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Major depressive disorder (MDD) is a leading cause of disability worldwide. Individuals with MDD exhibit decreased motivation and deficits in reward processing. In a subset of MDD patients, chronic dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis occurs, resulting in increased levels of the ‘stress hormone’ cortisol during the normal rest period (i.e., evening and night). However, the mechanistic relationship between chronically elevated resting cortisol and behavioral deficits in motivation and reward processing remains unclear. Given that women are diagnosed with MDD at twice the rate of men, it is important to understand whether the mechanisms linking cortisol to the symptoms of MDD differ by sex. In this study, we used subcutaneous implants to chronically elevate free plasma corticosterone (the rodent homolog of cortisol; ‘CORT’) during the rest period in male and female mice and examined changes in behavior and dopamine system function. We found that chronic CORT treatment impaired motivated reward-seeking in both sexes. In female but not male mice, CORT treatment reduced dopamine content in the dorsomedial striatum (DMS). In male but not female mice, CORT treatment impaired the function of the dopamine transporter (DAT) in DMS. From these studies, we conclude that chronic CORT dysregulation impairs motivation by impairing dopaminergic transmission in the DMS, but via different mechanisms in male and female mice. A better understanding of these sex-specific mechanisms could lead to new directions in MDD diagnosis and treatment.

BackgroundMajor depressive disorder (MDD) is a leading cause of disability worldwide. Individuals with MDD exhibit decreased motivation and deficits in reward processing along with chronically elevated levels of the ‘stress hormone,’ cortisol. However, the mechanistic relationship between chronically elevated cortisol and behavioral deficits in motivation and reward processing remains unclear. Given that women are diagnosed with MDD at twice the rate of men, it is important to understand whether the mechanisms linking chronically elevated cortisol to the symptoms of MDD differ by sex.MethodsWe used subcutaneous implants to chronically elevate free plasma corticosterone (rodent homolog of cortisol; ‘CORT’) in male and female mice and examined changes in behavior and dopamine system function. We used operant training to assay reward-guided motivation and high-performance liquid chromatography to quantify striatal dopamine content. We assessed dopamine transporter (DAT) function using ex vivo slice imaging and in vivo fiber photometry in combination with a fluorescent dopamine sensor, dLight1.3b. We further quantified DAT expression and phosphorylation using western blot.ResultsWe found that chronic CORT treatment impaired reward-seeking in both sexes. In female but not male mice, CORT treatment reduced dopamine content in the dorsomedial striatum (DMS). In male but not female mice, the function of the dopamine transporter (DAT) was impaired in DMS.ConclusionsChronic elevation of CORT impairs reward-seeking by impairing dopaminergic transmission in the DMS, but via different mechanisms in male and female mice. A better understanding of these sex-specific mechanisms could lead to new directions in MDD diagnosis and treatment.

The external globus pallidus (GPe) is a critical node within the basal ganglia circuit. Phasic changes in the activity of GPe neurons during movement and their alterations in Parkinson's disease (PD) argue that the GPe is important in motor control. Parvalbumin-positive (PV+) neurons and Npas1+neurons are the two principal neuron classes in the GPe. The distinct electrophysiological properties and axonal projection patterns argue that these two neuron classes serve different roles in regulating motor output. However, the causal relationship between GPe neuron classes and movement remains to be established. Here, by using optogenetic approaches in mice (both males and females), we showed that PV+neurons and Npas1+neurons promoted and suppressed locomotion, respectively. Moreover, PV+neurons and Npas1+neurons are under different synaptic influences from the subthalamic nucleus (STN). Additionally, we found a selective weakening of STN inputs to PV+neurons in the chronic 6-hydroxydopamine lesion model of PD. This finding reinforces the idea that the reciprocally connected GPe–STN network plays a key role in disease symptomatology and thus provides the basis for future circuit-based therapies.SIGNIFICANCE STATEMENTThe external pallidum is a key, yet an understudied component of the basal ganglia. Neural activity in the pallidum goes awry in neurologic diseases, such as Parkinson's disease. While this strongly argues that the pallidum plays a critical role in motor control, it has been difficult to establish the causal relationship between pallidal activity and motor function/dysfunction. This was in part because of the cellular complexity of the pallidum. Here, we showed that the two principal neuron types in the pallidum have opposing roles in motor control. In addition, we described the differences in their synaptic influence. Importantly, our research provides new insights into the cellular and circuit mechanisms that explain the hypokinetic features of Parkinson's disease.

Recent developments in intersectional strategies have greatly advanced our ability to precisely target brain cell types based on unique co-expression patterns. To accelerate the application of intersectional genetics, we perform a brain-wide characterization of 13 Flp and tTA mouse driver lines and selected seven for further analysis based on expression of vesicular neurotransmitter transporters. Using selective Cre driver lines, we created more than 10 Cre/tTA combinational lines for cell type targeting and circuit analysis. We then used VGLUT-Cre/VGAT-Flp combinational lines to identify and map 30 brain regions containing neurons that co-express vesicular glutamate and gamma-aminobutyric acid (GABA) transporters, followed by tracing their projections with intersectional viral vectors. Focusing on the lateral habenula (LHb) as a target, we identified glutamatergic, GABAergic, or co-glutamatergic/GABAergic innervations from ∼40 brain regions. These data provide an important resource for the future application of intersectional strategies and expand our understanding of the neuronal subtypes in the brain.

Fiber photometry (FP) is an adaptable method for recording in vivo neural activity in freely behaving animals. It has become a popular tool in neuroscience due to its ease of use, low cost, the ability to combine FP with freely moving behavior, among other advantages. However, analysis of FP data can be a challenge for new users, especially those with a limited programming background. Here, we present Guided Photometry Analysis in Python (GuPPy), a free and open-source FP analysis tool. GuPPy is provided as a Jupyter notebook, a well-commented interactive development environment (IDE) designed to operate across platforms. GuPPy presents the user with a set of graphic user interfaces (GUIs) to load data and provide input parameters. Graphs produced by GuPPy can be exported into various image formats for integration into scientific figures. As an open-source tool, GuPPy can be modified by users with knowledge of Python to fit their specific needs.

SUMMARY/ABSTRACTThe basal ganglia operate largely in closed parallel loops, including an associative circuit for goal-directed behavior originating from the dorsomedial striatum (DMS) and a somatosensory circuit important for habit formation originating from the dorsolateral striatum (DLS). An exception to this parallel circuit organization has been proposed to explain how information might be transferred between striatal subregions, for example from DMS to DLS during habit formation. The “ascending spiral hypothesis” proposes that DMS disinhibits dopamine signaling in DLS through a tri-synaptic, open-loop striato-nigro-striatal circuit. Here, we used transsynaptic and intersectional genetic tools to investigate both closed- and open-loop striato-nigro-striatal circuits. We found strong evidence for closed loops, which would allow striatal subregions to regulate their own dopamine release. We also found evidence for functional synapses in open loops. However, these synapses were unable to modulate tonic dopamine neuron firing, questioning the prominence of their role in mediating crosstalk between striatal subregions.

SUMMARYCompulsive behavior is a defining feature of disorders such as substance use disorder and obsessive-compulsive disorder. Current evidence suggests that corticostriatal circuits control the expression of established compulsions, but little is known about the mechanisms regulating the development of compulsions. We hypothesized that dopamine, a critical modulator of striatal synaptic plasticity, could control alterations in corticostriatal circuits leading to the development of compulsions (defined as continued reward-seeking in the face of punishment). We used dual-site fiber photometry to measure dopamine axon activity in the dorsomedial striatum (DMS) and the dorsolateral striatum (DLS) as compulsions emerged. Individual variability in the speed with which compulsions emerged was predicted by DMS dopamine axon activity. Amplifying this dopamine signal accelerated animals’ transitions to compulsion, whereas inhibition led to learning delays. In contrast, amplifying DLS dopamine signaling had no effect on the emergence of compulsions. These results establish DMS dopamine signaling as a key controller of the development of compulsive reward-seeking.

The external globus pallidus (GPe) is a critical node within the basal ganglia circuit. Phasic changes in the activity of GPe neurons during movement and their alterations in Parkinson’s disease (PD) argue that the GPe is important in motor control. PV+ neurons and Npas1+ neurons are the two principal neuron classes in the GPe. The distinct electrophysiological properties and axonal projection patterns argue that these two neuron classes serve different roles in regulating motor output. However, the causal relationship between GPe neuron classes and movement remains to be established. Here, by using optogenetic approaches in mice (both males and females), we showed that PV+ neurons and Npas1+ neurons promoted and suppressed locomotion, respectively. Moreover, PV+ neurons and Npas1+ neurons are under different synaptic influences from the subthalamic nucleus (STN). Additionally, we found a selective weakening of STN inputs to PV+ neurons in the chronic 6-hydroxydopamine lesion model of PD. This finding reinforces the idea that the reciprocally connected GPe-STN network plays a key role in disease symptomatology and thus provides the basis for future circuit-based therapies.Significance StatementThe external pallidum is a key, yet an understudied component of the basal ganglia. Neural activity in the pallidum goes awry in neurological diseases, such as Parkinson’s disease. While this strongly argues that the pallidum plays a critical role in motor control, it has been difficult to establish the causal relationship between pallidal activity and motor (dys)function. This was in part due to the cellular complexity of the pallidum. Here, we showed that the two principal neuron types in the pallidum have opposing roles in motor control. In addition, we described the differences in their synaptic influence. Importantly, our research provides new insights into the cellular and circuit mechanisms that explain the hypokinetic features of Parkinson’s disease.

The external globus pallidus (GPe) is a critical node within the basal ganglia circuit. Phasic changes in the activity of GPe neurons during movement and their alterations in Parkinson's disease (PD) argue that the GPe is important in motor control. Parvalbumin-positive (PV(+)) neurons and Npas1(+) neurons are the two principal neuron classes in the GPe. The distinct electrophysiological properties and axonal projection patterns argue that these two neuron classes serve different roles in regulating motor output. However, the causal relationship between GPe neuron classes and movement remains to be established. Here, by using optogenetic approaches in mice (both males and females), we showed that PV(+) neurons and Npas1(+) neurons promoted and suppressed locomotion, respectively. Moreover, PV(+) neurons and Npas1(+) neurons are under different synaptic influences from the subthalamic nucleus (STN). Additionally, we found a selective weakening of STN inputs to PV(+) neurons in the chronic 6-hydroxydopamine lesion model of PD. This finding reinforces the idea that the reciprocally connected GPe–STN network plays a key role in disease symptomatology and thus provides the basis for future circuit-based therapies. SIGNIFICANCE STATEMENT The external pallidum is a key, yet an understudied component of the basal ganglia. Neural activity in the pallidum goes awry in neurologic diseases, such as Parkinson's disease. While this strongly argues that the pallidum plays a critical role in motor control, it has been difficult to establish the causal relationship between pallidal activity and motor function/dysfunction. This was in part because of the cellular complexity of the pallidum. Here, we showed that the two principal neuron types in the pallidum have opposing roles in motor control. In addition, we described the differences in their synaptic influence. Importantly, our research provides new insights into the cellular and circuit mechanisms that explain the hypokinetic features of Parkinson's disease.

Compulsive behavior is a defining feature of disorders such as substance use disorders. Current evidence suggests that corticostriatal circuits control the expression of established compulsions, but little is known about the mechanisms regulating the development of compulsions. We hypothesized that dopamine, a critical modulator of striatal synaptic plasticity, could control alterations in corticostriatal circuits leading to the development of compulsions (defined here as continued reward seeking in the face of punishment). We used dual-site fiber photometry to measure dopamine axon activity in the dorsomedial striatum (DMS) and the dorsolateral striatum (DLS) as compulsions emerged. Individual variability in the speed with which compulsions emerged was predicted by DMS dopamine axon activity. Amplifying this dopamine signal accelerated animals' transitions to compulsion, whereas inhibition delayed it. In contrast, amplifying DLS dopamine signaling had no effect on the emergence of compulsions. These results establish DMS dopamine signaling as a key controller of the development of compulsive reward seeking.

Classical models of cerebellar learning posit that climbing fibers operate according to a supervised learning rule to instruct changes in motor output by signaling the occurrence of movement errors. However, cerebellar output is also associated with non-motor behaviors, and recently with modulating reward association pathways in the VTA. To test how the cerebellum processes reward related signals in the same type of classical conditioning behavior typically studied to evaluate reward processing in the VTA and striatum, we have used calcium imaging to visualize instructional signals carried by climbing fibers across the lateral cerebellum in mice before and after learning. We find distinct climbing fiber responses in three lateral cerebellar regions that can each signal reward prediction. These instructional signals are well suited to guide cerebellar learning based on reward expectation and enable a cerebellar contribution to reward driven behaviors, suggesting a broad role for the lateral cerebellum in reward-based learning.

Habits are an important mechanism by which organisms can automate the control of behavior to alleviate cognitive demand. However, transitions to habitual control are risky because they lead to inflexible responding in the face of change. The question of how the brain controls transitions into habit is thus an intriguing one. How do we regulate when our repeated actions become automated? When is it advantageous or disadvantageous to release actions from cognitive control? Decades of research have identified a variety of methods for eliciting habitual responding in animal models. Progress has also been made to understand which brain areas and neural circuits control transitions into habit. Here, I discuss existing research on behavioral and neural circuit models for habit formation (with an emphasis on striatal circuits), and discuss strategies for combining information from different paradigms and levels of analysis to prompt further progress in the field.

Summary Prolonged behavioral challenges can cause animals to switch from active to passive coping strategies to manage effort-expenditure during stress; such normally adaptive behavioral state transitions can become maladaptive in psychiatric disorders such as depression. The underlying neuronal dynamics and brainwide interactions important for passive coping have remained unclear. Here, we develop a paradigm to study these behavioral state transitions at cellular-resolution across the entire vertebrate brain. Using brainwide imaging in zebrafish, we observed that the transition to passive coping is manifested by progressive activation of neurons in the ventral (lateral) habenula. Activation of these ventral-habenula neurons suppressed downstream neurons in the serotonergic raphe nucleus and caused behavioral passivity, whereas inhibition of these neurons prevented passivity. Data-driven recurrent neural network modeling pointed to altered intra-habenula interactions as a contributory mechanism. These results demonstrate ongoing encoding of experience features in the habenula, which guides recruitment of downstream networks and imposes a passive coping behavioral strategy.

Communication, the effective delivery of information, is fundamental to life across all scales and species. Nervous systems (by necessity) may be most specifically adapted among biological tissues for high rate and complexity of information transmitted, and thus, the properties of neural tissue and principles of its organization into circuits may illuminate capabilities and limitations of biological communication. Here, we consider recent developments in tools for studying neural circuits with particular attention to defining neuronal cell types by input and output information streams--i.e., by how they communicate. Complementing approaches that define cell types by virtue of genetic promoter/enhancer properties, this communication-based approach to defining cell types operationally by input/output (I/O) relationships links structure and function, resolves difficulties associated with single-genetic-feature definitions, leverages technology for observing and testing significance of precisely these I/O relationships in intact brains, and maps onto processes through which behavior may be adapted during development, experience, and evolution.

The central amygdala (CeA) orchestrates adaptive responses to emotional events. While CeA substrates for defensive behaviors have been studied extensively, CeA circuits for appetitive behaviors and their relationship to threat-responsive circuits remain poorly defined. Here, we demonstrate that the CeA sends robust inhibitory projections to the lateral substantia nigra (SNL) that contribute to appetitive and aversive learning in mice. CeA→SNL neural responses to appetitive and aversive stimuli were modulated by expectation and magnitude consistent with a population-level salience signal, which was required for Pavlovian conditioned reward-seeking and defensive behaviors. CeA→SNL terminal activation elicited reinforcement when linked to voluntary actions but failed to support Pavlovian associations that rely on incentive value signals. Consistent with a disinhibitory mechanism, CeA inputs preferentially target SNL GABA neurons, and CeA→SNL and SNL dopamine neurons respond similarly to salient stimuli. Collectively, our results suggest that amygdala-nigra interactions represent a previously unappreciated mechanism for influencing emotional behaviors.

Differentially controlled dopamine signaling in the striatum may be critical to habit formation

Neuromodulatory systems exert profound influences on brain function. Understanding how these systems modify the operating mode of target circuits requires spatiotemporally precise measurement of neuromodulator release. We developed dLight1, an intensity-based genetically encoded dopamine indicator, to enable optical recording of dopamine dynamics with high spatiotemporal resolution in behaving mice. We demonstrated the utility of dLight1 by imaging dopamine dynamics simultaneously with pharmacological manipulation, electrophysiological or optogenetic stimulation, and calcium imaging of local neuronal activity. dLight1 enabled chronic tracking of learning-induced changes in millisecond dopamine transients in mouse striatum. Further, we used dLight1 to image spatially distinct, functionally heterogeneous dopamine transients relevant to learning and motor control in mouse cortex. We also validated our sensor design platform for developing norepinephrine, serotonin, melatonin, and opioid neuropeptide indicators.

SummaryRecent progress in understanding the diversity of midbrain dopamine neurons has highlighted the importance—and the challenges—of defining mammalian neuronal cell types. Although neurons may be best categorized using inclusive criteria spanning biophysical properties, wiring of inputs, wiring of outputs, and activity during behavior, linking all of these measurements to cell types within the intact brains of living mammals has been difficult. Here, using an array of intact-brain circuit interrogation tools, including CLARITY, COLM, optogenetics, viral tracing, and fiber photometry, we explore the diversity of dopamine neurons within the substantia nigra pars compacta (SNc). We identify two parallel nigrostriatal dopamine neuron subpopulations differing in biophysical properties, input wiring, output wiring to dorsomedial striatum (DMS) versus dorsolateral striatum (DLS), and natural activity patterns during free behavior. Our results reveal independently operating nigrostriatal information streams, with implications for understanding the logic of dopaminergic feedback circuits and the diversity of mammalian neuronal cell types.

Hippocampal oscillations are critical for information processing, and are strongly influenced by inputs from the medial septum. Hippocamposeptal neurons provide direct inhibitory feedback from the hippocampus onto septal cells, and are therefore likely to also play an important role in the circuit; these neurons fire at either low or high frequency, reflecting hippocampal network activity during theta oscillations or ripple events, respectively. Here, we optogenetically target the long-range GABAergic projection from the hippocampus to the medial septum in rats, and thereby simulate hippocampal input onto downstream septal cells in an acute slice preparation. In response to optogenetic activation of hippocamposeptal fibers at theta and ripple frequencies, we elicit postsynaptic GABAergic responses in a subset (24%) of septal cells, most predominantly in fast-spiking cells. In addition, in another subset of septal cells (19%) corresponding primarily to cholinergic cells, we observe a slow hyperpolarization of the resting membrane potential and a decrease in input resistance, particularly in response to prolonged high-frequency (ripple range) stimulation. This slow response is partially sensitive to GIRK channel and D2 dopamine receptor block. Our results suggest that two independent populations of septal cells distinctly encode hippocampal feedback, enabling the septum to monitor ongoing patterns of activity in the hippocampus.

SummaryPlasticity of excitatory synapses onto striatal projection neurons (MSNs) has the potential to regulate motor function by setting the gain on signals driving both direct- and indirect-pathway basal ganglia circuits. Endocannabinoid-dependent long-term depression (eCB-LTD) is the best characterized form of striatal plasticity, but the mechanisms governing its normal regulation and pathological dysregulation are not well understood. We characterized two distinct signaling pathways mediating eCB production in striatal indirect-pathway MSNs and found that both pathways were modulated by dopamine D2 and adenosine A2A receptors, acting through cAMP/PKA. We identified regulator of G protein signaling 4 (RGS4) as a key link between D2/A2A signaling and eCB mobilization pathways. In contrast to wild-type mice, RGS4−/− mice exhibited normal eCB-LTD after dopamine depletion and were significantly less impaired in the 6-OHDA model of Parkinson's disease. Taken together, these results suggest that inhibition of RGS4 may be an effective nondopaminergic strategy for treating Parkinson's disease.Video Abstract

Excitatory synapses onto projection neurons in the striatum, the input nucleus of the basal ganglia, play a key role in regulating basal ganglia circuit function and are a major site of long-term synaptic plasticity. Here, we review the mechanisms and regulation of both long-term potentiation and long-term depression at these synapses. In particular, we highlight the role that neuromodulators play in determining the strength and direction of plasticity, which ultimately shapes the balance of activity in basal ganglia circuits and regulates motor behavior.

Real-time activity measurements from multiple specific cell populations and projections are likely to be important for understanding the brain as a dynamical system. Here we developed frame-projected independent-fiber photometry (FIP), which we used to record fluorescence activity signals from many brain regions simultaneously in freely behaving mice. We explored the versatility of the FIP microscope by quantifying real-time activity relationships among many brain regions during social behavior, simultaneously recording activity along multiple axonal pathways during sensory experience, performing simultaneous two-color activity recording, and applying optical perturbation tuned to elicit dynamics that match naturally occurring patterns observed during behavior.

A marked bias towards risk aversion has been observed in nearly every species tested1–4. A minority of individuals, however, instead seem to prefer risk (repeatedly choosing uncertain large rewards over certain but smaller rewards), and even risk-averse individuals sometimes opt for riskier alternatives2,5. It is not known how neural activity underlies such important shifts in decision-making—either as a stable trait across individuals or at the level of variability within individuals. Here we describe a model of risk-preference in rats, in which stable individual differences, trial-by-trial choices, and responses to pharmacological agents all parallel human behaviour. By combining new genetic targeting strategies with optical recording of neural activity during behaviour in this model, we identify relevant temporally specific signals from a genetically and anatomically defined population of neurons. This activity occurred within dopamine receptor type-2 (D2R)-expressing cells in the nucleus accumbens (NAc), signalled unfavourable outcomes from the recent past at a time appropriate for influencing subsequent decisions, and also predicted subsequent choices made. Having uncovered this naturally occurring neural correlate of risk selection, we then mimicked the temporally specific signal with optogenetic control during decision-making and demonstrated its causal effect in driving risk-preference. Specifically, risk-preferring rats could be instantaneously converted to risk-averse rats with precisely timed phasic stimulation of NAc D2R cells. These findings suggest that individual differences in risk preference, as well as real-time risky decision-making, can be in large part explained by the encoding in D2R-expressing NAc cells of prior unfavourable outcomes during decision-making.

Anxiety-related conditions are among the most difficult neuropsychiatric diseases to treat pharmacologically, but respond to cognitive therapies. There has therefore been interest in identifying relevant top-down pathways from cognitive control regions in medial prefrontal cortex (mPFC). Identification of such pathways could contribute to our understanding of the cognitive regulation of affect, and provide pathways for intervention. Previous studies have suggested that dorsal and ventral mPFC subregions exert opposing effects on fear, as do subregions of other structures. However, precise causal targets for top-down connections among these diverse possibilities have not been established. Here we show that the basomedial amygdala (BMA) represents the major target of ventral mPFC in amygdala in mice. Moreover, BMA neurons differentiate safe and aversive environments, and BMA activation decreases fear-related freezing and high-anxiety states. Lastly, we show that the ventral mPFC-BMA projection implements top-down control of anxiety state and learned freezing, both at baseline and in stress-induced anxiety, defining a broadly relevant new top-down behavioural regulation pathway.

Adenosine A2Areceptor antagonists are psychomotor stimulants that also hold therapeutic promise for movement disorders. However, the molecular mechanisms underlying their stimulant properties are not well understood. Here, we show that the robust increase in locomotor activity induced by an A2Aantagonistin vivois greatly attenuated by antagonizing cannabinoid CB1receptor signaling or by administration to CB1−/−mice. To determine the locus of increased endocannabinoid signaling, we measured the amount of anandamide [AEA (N-arachidonoylethanolamine)] and 2-arachidonoylglycerol (2-AG) in brain tissue from striatum and cortex. We find that 2-AG is selectively increased in striatum after acute blockade of A2Areceptors, which are highly expressed by striatal indirect-pathway medium spiny neurons (MSNs). Using targeted whole-cell recordings from direct- and indirect-pathway MSNs, we demonstrate that A2Areceptor antagonists potentiate 2-AG release and induction of long-term depression at indirect-pathway MSNs, but not direct-pathway MSNs. Together, these data outline a molecular mechanism by which A2Aantagonists reduce excitatory synaptic drive on the indirect pathway through CB1receptor signaling, thus leading to increased psychomotor activation.