Although learning in response to extrinsic reinforcement is theorized to be driven by dopamine signals that encode the difference between expected and experienced rewards, skills that enable verbal or musical expression can be learned without extrinsic reinforcement. Instead, spontaneous execution of these skills is thought to be intrinsically reinforcing. Whether dopamine signals similarly guide learning of these intrinsically reinforced behaviours is unknown. In juvenile zebra finches learning from an adult tutor, dopamine signalling in a song-specialized basal ganglia region is required for successful song copying, a spontaneous, intrinsically reinforced process. Here we show that dopamine dynamics in the song basal ganglia faithfully track the learned quality of juvenile song performance on a rendition-by-rendition basis. Furthermore, dopamine release in the basal ganglia is driven not only by inputs from midbrain dopamine neurons classically associated with reinforcement learning but also by song premotor inputs, which act by means of local cholinergic signalling to elevate dopamine during singing. Although both cholinergic and dopaminergic signalling are necessary for juvenile song learning, only dopamine tracks the learned quality of song performance. Therefore, dopamine dynamics in the basal ganglia encode performance quality during self-directed, long-term learning of natural behaviours.

Linking activity in specific cell types with perception, cognition, and action, requires quantitative behavioral experiments in genetic model systems such as the mouse. In head-fixed primates, the combination of precise stimulus control, monitoring of motor output, and physiological recordings over large numbers of trials are the foundation on which many conceptually rich and quantitative studies have been built. Choice-based, quantitative behavioral paradigms for head-fixed mice have not been described previously. Here, we report a somatosensory absolute object localization task for head-fixed mice. Mice actively used their mystacial vibrissae (whiskers) to sense the location of a vertical pole presented to one side of the head and reported with licking whether the pole was in a target (go) or a distracter (no-go) location. Mice performed hundreds of trials with high performance (>90% correct) and localized to <0.95 mm (<6 degrees of azimuthal angle). Learning occurred over 1-2 weeks and was observed both within and across sessions. Mice could perform object localization with single whiskers. Silencing barrel cortex abolished performance to chance levels. We measured whisker movement and shape for thousands of trials. Mice moved their whiskers in a highly directed, asymmetric manner, focusing on the target location. Translation of the base of the whiskers along the face contributed substantially to whisker movements. Mice tended to maximize contact with the go (rewarded) stimulus while minimizing contact with the no-go stimulus. We conjecture that this may amplify differences in evoked neural activity between trial types.

The substantia nigra pars reticulata (SNr) functions as the principal inhibitory output of the basal ganglia, with the timing of its spikes critically controlling downstream disinhibition required for movement initiation. The external globus pallidus (GPe) and D1-expressing medium spiny neurons (D1-MSNs) in the striatum provide GABAergic inputs to the SNr that differ in their amplitude and kinetic properties. How these inputs interact with the intrinsic membrane currents that determine SNr firing is only partially understood. Using optogenetics, computational modeling, and electrophysiology in acute mouse brain slices, 47 animals of either sex were used for measurements, and we found an unexpected interaction between GABAergic inputs and hyperpolarization-activated currents (Ih) that tunes inhibitory efficacy in a pathway-specific manner. GPe inputs evoke fast, large IPSCs that transiently suppress SNr firing within a narrow window but whose rapid decay enables depolarization from Ih to restore firing after only a brief pause. In contrast, the slower decay kinetics of striatal IPSCs enables more sustained inhibition that counters the depolarizing drive from Ih to produce longer pauses, despite their lower conductance amplitudes. Pharmacological blockade of Ih with ZD7288 eliminated the rapid recovery of firing after GPe inhibition and equalized the inhibitory efficacy between GPe and striatal pathways. These findings establish an important interplay between synaptic kinetics and intrinsic membrane conductances in establishing pathway-specific inhibitory balance in the basal ganglia. Our study reveals that inhibitory pathways to the substantia nigra pars reticulata are differentially shaped by the interplay between synaptic kinetics and intrinsic membrane conductances. Using optogenetics, electrophysiology, and modeling, we showed that fast-decaying GABAergic inputs from the external globus pallidus are rapidly overcome by Ih, producing only brief pauses in SNr firing, whereas slower striatal inputs generate longer-lasting inhibition. Blocking Ih abolishes this difference, demonstrating that intrinsic currents tune inhibitory efficacy in a pathway-specific manner. These results identify a biophysical mechanism that helps set the balance of basal ganglia output essential for movement control.

Frontal Cortex (FC) plays a pivotal role in adaptively controlling actions and their dynamics in response to incoming sensory signals. We explored FC encoding of identical stimuli and their behavioral consequences when they signified diametrically opposite responses depending on task context. Two groups of female ferrets performed Go-NoGo auditory categorization tasks with opposite contingencies and rewards, and diverse stimuli. Remarkably, despite opposite stimulus-action associations, single-unit responses were similar across all tasks, being more sustained and stronger to Target sounds (signaling a change in action) than to Reference sounds (indicating maintenance of ongoing actions) especially during task engagement. Overall activity was composed of three distinct dynamic response profiles. Each corresponded to a separate neuronal cluster and exhibited a different role in relation to the succession of task events. Decoding based on the temporal structure of population responses revealed distinct decoders that were aligned to different task events. Similar to single unit findings, the β-band power extracted from the FC local field potentials (LFPs) was strongly and similarly modulated during Target stimuli across all tasks despite opposite behavioral actions. In contrast, power in all other LFP frequency bands varied significantly across task stimuli and actions. Based on these findings, we propose the FC encodes a common, highly abstract representation of all the different behavioral tasks. We further outline a hypothetical model of pathway-specific functional projections from the tripartite FC neuronal clusters to the basal ganglia, consistent with previous evidence for the conjoint roles of the FC and striatum in adaptive motor control. The frontal cortex (FC) encodes an abstract representation of perception and action with associated rewards and cognitive functions. Thus, even when ferrets perform opposite Go/NoGo behaviors, FC responses exhibit similar sequences of dynamic patterns from 3 cell clusters. The first component is phasic encoding stimulus category and the decision to maintain or change ongoing actions. The second is a rapid response suppression, initiated if the animal switches to a new action. The third is a buildup of excitatory activity as the animal sustains its new action. We propose a model for how such an abstract FC representation may emerge from separate functional projections from the FC clusters to the striatum, offering new insights into the FC role in behavioral control.

Acetylcholine (ACh) release in the medial prefrontal cortex (mPFC) is a major contributor to the balance between attention and inhibitory control, which is crucial for the execution of adaptive goal-directed behaviors. Yet, our understanding of the functional heterogeneity within the mPFC, particularly how distinct cholinergic afferences and circuits regulate these processes, remains limited. Here, we used in vivo fiber photometry, neuronal tracing, and chemogenetics manipulations to demonstrate the role of the prelimbic sub-region of the mPFC (PrL) and its ascending cholinergic projections in a cued-Fixed Consecutive Number task (FCNcue task) in male mice. We found that following a transient activation at the initiation of the behavioral response (cue detection), persistent inhibition of PrL neuronal activity, measured by fiber photometry, may be necessary to maintain engagement in the task and completion of the chain of required responses (i.e., optimal responses). Moreover, we found that the PrL receives dense ACh projections almost exclusively from the most anterior-medial part of the basal forebrain (BF) comprising the horizontal and ventral parts of the diagonal band of Broca (HDB and VDB) and the substantia innominata (SI) nuclei. Finally, chemogenetic activation of this ACh pathway inhibited PrL activity and enhanced behavioral performance of the mice by increasing the percentage of optimal responses. Overall, this study provides insights into the spatial and temporal dynamics of cholinergic signaling to the PrL and its causal role on attentional control.

Midbrain dopamine (DA) neurons are diverse with distinct subpopulations being essential for key functions of the brain: nigrostriatal DA neurons for voluntary movement and mesolimbic DA neurons for learning from reward prediction errors. In addition to being primarily associated with distinct senso-motor or limbic cortical-striatal circuits, DA subpopulations also directly communicate with each other via local DA release in the midbrain. While the inhibitory synaptic nature of this dopamine-to-dopamine signaling has been well established, the pre- and postsynaptic identity and logic of connectivity among DA subpopulations are still unresolved. To fill this gap, we combined retrograde tracing with projection-specific optogenetic stimulation of DA neurons and patch-clamp recordings in the adult mouse of either sex. We functionally identified a unidirectional, motor-to-limbic design of the DA synapse in the midbrain. This motor-to-limbic negative feedback connection in the midbrain was independently confirmed by monosynaptic rabies tracing of projection-defined DA subpopulations. This DA synapse might complement the limbic-to-motor striato-nigro-striatal feedforward architecture of the basal ganglia. We identified the pre- and postsynaptic partners of the dopamine-to-dopamine synapse in the midbrain, independently by functional in vitro patch clamp recordings and monosynaptic rabies tracing of identified dopamine subpopulations. This DA synapse is surprisingly circuit-specific with presynaptic DA neurons projecting to the dorsal striatum and post-synaptic DA neurons projecting to the lateral shell of the nucleus accumbens. Thus, this DA synapse establishes a unidirectional, direct communication between the nigro-striatal and the meso-limbic dopamine systems.

The GABAergic striatal outputs forming the direct and indirect pathways of the basal ganglia (BG), classically defined by Drd1 and Drd2 expression, respectively, are crucial in the control of voluntary movement. Recent evidence has identified parallel direct and indirect pathways within striosomes, that exert opposing control over striatal DA release and thereby influencing locomotor behavior. These findings refine our understanding of how discrete BG pathways coordinate voluntary actions.

We describe a novel method for adapting a two-photon scanning microscope to enable simultaneous detection of two-photon-generated visible fluorescence and single-photon-generated near-infrared (nIR) fluorescence. In this configuration, nIR fluorescence is routed through a single-mode optical fiber before detection by a photomultiplier tube. This fiber coupling offers two advantages: first, the optical fiber functions as a pinhole aperture, allowing for improved optical sectioning of the nIR signal; second, it minimizes nIR background fluorescence. To validate the effectiveness of this design, we conducted two sets of experiments in male and female C57B/6 mice. First, we compare two fluorescence indicators of the neurotransmitter dopamine: the genetically encoded indicator GRAB and single walled carbon nanotube based optical nanosensors (nIRCats). Although nIRCats exhibit lower affinity for dopamine than GRAB, this property allows for identification of high concentration release sites in the striatum. Second, we simultaneously imaged depolarization-induced calcium changes and dopamine release in the retina. Together, these results demonstrate the utility of integrating confocal nIR detection into a two-photon platform for simultaneous functional imaging across complementary spectral channels. Dual-color, real-time imaging is a powerful technique in biomedical imaging, including neuroscience. Here, we present a widely applicable modification to a standard two-photon scanning microscope that adds a near-infrared detection capability, a wavelength range that minimizes photon scattering and autofluorescence from biological samples. Using this microscope, we demonstrate the first direct comparison of two dopamine sensors: the genetically encoded sensor GRAB detected in the visible channel and carbon-nanotube-based sensors detected in the near-infrared channel. We further demonstrate simultaneous imaging calcium activity and dopamine signaling in the developing retina. While we focused on dopamine sensors in this study, this platform is broadly applicable to a wide range of fluorophores and can be implemented on existing two-photon microscopes.

The brain's reward-processing circuitry remains sensitive to experience throughout early life and into adulthood, allowing individuals to adapt to their unique environments. Adverse experiences early in life can increase vulnerability to substance use disorders, likely through alterations to this circuitry. Yet, the precise neurobiological mechanisms by which early life adversity acts are incompletely characterized. In this study, we used a limited bedding and nesting (LBN) paradigm as a translationally relevant model of early life adversity in isogenic C57BL/6J mice. After LBN-rearing, we assessed the lasting behavioral and neurobiological impacts of this experience in adulthood. In robust sample sizes, our results validated previous findings of increased risk avoidance, enhanced acute locomotor response to alcohol, and greater voluntary alcohol drinking in socially-housed LBN-reared mice, especially males. Further, using autoradiography, we found LBN-reared mice had increased striatal D1-like receptor binding, skewing D1- to D2-like receptor balance relative to cross-fostered controls. However, after voluntary alcohol drinking, we found a strong downregulation in D1-like, and some D2-like, receptor binding, negating pre-existing differences in striatal dopamine receptor binding. We posit that via both transcriptional and post-transcriptional mechanisms, LBN-rearing upregulates striatal D1-receptor density and alters risk avoidance and acute alcohol stimulation to promote alcohol drinking among adversity-exposed mice. Together, these findings reveal specific neurobiological mechanisms that promote alcohol consumption following early life adversity and suggest complex interactions between early life adversity, sex-related factors, and dopamine receptor regulation in contributing to alcohol use disorder (AUD) vulnerability.

Genetically encoded fluorescent voltage indicators are ideally suited to reveal the millisecond-scale interactions among and between targeted cell populations. However, current indicators lack the requisite sensitivity for in vivo multipopulation imaging. We describe next-generation green and red voltage sensors, Ace-mNeon2 and VARNAM2, and their reverse response-polarity variants pAce and pAceR. Our indicators enable 0.4- to 1-kilohertz voltage recordings from >50 spiking neurons per field of view in awake mice and ~30-minute continuous imaging in flies. Using dual-polarity multiplexed imaging, we uncovered brain state-dependent antagonism between neocortical somatostatin-expressing (SST) and vasoactive intestinal peptide-expressing (VIP) interneurons and contributions to hippocampal field potentials from cell ensembles with distinct axonal projections. By combining three mutually compatible indicators, we performed simultaneous triple-population imaging. These approaches will empower investigations of the dynamic interplay between neuronal subclasses at single-spike resolution.

Dopamine critically modulates prefrontal circuits underlying cognitive control, but how D1-type (D1R) and D2-type (D2R) receptors influence abstract decision coding is unclear. We recorded single-neuron activity in two monkeys performing a number comparison task, in which abstract decisions about sequentially presented dot displays were dissociated from motor responses, while locally stimulating D1R or D2R via microiontophoresis. D1R stimulation suppressed, whereas D2R stimulation enhanced, the decision-coding strength of individual neurons, effects mirrored at the population level in decoding accuracy. Interestingly, dopamine receptors also bidirectionally modulated the temporal structure of population activity: D1R stimulation reduced the temporal generalizability of neuronal decision selectivity, suggesting more transient tuning and a shift toward a more dynamic coding regime. Conversely, D2R stimulation increased temporal generalizability of decision selectivity, implying more sustained tuning and a shift toward a more static coding regime. These findings suggest that D1- and D2-mediated mechanisms in the prefrontal cortex provide a receptor-specific substrate for balancing cognitive flexibility and stability in abstract decision-making. This pattern may reflect task-dependent deviations from classical dual-state models, in which D1 receptor activity stabilizes working memory representations whereas D2 receptor activity supports flexible coding-a relationship that appears reversed in the context of abstract decision formation.

The striatum is one of the first brain regions affected in Parkinson's disease (PD), where dopaminergic axons projecting from the substantia nigra undergo dying-back degeneration. Growing evidence shows that dopamine depletion triggers network-level remodeling in the striatum, whose pathological significance extends far beyond acute changes in neuronal excitability. Striatal parvalbumin interneurons (PVINs) have recently been recognized as unique integrators of dopaminergic, neuroinflammatory and electrical network signals and as the principal striatal source of glial-cell-line-derived neurotrophic factor (GDNF). This integrative capacity renders PVINs early targets of parkinsonian injury, yet also allows them to orchestrate compensatory plasticity that shapes subsequent disease progression. Here we review how PVINs, via receptor-specific signaling, drive network reorganization in response to dopaminergic degeneration. We propose that these cells follow a compensatory-to-degenerative trajectory that canalizes abnormal synaptic plasticity and thereby exerts a maladaptive influence on PD pathogenesis. Finally, we discuss the therapeutic potential of interventions targeting these adaptive mechanisms.

Rhythmic neural network activity has been broadly linked to behavior. However, it is unclear how membrane potentials of individual neurons track behavioral rhythms, even though many neurons exhibit pace-making properties in isolated brain circuits. To examine whether single-cell voltage rhythmicity is coupled to behavioral rhythms, we focused on delta-frequencies (1-4 Hz) that are known to occur at both the neural network and behavioral levels. We performed membrane voltage imaging of individual striatal neurons simultaneously with network-level local field potential recordings in mice during voluntary movement. We report sustained delta oscillations in the membrane potentials of many striatal neurons, particularly cholinergic interneurons, which organize spikes and network oscillations at beta-frequencies (20-40 Hz) associated with locomotion. Furthermore, the delta-frequency patterned cellular dynamics are coupled to animals' stepping cycles. Thus, delta-rhythmic cellular dynamics in cholinergic interneurons, known for their autonomous pace-making capabilities, play an important role in regulating network rhythmicity and movement patterning.

Dopamine affects voluntary movement by modulating basal ganglia function. However, the contribution of dopamine on striatopallidal synapses, an initial hub in the indirect pathway connecting the striatum to the GPe, remains poorly understood because of the sparse dopaminergic innervation. Here, we combine optogenetic projection targeting, whole cell patch clamp recordings in acute brain slices from mice, and computational modeling to overcome this limitation. We show that dopamine activates D2 receptors (D2Rs) and D4 receptors (D4Rs) differentially in distinct GPe subregions. In a pinwheel-like fashion, dorsolateral and ventromedial GPe expresses high levels of D2Rs, which exert presynaptic inhibition, while in dorsomedial and ventrolateral GPe D4Rs cause postsynaptic inhibition. Dopamine depletion by 6-OHDA reshapes the region-specific effect of dopamine, shifting it in the opposite direction and contributing to hypokinesia. These findings reveal the mechanism by which the different modality information conveyed spatially through the indirect pathway is differentially modulated by dopamine at striatopallidal synapses.

Advances in optical techniques and two-photon (2P) sensitive genetic voltage indicators (GEVIs) enabled in-depth voltage imaging at single-spike and single-cell resolution. These results were achieved using ASAP-type sensors, while rhodopsin-based GEVIs were mainly used with one-photon (1P) illumination. Here, we demonstrate compatibility of rhodopsin-based GEVIs with 2P illumination. We rationally engineer a fully genetically encoded, rhodopsin-based GEVI, just another voltage indicating sensor (Jarvis), and demonstrate its utility under 1P and 2P illumination. We further show 2P usability of the fluorescence resonance energy transfer (FRET)-opsin GEVIs pAce and Voltron2. Comparing 2P scanless with fast 2P scanning illumination revealed that responses are resolved with both approaches, but FRET-opsin GEVIs show improved signal-to-noise ratio (SNR) with low irradiance, inherent to scanless illumination. Utilizing Jarvis and pAce, we establish high-SNR action potential detection at kilohertz imaging rates in mouse hippocampal slices, zebrafish larvae, and the cortex of awake mice, demonstrating high-contrast action potential detection under 2P illumination with rhodopsin-based GEVIs in vitro and in vivo.