Top-down control of flight by a non-canonical cortico-amygdala pathway.

Survival requires the selection of appropriate behaviour in response to threats, and dysregulated defensive reactions are associated with psychiatric illnesses such as post-traumatic stress and panic disorder1. Threat-induced behaviours, including freezing and flight, are controlled by neuronal circuits in the central amygdala (CeA)2; however, the source of neuronal excitation of the CeA that contributes to high-intensity defensive responses is unknown. Here we used a combination of neuroanatomical mapping, in vivo calcium imaging, functional manipulations and electrophysiology to characterize a previously unknown projection from the dorsal peduncular (DP) prefrontal cortex to the CeA. DP-to-CeA neurons are glutamatergic and specifically target the medial CeA, the main amygdalar output nucleus mediating conditioned responses to threat. Using a behavioural paradigm that elicits both conditioned freezing and flight, we found that CeA-projecting DP neurons are activated by high-intensity threats in a context-dependent manner. Functional manipulations revealed that the DP-to-CeA pathway is necessary and sufficient for both avoidance behaviour and flight. Furthermore, we found that DP neurons synapse onto neurons within the medial CeA that project to midbrain flight centres. These results elucidate a non-canonical top-down pathway regulating defensive responses.

Sucrose overconsumption impairs AgRP neuron dynamics and promotes palatable food intake.

Rapid gut-brain communication is critical to maintain energy balance and is disrupted in diet-induced obesity. In particular, the role of carbohydrate overconsumption in the regulation of interoceptive circuits in vivo requires further investigation. Here, we report that an obesogenic high-sucrose diet (HSD) selectively blunts silencing of hunger-promoting agouti-related protein (AgRP) neurons following intragastric delivery of glucose, whereas we previously showed that overconsumption of a high-fat diet (HFD) selectively attenuates lipid-induced neural silencing. By contrast, both HSD and HFD reversibly dampen rapid AgRP neuron inhibition following chow presentation and promote intake of more palatable foods. Our findings reveal that excess sugar and fat pathologically modulate feeding circuit activity in both macronutrient-dependent and -independent ways and thus may additively exacerbate obesity.

Antipsychotic drug efficacy correlates with the modulation of D1 rather than D2 receptor-expressing striatal projection neurons.

Elevated dopamine transmission in psychosis is assumed to unbalance striatal output through D1- and D2-receptor-expressing spiny-projection neurons (SPNs). Antipsychotic drugs are thought to re-balance this output by blocking D2 receptors (D2Rs). In this study, we found that amphetamine-driven dopamine release unbalanced D1-SPN and D2-SPN Ca2+ activity in mice, but that antipsychotic efficacy was associated with the reversal of abnormal D1-SPN, rather than D2-SPN, dynamics, even for drugs that are D2R selective or lacking any dopamine receptor affinity. By contrast, a clinically ineffective drug normalized D2-SPN dynamics but exacerbated D1-SPN dynamics under hyperdopaminergic conditions. Consistent with antipsychotic effect, selective D1-SPN inhibition attenuated amphetamine-driven changes in locomotion, sensorimotor gating and hallucination-like perception. Notably, antipsychotic efficacy correlated with the selective inhibition of D1-SPNs only under hyperdopaminergic conditions-a dopamine-state-dependence exhibited by D1R partial agonism but not non-antipsychotic D1R antagonists. Our findings provide new insights into antipsychotic drug mechanism and reveal an important role for D1-SPN modulation.

The effect of selective nigrostriatal dopamine excess on behaviors linked to the cognitive and negative symptoms of schizophrenia.

Excess dopamine release in the dorsal striatum (DS) is linked to psychosis. Antipsychotics are thought to work by blocking striatal D2 dopamine receptors, but they lack efficacy for the negative and cognitive symptoms of schizophrenia. These observations and the fact that increasing brain-wide dopamine improves cognition have fueled the dogma that excess dopamine is not involved in negative and cognitive symptoms. However, this idea has never been explicitly tested with DS-pathway specificity. To determine if excess DS dopamine is involved in cognitive and negative symptoms, we selectively re-expressed excitatory TRPV1 receptors in DS-projecting dopamine neurons of Trpv1 knockout mice. We treated these mice with capsaicin (TRPV1 agonist) to selectively activate these neurons, validated this approach with fiber photometry, and assessed its effects on social interaction and working memory, behavioral constructs related to negative and cognitive symptoms. We combined this manipulation with antipsychotic treatment (haloperidol) and compared it to brain-wide dopamine release via amphetamine treatment. We found that selectively activating DS-projecting dopamine neurons increased DS (but not cortical) dopamine release and increased locomotor activity. Surprisingly, this manipulation also impaired social interaction and working memory. Haloperidol normalized locomotion, but only partially rescued working memory and had no effect on social interaction. By contrast, amphetamine increased locomotion but did not impair social interaction or working memory. These results suggest that excess dopamine release, when restricted to the DS, causes behavioral deficits linked to negative and cognitive symptoms. Future therapies should address this disregarded role for excess striatal dopamine in the treatment-resistant symptoms of psychosis.

Fiber photometry in striatum reflects primarily nonsomatic changes in calcium.

Fiber photometry enables recording of population neuronal calcium dynamics in awake mice. While the popularity of fiber photometry has grown in recent years, it remains unclear whether photometry reflects changes in action potential firing (that is, 'spiking') or other changes in neuronal calcium. In microscope-based calcium imaging, optical and analytical approaches can help differentiate somatic from neuropil calcium. However, these approaches cannot be readily applied to fiber photometry. As such, it remains unclear whether the fiber photometry signal reflects changes in somatic calcium, changes in nonsomatic calcium or a combination of the two. Here, using simultaneous in vivo extracellular electrophysiology and fiber photometry, along with in vivo endoscopic one-photon and two-photon calcium imaging, we determined that the striatal fiber photometry does not reflect spiking-related changes in calcium and instead primarily reflects nonsomatic changes in calcium.

Calcium imaging in freely-moving mice during electrical stimulation of deep brain structures.

After decades of study in humans and animal models, there remains a lack of consensus regarding how the action of electrical stimulation on neuronal and non-neuronal elements - e.g. neuropil, cell bodies, glial cells, etc. - leads to the therapeutic effects of neuromodulation therapies. To further our understanding of neuromodulation therapies, there is a critical need for novel methodological approaches using state-of-the-art neuroscience tools to study neuromodulation therapy in preclinical models of disease. In this manuscript we outline one such approach combining chronic behaving single-photon microendoscope recordings in a pathological mouse model with electrical stimulation of a common deep brain stimulation (DBS) target. We describe in detail the steps necessary to realize this approach, as well as discuss key considerations for extending this experimental paradigm to other DBS targets for different therapeutic indications. Additionally, we make recommendations from our experience on implementing and validating the required combination of procedures that includes: the induction of a pathological model (6-OHDA model of Parkinson's disease) through an injection procedure, the injection of the viral vector to induce GCaMP expression, the implantation of the GRIN lens and stimulation electrode, and the installation of a baseplate for mounting the microendoscope. We proactively identify unique data analysis confounds occurring due to the combination of electrical stimulation and optical recordings and outline an approach to address these confounds. In order to validate the technical feasibility of this unique combination of experimental methods, we present data to demonstrate that 1) despite the complex multifaceted surgical procedures, chronic optical recordings of hundreds of cells combined with stimulation is achievable over week long periods 2) this approach enables measurement of differences in DBS evoked neural activity between anesthetized and awake conditions and 3) this combination of techniques can be used to measure electrical stimulation induced changes in neural activity during behavior in a pathological mouse model. These findings are presented to underscore the feasibility and potential utility of minimally constrained optical recordings to elucidate the mechanisms of DBS therapies in animal models of disease.

Diametric neural ensemble dynamics in parkinsonian and dyskinetic states.

Loss of dopamine in Parkinson's disease is hypothesized to impede movement by inducing hypo- and hyperactivity in striatal spiny projection neurons (SPNs) of the direct (dSPNs) and indirect (iSPNs) pathways in the basal ganglia, respectively. The opposite imbalance might underlie hyperkinetic abnormalities, such as dyskinesia caused by treatment of Parkinson's disease with the dopamine precursor L-DOPA. Here we monitored thousands of SPNs in behaving mice, before and after dopamine depletion and during L-DOPA-induced dyskinesia. Normally, intermingled clusters of dSPNs and iSPNs coactivated before movement. Dopamine depletion unbalanced SPN activity rates and disrupted the movement-encoding iSPN clusters. Matching their clinical efficacy, L-DOPA or agonism of the D2 dopamine receptor reversed these abnormalities more effectively than agonism of the D1 dopamine receptor. The opposite pathophysiology arose in L-DOPA-induced dyskinesia, during which iSPNs showed hypoactivity and dSPNs showed unclustered hyperactivity. Therefore, both the spatiotemporal profiles and rates of SPN activity appear crucial to striatal function, and next-generation treatments for basal ganglia disorders should target both facets of striatal activity.

Neural ensemble dynamics underlying a long-term associative memory.

The brain's ability to associate different stimuli is vital for long-term memory, but how neural ensembles encode associative memories is unknown. Here we studied how cell ensembles in the basal and lateral amygdala encode associations between conditioned and unconditioned stimuli (CS and US, respectively). Using a miniature fluorescence microscope, we tracked the Ca2+ dynamics of ensembles of amygdalar neurons during fear learning and extinction over 6 days in behaving mice. Fear conditioning induced both up- and down-regulation of individual cells' CS-evoked responses. This bi-directional plasticity mainly occurred after conditioning, and reshaped the neural ensemble representation of the CS to become more similar to the US representation. During extinction training with repetitive CS presentations, the CS representation became more distinctive without reverting to its original form. Throughout the experiments, the strength of the ensemble-encoded CS-US association predicted the level of behavioural conditioning in each mouse. These findings support a supervised learning model in which activation of the US representation guides the transformation of the CS representation.

Cellular level brain imaging in behaving mammals: an engineering approach.

Fluorescence imaging offers expanding capabilities for recording neural dynamics in behaving mammals, including the means to monitor hundreds of cells targeted by genetic type or connectivity, track cells over weeks, densely sample neurons within local microcircuits, study cells too inactive to isolate in extracellular electrical recordings, and visualize activity in dendrites, axons, or dendritic spines. We discuss recent progress and future directions for imaging in behaving mammals from a systems engineering perspective, which seeks holistic consideration of fluorescent indicators, optical instrumentation, and computational analyses. Today, genetically encoded indicators of neural Ca(2+) dynamics are widely used, and those of trans-membrane voltage are rapidly improving. Two complementary imaging paradigms involve conventional microscopes for studying head-restrained animals and head-mounted miniature microscopes for imaging in freely behaving animals. Overall, the field has attained sufficient sophistication that increased cooperation between those designing new indicators, light sources, microscopes, and computational analyses would greatly benefit future progress.

Transient activation of specific neurons in mice by selective expression of the capsaicin receptor.

The ability to control the electrical activity of a neuronal subtype is a valuable tool in deciphering the role of discreet cell populations in complex neural circuits. Recent techniques that allow remote control of neurons are either labor intensive and invasive or indirectly coupled to neural electrical potential with low temporal resolution. Here we show the rapid, reversible and direct activation of genetically identified neuronal subpopulations by generating two inducible transgenic mouse models. Confined expression of the capsaicin receptor, TRPV1, allows cell-specific activation after peripheral or oral delivery of ligand in freely moving mice. Capsaicin-induced activation of dopaminergic or serotonergic neurons reversibly alters both physiological and behavioural responses within minutes, and lasts ~10 min. These models showcase a robust and remotely controllable genetic tool that modulates a distinct cell population without the need for invasive and labour-intensive approaches.

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.

The contribution of NMDA receptor signaling in the corticobasal ganglia reward network to appetitive Pavlovian learning.

NMDA receptors (NMDARs) contribute to phasic transmission and synaptic plasticity and are thought to be important for learning. To better understand where NMDAR signaling is necessary for learning, we combined viral genetic strategies with genetic mouse models to investigate the contribution of NMDARs in the dopamine system to appetitive Pavlovian conditioning. NMDAR signaling in dopamine neurons was not required for Pavlovian conditioning; however, NMDARs in D(1) dopamine receptor (D(1)R)-expressing medium spiny neurons (MSNs), which receive input from dopamine neurons, were critical for this type of learning. NMDAR signaling was also required in brain regions that project to dopamine neurons, because removing NMDARs from afferent neurons to the ventral tegmental area (VTA) also prevented learning. This effect was likely attributable to loss of NMDAR signaling in the neurons of the prefrontal cortex (PFC), because learning could be restored in these animals by rescuing NMDAR expression in the PFC. Moreover, removing NMDARs exclusively from the PFC also prevented learning. Our findings suggest that NMDARs in neurons that project to and receive projections from the VTA are necessary for Pavlovian conditioning and specifically implicate the PFC and D(1)R-expressing MSNs in associative learning.

A behavioral genetics approach to understanding D1 receptor involvement in phasic dopamine signaling.

Dopamine-producing neurons fire with both basal level tonic patterns and phasic bursts. Varying affinities of the five dopamine receptors have led to a hypothesis that higher affinity receptors are primarily activated by basal level tonic dopamine, while lower affinity receptors may be tuned to be sensitive to higher levels caused by phasic bursts. Genetically modified mice provide a method to begin to probe this hypothesis. Here we discuss three mouse models. Dopamine-deficient mice were used to determine which behaviors require dopamine. These behaviors were then analyzed in mice lacking D1 receptors and in mice with reduced phasic dopamine release. Comparison of the latter two mouse models revealed a similar failure to learn about and respond normally to cues that indicate either a positive or negative outcome, giving support to the hypothesis that phasic dopamine release and the D1 receptor act in the same pathway. However, the D1 receptor likely has additional roles beyond those of phasic dopamine detection, because D1 receptor knockout mice have deficits in addition to what has been observed in mice with reduced phasic dopamine release.

Absence of NMDA receptors in dopamine neurons attenuates dopamine release but not conditioned approach during Pavlovian conditioning.

During Pavlovian conditioning, phasic dopamine (DA) responses emerge to reward-predictive stimuli as the subject learns to anticipate reward delivery. This observation has led to the hypothesis that phasic dopamine signaling is important for learning. To assess the ability of mice to develop anticipatory behavior and to characterize the contribution of dopamine, we used a food-reinforced Pavlovian conditioning paradigm. As mice learned the cue-reward association, they increased their head entries to the food receptacle in a pattern that was consistent with conditioned anticipatory behavior. D1-receptor knockout (D1R-KO) mice had impaired acquisition, and systemic administration of a D1R antagonist blocked both the acquisition and expression of conditioned approach in wild-type mice. To assess the specific contribution of phasic dopamine transmission, we tested mice lacking NMDA-type glutamate receptors (NMDARs) exclusively in dopamine neurons (NR1-KO mice). Surprisingly, NR1-KO mice learned at the same rate as their littermate controls. To evaluate the contribution of NMDARs to phasic dopamine release in this paradigm, we performed fast-scan cyclic voltammetry in the nucleus accumbens of awake mice. Despite having significantly attenuated phasic dopamine release following reward delivery, KO mice developed cue-evoked dopamine release at the same rate as controls. We conclude that NMDARs in dopamine neurons enhance but are not critical for phasic dopamine release to behaviorally relevant stimuli; furthermore, their contribution to phasic dopamine signaling is not necessary for the development of cue-evoked dopamine or anticipatory activity in a D1R-dependent Pavlovian conditioning paradigm.

Chronic microsensors for longitudinal, subsecond dopamine detection in behaving animals.

Neurotransmission operates on a millisecond timescale but is changed by normal experience or neuropathology over days to months. Despite the importance of long-term neurotransmitter dynamics, no technique exists to track these changes in a subject from day to day over extended periods of time. Here we describe and characterize a microsensor that can detect the neurotransmitter dopamine with subsecond temporal resolution over months in vivo in rats and mice.

Disruption of NMDAR-dependent burst firing by dopamine neurons provides selective assessment of phasic dopamine-dependent behavior.

Midbrain dopamine (DA) neurons fire in 2 characteristic modes, tonic and phasic, which are thought to modulate distinct aspects of behavior. However, the inability to selectively disrupt these patterns of activity has hampered the precise definition of the function of these modes of signaling. Here, we addressed the role of phasic DA in learning and other DA-dependent behaviors by attenuating DA neuron burst firing and subsequent DA release, without altering tonic neural activity. Disruption of phasic DA was achieved by selective genetic inactivation of NMDA-type, ionotropic glutamate receptors in DA neurons. Disruption of phasic DA neuron activity impaired the acquisition of numerous conditioned behavioral responses, and dramatically attenuated learning about cues that predicted rewarding and aversive events while leaving many other DA-dependent behaviors unaffected.