Behavioral Neuroscience publications

The presence of valence coding neurons in the basolateral amygdala (BLA) that form distinct projections to other brain regions implies functional opposition between aversion and reward during learning. However, evidence for opponent interactions in fear learning is sparse and may only be apparent under certain conditions. Here we test this possibility by studying the roles of the BLA→central amygdala (CeA) and BLA→nucleus accumbens (Acb) pathways in fear learning in male rats. First, we assessed the organisation of these pathways in the rat brain. BLA→CeA and BLA→Acb pathways were largely segregated in the BLA but shared overlapping molecular profiles. Then we assessed activity of the BLA→CeA and BLA→Acb pathways during two different forms of fear learning - fear learning in a neutral context and fear learning in a reward context. BLA → CeA neurons were robustly recruited by footshock regardless of where fear learning occurred whereas recruitment of BLA→Acb neurons was state-dependent because footshock only recruited this pathway in a reward context. Finally, we assessed the causal roles of activity in these pathways in fear learning. Photoinhibition of the BLA→CeA pathway during the footshock US impaired fear learning, regardless of where fear learning occurred. In contrast, photoinhibition of the BLA→Acb pathway augmented fear learning, but only in the reward context. Taken together, our findings show circuit- and state-dependent opponent processing of fear. Footshock activity in the BLA → Acb pathway limits how much fear is learned. Significance Statement Here we identify a fear opponent process in the brain. We show that an aversive event can recruit distinct populations of neurons in the rat basolateral amygdala. One population projects to the central amygdala to promote fear learning. A second population projects to the nucleus accumbens to oppose fear learning. These nucleus accumbens projecting neurons limit how much fear is learned and are candidates for therapeutic targeting to minimize the amount of fear learned after a traumatic experience.

Reactive astrocytes are associated with neuroinflammation and cognitive decline in diverse neuropathologies; however, the underlying mechanisms are unclear. We used optogenetic and chemogenetic tools to identify the crucial roles of the hippocampal CA1 astrocytes in cognitive decline. Our results showed that repeated optogenetic stimulation of the hippocampal CA1 astrocytes induced cognitive impairment in mice and decreased synaptic long-term potentiation (LTP), which was accompanied by the appearance of inflammatory astrocytes. Mechanistic studies conducted using knockout animal models and hippocampal neuronal cultures showed that lipocalin-2 (LCN2), derived from reactive astrocytes, mediated neuroinflammation and induced cognitive impairment by decreasing the LTP through the reduction of neuronal NMDA receptors. Sustained chemogenetic stimulation of hippocampal astrocytes provided similar results. Conversely, these phenomena were attenuated by a metabolic inhibitor of astrocytes. Fiber photometry using GCaMP revealed a high level of hippocampal astrocyte activation in the neuroinflammation model. Our findings suggest that reactive astrocytes in the hippocampus are sufficient and required to induce cognitive decline through LCN2 release and synaptic modulation. This abnormal glial–neuron interaction may contribute to the pathogenesis of cognitive disturbances in neuroinflammation-associated brain conditions.

The synaptojanin-1 ( SYNJ1 ) gene is known to be important for dopamine-related disorders. Recent evidence has demonstrated that Synj1 deficient mice ( Synj1 +/− ) have impairments in dopaminergic synaptic vesicular recycling. However, less is known about how Synj1 deficits affect the mesolimbic system, reward processing, and motivated behavior. To examine the role of the Synj1 gene in motivated behavior, we subjected male and female Synj1 +/− and Synj1 +/+ mice to a battery of behavioral tests evaluating hedonic responses, effortful responding, and responses to psychomotor stimulants. We observed that Synj1 +/− mice exhibit few differences in reward processing and motivated behavior, with normal hedonic responses and motivated responding for sucrose. However, male but not female Synj1 +/− demonstrated an attenuated conditioned place preference for cocaine that could not be attributed to deficits in spatial memory. To further understand the dopamine signaling underlying the attenuated response to cocaine in these mutant mice, we recorded nucleus accumbens dopamine in response to cocaine and observed that Synj1 +/− male and female mice took longer to reach peak dopamine release following experimenter-administered cocaine. However, female mice also showed slower decay in accumbens dopamine that appear to be linked to differences in cocaine-induced DAT responses. These findings demonstrate that SYNJ1 deficiencies result in abnormal mesolimbic DA signaling which has not previously been demonstrated. Our work also highlights the need to develop targeted therapeutics capable of restoring deficits in DAT function, which may be effective for reversing the pathologies associated with Synj1 mutations.

Genetically-encoded dopamine (DA) sensors enable high-resolution imaging of DA release, but their ability to detect a wide range of extracellular DA levels, especially tonic versus phasic DA release, is limited by their intrinsic affinity. Here we show that a human-selective dopamine receptor positive allosteric modulator (PAM) can be used to boost sensor affinity on-demand. The PAM enhances DA detection sensitivity across experimental preparations (in vitro, ex vivo and in vivo) via one-photon or two-photon imaging. In vivo photometry-based detection of optogenetically-evoked DA release revealed that DETQ administration produces a stable 31 minutes window of potentiation without effects on animal behavior. The use of the PAM revealed region-specific and metabolic state-dependent differences in tonic DA levels and enhanced single-trial detection of behavior-evoked phasic DA release in cortex and striatum. Our chemogenetic strategy can potently and flexibly tune DA imaging sensitivity and reveal multi-modal (tonic/phasic) DA signaling across preparations and imaging approaches.

Failure to appropriately predict and titrate reactivity to threat is a core feature of fear and anxiety-related disorders and is common following early life adversity (ELA). A population of neurons in the lateral central amygdala (CeAL) expressing corticotropin releasing factor (CRF) have been proposed to be key in processing threat of different intensities to mediate active fear expression. Here, we use in vivo fiber photometry to show that ELA results in sex-specific changes in the activity of CeAL CRF+ neurons, yielding divergent mechanisms underlying the augmented startle in ELA mice, a translationally relevant behavior indicative of heightened threat reactivity and hypervigilance. Further, chemogenic inhibition of CeAL CRF+ neurons selectively diminishes startle and produces a long-lasting suppression of threat reactivity. These findings identify a mechanism for sex-differences in susceptibility for anxiety following ELA and have broad implications for understanding the neural circuitry that encodes and gates the behavioral expression of fear.

The claustrum has been linked to attention and sleep. We hypothesized that this reflects a shared function, determining responsiveness to stimuli, which spans the axis of engagement. To test this hypothesis, we recorded claustrum population dynamics from male mice during both sleep and an attentional task (‘ENGAGE’). Heightened activity in claustrum neurons projecting to the anterior cingulate cortex (ACCp) corresponded to reduced sensory responsiveness during sleep. Similarly, in the ENGAGE task, heightened ACCp activity correlated with disengagement and behavioral lapses, while low ACCp activity correlated with hyper-engagement and impulsive errors. Chemogenetic elevation of ACCp activity reduced both awakenings during sleep and impulsive errors in the ENGAGE task. Furthermore, mice employing an exploration strategy in the task showed a stronger correlation between ACCp activity and performance compared to mice employing an exploitation strategy which reduced task complexity. Our results implicate ACCp claustrum neurons in restricting engagement during sleep and goal-directed behavior.

Nociceptin/orphanin-FQ (N/OFQ) is a recently appreciated critical opioid peptide with key regulatory functions in several central behavioral processes including motivation, stress, feeding, and sleep. The functional relevance of N/OFQ action in the mammalian brain remains unclear due to a lack of high-resolution approaches to detect this neuropeptide with appropriate spatial and temporal resolution. Here we develop and characterize NOPLight, a genetically encoded sensor that sensitively reports changes in endogenous N/OFQ release. We characterized the affinity, pharmacological profile, spectral properties, kinetics, ligand selectivity, and potential interaction with intracellular signal transducers of NOPLight in vitro. Its functionality was established in acute brain slices by exogeneous N/OFQ application and chemogenetic induction of endogenous N/OFQ release from PNOC neurons. In vivo studies with fibre photometry enabled direct recording of NOPLight binding to exogenous N/OFQ receptor ligands, as well as detection of endogenous N/OFQ release within the paranigral ventral tegmental area (pnVTA) during natural behaviors and chemogenetic activation of PNOC neurons. In summary, we show here that NOPLight can be used to detect N/OFQ opioid peptide signal dynamics in tissue and freely behaving animals.

All drugs of abuse induce long-lasting changes in synaptic transmission and neural circuit function that underlie substance-use disorders 1,2 . Another recently appreciated mechanism of neural circuit plasticity is mediated through activity-regulated changes in myelin that can tune circuit function and influence cognitive behaviour 3–7 . Here we explore the role of myelin plasticity in dopaminergic circuitry and reward learning. We demonstrate that dopaminergic neuronal activity-regulated myelin plasticity is a key modulator of dopaminergic circuit function and opioid reward. Oligodendroglial lineage cells respond to dopaminergic neuronal activity evoked by optogenetic stimulation of dopaminergic neurons, optogenetic inhibition of GABAergic neurons, or administration of morphine. These oligodendroglial changes are evident selectively within the ventral tegmental area but not along the axonal projections in the medial forebrain bundle nor within the target nucleus accumbens. Genetic blockade of oligodendrogenesis dampens dopamine release dynamics in nucleus accumbens and impairs behavioural conditioning to morphine. Taken together, these findings underscore a critical role for oligodendrogenesis in reward learning and identify dopaminergic neuronal activity-regulated myelin plasticity as an important circuit modification that is required for opioid reward.

Learning causal relationships relies on understanding how often one event precedes another. To investigate how dopamine neuron activity and neurotransmitter release change when a retrospective relationship is degraded for a specific pair of events, we used outcome-selective Pavlovian contingency degradation in rats. Conditioned responding was attenuated for the cue-reward contingency that was degraded, as was dopamine neuron activity in the midbrain and dopamine release in the ventral striatum in response to the cue and subsequent reward. Contingency degradation also abolished the trial-by-trial history dependence of the dopamine responses at the time of trial outcome. This profile of changes in cue- and reward-evoked responding is not easily explained by a standard reinforcement learning model. An alternative model based on learning causal relationships was better able to capture dopamine responses during contingency degradation, as well as conditioned behavior following optogenetic manipulations of dopamine during noncontingent rewards. Our results suggest that mesostriatal dopamine encodes the contingencies between meaningful events during learning.

Information is transmitted between brain regions through the release of neurotransmitters from long-range projecting axons. Understanding how the activity of such long-range connections contributes to behavior requires efficient methods for reversibly manipulating their function. Chemogenetic and optogenetic tools, acting through endogenous G-protein-coupled receptor pathways, can be used to modulate synaptic transmission, but existing tools are limited in sensitivity, spatiotemporal precision or spectral multiplexing capabilities. Here we systematically evaluated multiple bistable opsins for optogenetic applications and found that the Platynereis dumerilii ciliary opsin ( Pd CO) is an efficient, versatile, light-activated bistable G-protein-coupled receptor that can suppress synaptic transmission in mammalian neurons with high temporal precision in vivo. Pd CO has useful biophysical properties that enable spectral multiplexing with other optogenetic actuators and reporters. We demonstrate that Pd CO can be used to conduct reversible loss-of-function experiments in long-range projections of behaving animals, thereby enabling detailed synapse-specific functional circuit mapping.

Binge alcohol consumption during adolescence can produce lasting deficits in learning and memory while also increasing the susceptibility to substance use disorders. The adolescent intermittent ethanol (AIE) rodent model mimics human adolescent binge drinking and has identified the nucleus basalis magnocellularis (NbM) as a key site of pathology. The NbM is a critical regulator of prefrontal cortical (PFC) cholinergic function and attention. The cholinergic phenotype is controlled pro/mature neurotrophin receptor activation. We sought to determine if p75NTR activity contributes to the loss of cholinergic phenotype in AIE by using a p75NTR modulator (LM11A-31) to inhibit prodegenerative signaling during ethanol exposure. Male and female rats underwent 5 g/kg ethanol (AIE) or water (CON) exposure following 2-day-on 2-day-off cycles from postnatal day 25–57. A subset of these groups also received a protective dose of LM11A-31 (50 mg/kg) during adolescence. Rats were trained on a sustained attention task (SAT) and behaviorally relevant acetylcholine (ACh) activity was recorded in the PFC with a fluorescent indicator (AChGRAB 3.0). AIE produced learning deficits on the SAT, which were spared with LM11A-31. In addition, PFC ACh activity was blunted by AIE, which LM11A-31 corrected. Investigation of NbM ChAT+ and TrkA+ neuronal expression found that AIE led to a reduction of ChAT+TrkA+ neurons, which again LM11A-31 protected. Taken together, these findings demonstrate the p75NTR activity during AIE treatment is a key regulator of cholinergic degeneration.

Fentanyl is a powerful painkiller that elicits euphoria and positive reinforcement 1 . Fentanyl also leads to dependence, defined by the aversive withdrawal syndrome, which fuels negative reinforcement 2,3 (that is, individuals retake the drug to avoid withdrawal). Positive and negative reinforcement maintain opioid consumption, which leads to addiction in one-fourth of users, the largest fraction for all addictive drugs 4 . Among the opioid receptors, µ-opioid receptors have a key role 5 , yet the induction loci of circuit adaptations that eventually lead to addiction remain unknown. Here we injected mice with fentanyl to acutely inhibit γ-aminobutyric acid-expressing neurons in the ventral tegmental area (VTA), causing disinhibition of dopamine neurons, which eventually increased dopamine in the nucleus accumbens. Knockdown of µ-opioid receptors in VTA abolished dopamine transients and positive reinforcement, but withdrawal remained unchanged. We identified neurons expressing µ-opioid receptors in the central amygdala (CeA) whose activity was enhanced during withdrawal. Knockdown of µ-opioid receptors in CeA eliminated aversive symptoms, suggesting that they mediate negative reinforcement. Thus, optogenetic stimulation caused place aversion, and mice readily learned to press a lever to pause optogenetic stimulation of CeA neurons that express µ-opioid receptors. Our study parses the neuronal populations that trigger positive and negative reinforcement in VTA and CeA, respectively. We lay out the circuit organization to develop interventions for reducing fentanyl addiction and facilitating rehabilitation.

The ventral pallidum (VP) contains GABA and glutamate neurons projecting to ventral tegmental area (VTA) whose stimulation drives approach and avoidance, respectively. Yet little is known about the mechanisms by which VP cell types shape VTA activity and drive behavior. Here, we found that both VP GABA and glutamate neurons were activated during approach to reward or by delivery of an aversive stimulus. Stimulation of VP GABA neurons inhibited VTA GABA, but activated dopamine and glutamate neurons. Remarkably, stimulation-evoked activation was behavior-contingent such that VTA recruitment was inhibited when evoked by the subject’s own action. Conversely, VP glutamate neurons activated VTA GABA, as well as dopamine and glutamate neurons, despite driving aversion. However, VP glutamate neurons evoked dopamine in aversion-associated ventromedial nucleus accumbens (NAc), but reduced dopamine release in reward-associated dorsomedial NAc. These findings show how heterogeneous VP projections to VTA can be engaged to shape approach and avoidance behaviors.

Dopamine neurons in the ventral tegmental area support intracranial self-stimulation (ICSS), yet the cognitive representations underlying this phenomenon remain unclear. Here, 20-Hz stimulation of dopamine neurons, which approximates a physiologically relevant prediction error, was not sufficient to support ICSS beyond a continuously reinforced schedule and did not endow cues with a general or specific value. However, 50-Hz stimulation of dopamine neurons was sufficient to drive robust ICSS and was represented as a specific reward to motivate behavior. The frequency dependence of this effect is due to the rate (not the number) of action potentials produced by dopamine neurons, which differently modulates dopamine release downstream.

Puberty is a crucial phase for the development of female sexual behavior. Growing evidence suggests that stress during this period may interfere with the development of sexual behavior. However, the neural circuits involved in this alteration remain elusive. Here, we demonstrated in mice that pubertal stress permanently disrupted sexual performance without affecting sexual preference. This was associated with a reduced expression and activation of neuronal nitric oxide synthase (nNOS) in the ventrolateral part of the ventromedial hypothalamus (VMHvl). Fiber photometry revealed that VMHvl nNOS neurons are strongly responsive to male olfactory cues with this activation being substantially reduced in pubertally stressed females. Finally, treatment with a NO donor partially restored sexual performance in pubertally stressed females. This study provides insights into the involvement of VMHvl nNOS in the processing of olfactory cues important for the expression of female sexual behavior. In addition, exposure to stress during puberty disrupts the integration of male olfactory cues leading to reduced sexual behavior.

MDMA (3,4-methylenedioxymethamphetamine) is a psychoactive drug with powerful prosocial effects. While MDMA is sometimes termed an “empathogen,” empirical studies have struggled to clearly demonstrate these effects or pinpoint underlying mechanisms. Here, we paired the social transfer of pain and analgesia—behavioral tests modeling empathy in mice—with region-specific neuropharmacology, optogenetics, and transgenic manipulations to explore MDMA’s action as an empathogen. We report that MDMA, given intraperitoneally or infused directly into the nucleus accumbens (NAc), robustly enhances the social transfer of pain and analgesia. Optogenetic stimulation of 5-HT release in the NAc recapitulates the effects of MDMA, implicating 5-HT signaling as a core mechanism. Last, we demonstrate that systemic MDMA or optogenetic stimulation of NAc 5-HT inputs restores deficits in empathy-like behaviors in the Shank3- deficient mouse model of autism. These findings demonstrate enhancement of empathy-related behaviors by MDMA and implicate 5-HT signaling in the NAc as a core mechanism mediating MDMA’s empathogenic effects.

Episodic memory function is predicated upon the precise coordination between the hippocampus and widespread cortical regions. However, our understanding of the neural mechanisms involved in this process is incomplete. In this study, human subjects undergoing intracranial electroencephalography (iEEG) monitoring performed a list learning task. We show sharp-wave ripple (SWR)-locked reactivation of specific semantic processing regions during free recall. This cortical activation consists of both broadband high frequency (non-oscillatory) and cortical ripple (oscillatory) activity. SWRs and cortical ripples in the anterior temporal lobe, a major semantic hub, co-occur and increase in rate prior to recall. Coincident hippocampal-ATL ripples are associated with a greater increase in cortical reactivation, show specificity in location based on recall content, and are preceded by cortical theta oscillations. These findings may represent a reactivation of hippocampus and cortical semantic regions orchestrated by an interplay between hippocampal SWRs, cortical ripples, and theta oscillations.

Dopamine and orexins (hypocretins) play important roles in regulating reward-seeking behaviors. It is known that hypothalamic orexinergic neurons project to dopamine neurons in the ventral tegmental area (VTA), where they can stimulate dopaminergic neuronal activity. Although there are reciprocal connections between dopaminergic and orexinergic systems, whether and how dopamine regulates the activity of orexin neurons is currently not known. Here we implemented an opto-Pavlovian task in which mice learn to associate a sensory cue with optogenetic dopamine neuron stimulation to investigate the relationship between dopamine release and orexin neuron activity in the lateral hypothalamus (LH). We found that dopamine release can be evoked in LH upon optogenetic stimulation of VTA dopamine neurons and is also naturally evoked by cue presentation after opto-Pavlovian learning. Furthermore, orexin neuron activity could also be upregulated by local stimulation of dopaminergic terminals in the LH in a way that is partially dependent on dopamine D2 receptors (DRD2). Our results reveal previously unknown orexinergic coding of reward expectation and unveil an orexin-regulatory axis mediated by local dopamine inputs in the LH.

Psychedelics produce lasting therapeutic responses in neuropsychiatric diseases suggesting they may disrupt entrenched associations and catalyze learning. Here, we examine psychedelic effects on dopamine signaling in the nucleus accumbens (NAc) core, a region extensively linked to reward learning, motivation, and drug-seeking. We measure phasic dopamine transients following acute psychedelic administration during well learned Pavlovian tasks in which sequential cues predict rewards. We find that the psychedelic 5-HT 2A/2C agonist, DOI, increases dopamine signaling to rewards and proximal reward cues but not to the distal cues that predict these events. We determine that the elevated dopamine produced by psychedelics to reward cues occurs independently of psychedelic-induced changes in reward value. The increased dopamine associated with predictable reward cues supports psychedelic-induced increases in prediction error signaling. These findings lay a foundation for developing psychedelic strategies aimed at engaging error-driven learning mechanisms to disrupt entrenched associations or produce new associations.

The entorhinal cortex is involved in establishing enduring visuo-auditory associative memory in the neocortex. Here we explored the mechanisms underlying this synaptic plasticity related to projections from the visual and entorhinal cortices to the auditory cortex in mice using optogenetics of dual pathways. High-frequency laser stimulation (HFS laser) of the visuo-auditory projection did not induce long-term potentiation. However, after pairing with sound stimulus, the visuo-auditory inputs were potentiated following either infusion of cholecystokinin (CCK) or HFS laser of the entorhino-auditory CCK-expressing projection. Combining retrograde tracing and RNAscope in situ hybridization, we show that Cck expression is higher in entorhinal cortex neurons projecting to the auditory cortex than in those originating from the visual cortex. In the presence of CCK, potentiation in the neocortex occurred when the presynaptic input arrived 200 ms before postsynaptic firing, even after just five trials of pairing. Behaviorally, inactivation of the CCK + projection from the entorhinal cortex to the auditory cortex blocked the formation of visuo-auditory associative memory. Our results indicate that neocortical visuo-auditory association is formed through heterosynaptic plasticity, which depends on release of CCK in the neocortex mostly from entorhinal afferents.