that utilizes distinct DNA recombinases to independently facilitate CRISPR-Cas9 mutagenesis2 of neurotransmitter- and neuropeptide-related genes in distinct cell types in two different brain regions simultaneously. We demonstrate that neurons within the lateral hypothalamus that produce the stimulatory neuropeptide neurotensin and the inhibitory neurotransmitter GABA (γ-aminobutyric acid) utilize these signals to coordinately activate dopamine-producing neurons of the ventral tegmental area. We show that GABA release from lateral hypothalamus neurotensin neurons inhibits GABA neurons within the ventral tegmental area, disinhibiting dopamine neurons and causing a rapid rise in calcium, whereas neurotensin directly generates a slow inactivating calcium signal in dopamine neurons that is dependent on the expression of neurotensin receptor 1 (Ntsr1). We further show that these two signals work together to regulate dopamine neuron responses to maximize behavioural responding. Thus, a neurotransmitter and a neuropeptide with opposing signals can act on distinct timescales through different cell types to enhance circuit output and optimize behaviour.

The potassium channel auxiliary subunit Kvß2 (Kcnab2) regulates Kv1 channels and dopamine neuron firing.

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 Kvß2 (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 Kvß2 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 Kvß2, an auxiliary subunit that forms a part of Kv1 channel complexes. We find that the absence of Kvß2 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 Kvß2 subunits and Kv1 complexes in regulating dopamine neuron activity.

Genetic identification of a neural circuit that suppresses appetite.

Appetite suppression occurs after a meal and in conditions when it is unfavourable to eat, such as during illness or exposure to toxins. A brain region proposed to play a role in appetite suppression is the parabrachial nucleus, a heterogeneous population of neurons surrounding the superior cerebellar peduncle in the brainstem. The parabrachial nucleus is thought to mediate the suppression of appetite induced by the anorectic hormones amylin and cholecystokinin, as well as by lithium chloride and lipopolysaccharide, compounds that mimic the effects of toxic foods and bacterial infections, respectively. Hyperactivity of the parabrachial nucleus is also thought to cause starvation after ablation of orexigenic agouti-related peptide neurons in adult mice. However, the identities of neurons in the parabrachial nucleus that regulate feeding are unknown, as are the functionally relevant downstream projections. Here we identify calcitonin gene-related peptide-expressing neurons in the outer external lateral subdivision of the parabrachial nucleus that project to the laterocapsular division of the central nucleus of the amygdala as forming a functionally important circuit for suppressing appetite. Using genetically encoded anatomical, optogenetic and pharmacogenetic tools, we demonstrate that activation of these neurons projecting to the central nucleus of the amygdala suppresses appetite. In contrast, inhibition of these neurons increases food intake in circumstances when mice do not normally eat and prevents starvation in adult mice whose agouti-related peptide neurons are ablated. Taken together, our data demonstrate that this neural circuit from the parabrachial nucleus to the central nucleus of the amygdala mediates appetite suppression in conditions when it is unfavourable to eat. This neural circuit may provide targets for therapeutic intervention to overcome or promote appetite.

Reversal of Alcohol-Induced Dysregulation in Dopamine Network Dynamics May Rescue Maladaptive Decision-making.

Alcohol is the most commonly abused substance among adolescents, promoting the development of substance use disorders and compromised decision-making in adulthood. We have previously demonstrated, with a preclinical model in rodents, that adolescent alcohol use results in adult risk-taking behavior that positively correlates with phasic dopamine transmission in response to risky options, but the underlying mechanisms remain unknown. Here, we show that adolescent alcohol use may produce maladaptive decision-making through a disruption in dopamine network dynamics via increased GABAergic transmission within the ventral tegmental area (VTA). Indeed, we find that increased phasic dopamine signaling after adolescent alcohol use is attributable to a midbrain circuit, including the input from the pedunculopontine tegmentum to the VTA. Moreover, we demonstrate that VTA dopamine neurons from adult rats exhibit enhanced IPSCs after adolescent alcohol exposure corresponding to decreased basal dopamine levels in adulthood that negatively correlate with risk-taking. Building on these findings, we develop a model where increased inhibitory tone on dopamine neurons leads to a persistent decrease in tonic dopamine levels and results in a potentiation of stimulus-evoked phasic dopamine release that may drive risky choice behavior. Based on this model, we take a pharmacological approach to the reversal of risk-taking behavior through normalization of this pattern in dopamine transmission. These results isolate the underlying circuitry involved in alcohol-induced maladaptive decision-making and identify a novel therapeutic target. SIGNIFICANCE STATEMENT: One of the primary problems resulting from chronic alcohol use is persistent, maladaptive decision-making that is associated with ongoing addiction vulnerability and relapse. Indeed, studies with the Iowa Gambling Task, a standard measure of risk-based decision-making, have reliably shown that alcohol-dependent individuals make riskier, more maladaptive choices than nondependent individuals, even after periods of prolonged abstinence. Using a preclinical model, in the current work, we identify a selective disruption in dopamine network dynamics that may promote maladaptive decision-making after chronic adolescent alcohol use and demonstrate its pharmacological reversal in adulthood. Together, these results highlight a novel neural mechanism underlying heightened risk-taking behavior in alcohol-dependent individuals and provide a potential therapeutic target for further investigation.

Visualization of plasticity in fear-evoked calcium signals in midbrain dopamine neurons.

Dopamine is broadly implicated in fear-related processes, yet we know very little about signaling dynamics in these neurons during active fear conditioning. We describe the direct imaging of calcium signals of dopamine neurons during Pavlovian fear conditioning using fiber-optic confocal microscopy coupled with the genetically encoded calcium indicator GCaMP3. We observed calcium transients in a subset of dopamine neurons to an unconditioned fear stimulus on the first day of Pavlovian fear conditioning. On the second day, calcium transients occurred in response to conditioned and unconditioned stimuli. These results demonstrate plasticity in dopamine neuron calcium signals and the occurrence of activity-dependent processes in these neurons during fear conditioning.

Opto-seq reveals input-specific immediate-early gene induction in ventral tegmental area cell types.

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.

Netrin-1 regulates the balance of synaptic glutamate signaling in the adult ventral tegmental area.

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.

Cocaine Seeking And Taking Are Oppositely Regulated By Dopamine.

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.

Temporal scaling of dopamine neuron firing and dopamine release by distinct ion channels shape behavior.

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.

Attenuating GABA(A) receptor signaling in dopamine neurons selectively enhances reward learning and alters risk preference in mice.

Phasic dopamine (DA) transmission encodes the value of reward-predictive stimuli and influences both learning and decision-making. Altered DA signaling is associated with psychiatric conditions characterized by risky choices such as pathological gambling. These observations highlight the importance of understanding how DA neuron activity is modulated. While excitatory drive onto DA neurons is critical for generating phasic DA responses, emerging evidence suggests that inhibitory signaling also modulates these responses. To address the functional importance of inhibitory signaling in DA neurons, we generated mice lacking the ß3 subunit of the GABA(A) receptor specifically in DA neurons (ß3-KO mice) and examined their behavior in tasks that assessed appetitive learning, aversive learning, and risk preference. DA neurons in midbrain slices from ß3-KO mice exhibited attenuated GABA-evoked IPSCs. Furthermore, electrical stimulation of excitatory afferents to DA neurons elicited more DA release in the nucleus accumbens of ß3-KO mice as measured by fast-scan cyclic voltammetry. ß3-KO mice were more active than controls when given morphine, which correlated with potential compensatory upregulation of GABAergic tone onto DA neurons. ß3-KO mice learned faster in two food-reinforced learning paradigms, but extinguished their learned behavior normally. Enhanced learning was specific for appetitive tasks, as aversive learning was unaffected in ß3-KO mice. Finally, we found that ß3-KO mice had enhanced risk preference in a probabilistic selection task that required mice to choose between a small certain reward and a larger uncertain reward. Collectively, these findings identify a selective role for GABA(A) signaling in DA neurons in appetitive learning and decision-making.

Distinct Encoding of Reward and Aversion by Peptidergic BNST Inputs to the VTA.

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.

Fragile X protein FMRP is required for homeostatic plasticity and regulation of synaptic strength by retinoic acid.

Homeostatic synaptic plasticity adjusts the strength of synapses during global changes in neural activity, thereby stabilizing the overall activity of neural networks. Suppression of synaptic activity increases synaptic strength by inducing synthesis of retinoic acid (RA), which activates postsynaptic synthesis of AMPA-type glutamate receptors (AMPARs) in dendrites and promotes synaptic insertion of newly synthesized AMPARs. Here, we show that fragile X mental retardation protein (FMRP), an RNA-binding protein that regulates dendritic protein synthesis, is essential for increases in synaptic strength induced by RA or by blockade of neural activity in the mouse hippocampus. Although activity-dependent RA synthesis is maintained in Fmr1 knock-out neurons, RA-dependent dendritic translation of GluR1-type AMPA receptors is impaired. Intriguingly, FMRP is only required for the form of homeostatic plasticity that is dependent on both RA signaling and local protein synthesis. Postsynaptic expression of wild-type or mutant FMRP(I304N) in knock-out neurons reduced the total, surface, and synaptic levels of AMPARs, implying a role for FMRP in regulating AMPAR abundance. Expression of FMRP lacking the RGG box RNA-binding domain had no effect on AMPAR levels. Importantly, postsynaptic expression of wild-type FMRP, but not FMRP(I304N) or FMRPΔRGG, restored synaptic scaling when expressed in knock-out neurons. Together, these findings identify an unanticipated role for FMRP in regulating homeostatic synaptic plasticity downstream of RA. Our results raise the possibility that at least some of the symptoms of fragile X syndrome reflect impaired homeostatic plasticity and impaired RA signaling.

A midbrain dynorphin circuit promotes threat generalization.

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.

Conditional Single Vector CRISPR/SaCas9 Viruses for Efficient Mutagenesis in the Adult Mouse Nervous System.

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.

Synergy of Distinct Dopamine Projection Populations in Behavioral Reinforcement.

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.

Anatomic resolution of neurotransmitter-specific projections to the VTA reveals diversity of GABAergic inputs.

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.

Dentate granule cell recruitment of feedforward inhibition governs engram maintenance and remote memory generalization.

Memories become less precise and generalized over time as memory traces reorganize in hippocampal-cortical networks. Increased time-dependent loss of memory precision is characterized by an overgeneralization of fear in individuals with post-traumatic stress disorder (PTSD) or age-related cognitive impairments. In the hippocampal dentate gyrus (DG), memories are thought to be encoded by so-called 'engram-bearing' dentate granule cells (eDGCs). Here we show, using rodents, that contextual fear conditioning increases connectivity between eDGCs and inhibitory interneurons (INs) in the downstream hippocampal CA3 region. We identify actin-binding LIM protein 3 (ABLIM3) as a mossy-fiber-terminal-localized cytoskeletal factor whose levels decrease after learning. Downregulation of ABLIM3 expression in DGCs was sufficient to increase connectivity with CA3 stratum lucidum INs (SLINs), promote parvalbumin (PV)-expressing SLIN activation, enhance feedforward inhibition onto CA3 and maintain a fear memory engram in the DG over time. Furthermore, downregulation of ABLIM3 expression in DGCs conferred conditioned context-specific reactivation of memory traces in hippocampal-cortical and amygdalar networks and decreased fear memory generalization at remote (i.e., distal) time points. Consistent with the observation of age-related hyperactivity of CA3, learning failed to increase DGC-SLIN connectivity in 17-month-old mice, whereas downregulation of ABLIM3 expression was sufficient to restore DGC-SLIN connectivity, increase PV+ SLIN activation and improve the precision of remote memories. These studies exemplify a connectivity-based strategy that targets a molecular brake of feedforward inhibition in DG-CA3 and may be harnessed to decrease time-dependent memory generalization in individuals with PTSD and improve memory precision in aging individuals.

Dopamine D1 Receptor-Positive Neurons in the Lateral Nucleus of the Cerebellum Contribute to Cognitive Behavior.

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.