Selective Modulation of Orbitofrontal Network Activity during Negative Occasion Setting.

Discrete cues can gain powerful control over behavior to help an animal anticipate and cope with upcoming events. This is important in conditions where understanding the relationship between complex stimuli provides a means to resolving situational ambiguity. However, it is unclear how cortical circuits generate and maintain these signals that conditionally regulate behavior. To address this, we established a Pavlovian serial feature-negative conditioning paradigm, where male mice are trained on a trial in which a conditioned stimulus (CS) is presented alone and followed by reward, or a feature-negative trial in which the CS is preceded by a feature cue indicating there is no reward. Mice learn to respond with anticipatory licking to a solitary CS, but significantly suppress their responding to the same cue during feature-negative trials. We show that the feature cue forms a selective association with its paired CS, because the ability of the feature to transfer its suppressive properties to a separately rewarded cue is limited. Next, to examine the underlying neural dynamics, we conduct recordings in the orbitofrontal cortex (OFC). We find that the feature cue significantly and selectively inhibits CS-evoked activity. Finally, we find that the feature triggers a distinct OFC network state during the delay period between the feature and CS, establishing a potential link between the feature and future events. Together, our findings suggest that OFC dynamics are modulated by the feature cue and its associated conditioned stimulus in a manner consistent with an occasion setting model.SIGNIFICANCE STATEMENT The ability of patterned cues to form an inhibitory relationship with ambiguously rewarded outcomes has been appreciated since early studies on learning and memory. However, it was often assumed that these cues, despite their hierarchical nature, still made direct associative links with neural rewarding events. This model was significantly challenged, largely by the work of Holland and colleagues, who demonstrated that under certain conditions cues can inherit occasion setting properties whereby they modulate the ability of a paired cue to elicit its conditioned response. Here we provide some of the first evidence that the activity of a cortical circuit is selectively modulated by such cues, thereby providing insight into the mechanisms of higher order learning.

Differential Encoding of Time by Prefrontal and Striatal Network Dynamics.

Telling time is fundamental to many forms of learning and behavior, including the anticipation of rewarding events. Although the neural mechanisms underlying timing remain unknown, computational models have proposed that the brain represents time in the dynamics of neural networks. Consistent with this hypothesis, changing patterns of neural activity dynamically in a number of brain areas-including the striatum and cortex-has been shown to encode elapsed time. To date, however, no studies have explicitly quantified and contrasted how well different areas encode time by recording large numbers of units simultaneously from more than one area. Here, we performed large-scale extracellular recordings in the striatum and orbitofrontal cortex of mice that learned the temporal relationship between a stimulus and a reward and reported their response with anticipatory licking. We used a machine-learning algorithm to quantify how well populations of neurons encoded elapsed time from stimulus onset. Both the striatal and cortical networks encoded time, but the striatal network outperformed the orbitofrontal cortex, a finding replicated both in simultaneously and nonsimultaneously recorded corticostriatal datasets. The striatal network was also more reliable in predicting when the animals would lick up to ∼1 s before the actual lick occurred. Our results are consistent with the hypothesis that temporal information is encoded in a widely distributed manner throughout multiple brain areas, but that the striatum may have a privileged role in timing because it has a more accurate "clock" as it integrates information across multiple cortical areas. SIGNIFICANCE STATEMENT: The neural representation of time is thought to be distributed across multiple functionally specialized brain structures, including the striatum and cortex. However, until now, the neural code for time has not been compared quantitatively between these areas. Here, we performed large-scale recordings in the striatum and orbitofrontal cortex of mice trained on a stimulus-reward association task involving a delay period and used a machine-learning algorithm to quantify how well populations of simultaneously recorded neurons encoded elapsed time from stimulus onset. We found that, although both areas encoded time, the striatum consistently outperformed the orbitofrontal cortex. These results suggest that the striatum may refine the code for time by integrating information from multiple inputs.

Temporal correlations among functionally specialized striatal neural ensembles in reward-conditioned mice.

As the major input to the basal ganglia, the striatum is innervated by a wide range of other areas. Overlapping input from these regions is speculated to influence temporal correlations among striatal ensembles. However, the network dynamics among behaviorally related neural populations in the striatum has not been extensively studied. We used large-scale neural recordings to monitor activity from striatal ensembles in mice undergoing Pavlovian reward conditioning. A subpopulation of putative medium spiny projection neurons (MSNs) was found to discriminate between cues that predicted the delivery of a reward and cues that predicted no specific outcome. These cells were preferentially located in lateral subregions of the striatum. Discriminating MSNs were more spontaneously active and more correlated than their nondiscriminating counterparts. Furthermore, discriminating fast spiking interneurons (FSIs) represented a highly prevalent group in the recordings, which formed a strongly correlated network with discriminating MSNs. Spike time cross-correlation analysis showed the existence of synchronized activity among FSIs and feedforward inhibitory modulation of MSN spiking by FSIs. These findings suggest that populations of functionally specialized (cue-discriminating) striatal neurons have distinct network dynamics that sets them apart from nondiscriminating cells, potentially to facilitate accurate behavioral responding during associative reward learning.

Brain activity mapping at multiple scales with silicon microprobes containing 1,024 electrodes.

The coordinated activity of neural ensembles across multiple interconnected regions has been challenging to study in the mammalian brain with cellular resolution using conventional recording tools. For instance, neural systems regulating learned behaviors often encompass multiple distinct structures that span the brain. To address this challenge we developed a three-dimensional (3D) silicon microprobe capable of simultaneously measuring extracellular spike and local field potential activity from 1,024 electrodes. The microprobe geometry can be precisely configured during assembly to target virtually any combination of four spatially distinct neuroanatomical planes. Here we report on the operation of such a device built for high-throughput monitoring of neural signals in the orbitofrontal cortex and several nuclei in the basal ganglia. We perform analysis on systems-level dynamics and correlations during periods of conditioned behavioral responding and rest, demonstrating the technology's ability to reveal functional organization at multiple scales in parallel in the mouse brain.

Force tuning explains changes in phasic dopamine signaling during stimulus-reward learning.

According to a popular hypothesis, phasic dopamine (DA) activity encodes a reward prediction error (RPE) necessary for reinforcement learning. However, recent work showed that DA neurons are necessary for performance rather than learning. One limitation of previous work on phasic DA signaling and RPE is the limited behavioral measures. Here, we measured subtle force exertion while recording and manipulating DA activity in the ventral tegmental area (VTA) during stimulus-reward learning. We found two major populations of DA neurons that increased firing before forward and backward force exertion. Force tuning is the same regardless of learning, reward predictability, or outcome valence. Changes in the pattern of force exertion can explain results traditionally used to support the RPE hypothesis, such as modulation by reward magnitude, probability, and unpredicted reward delivery or omission. Thus VTA DA neurons are not used to signal RPE but to regulate force exertion during motivated behavior.

Tendons of myostatin-deficient mice are small, brittle, and hypocellular.

Tendons play a significant role in the modulation of forces transmitted between bones and skeletal muscles and consequently protect muscle fibers from contraction-induced, or high-strain, injuries. Myostatin (GDF-8) is a negative regulator of muscle mass. Inhibition of myostatin not only increases the mass and maximum isometric force of muscles, but also increases the susceptibility of muscle fibers to contraction-induced injury. We hypothesized that myostatin would regulate the morphology and mechanical properties of tendons. The expression of myostatin and the myostatin receptors ACVR2B and ACVRB was detectable in tendons. Surprisingly, compared with wild type (MSTN(+/+)) mice, the tendons of myostatin-null mice (MSTN(-/-)) were smaller and had a decrease in fibroblast density and a decrease in the expression of type I collagen. Tendons of MSTN(-/-) mice also had a decrease in the expression of two genes that promote tendon fibroblast proliferation: scleraxis and tenomodulin. Treatment of tendon fibroblasts with myostatin activated the p38 MAPK and Smad2/3 signaling cascades, increased cell proliferation, and increased the expression of type I collagen, scleraxis, and tenomodulin. Compared with the tendons of MSTN(+/+) mice, the mechanical properties of tibialis anterior tendons from MSTN(-/-) mice had a greater peak stress, a lower peak strain, and increased stiffness. We conclude that, in addition to the regulation of muscle mass and force, myostatin regulates the structure and function of tendon tissues.

Protocol for Recording from Ventral Tegmental Area Dopamine Neurons in Mice while Measuring Force during Head-Fixation.

Many studies in systems neuroscience use head-fixation preparations for in vivo experimentation. While head-fixation confers several advantages, one major limitation is the lack of behavioral measures that quantify whole-body movements. Here, we detail a step-by-step protocol for using a novel head-fixation device that measures the forces exerted by head-fixed mice in multiple dimensions. We further detail how this system can be used in conjunction with in vivo electrophysiology and optogenetics to study dopamine neurons in the ventral tegmental area. For complete details on the use and execution of this protocol, please refer to Hughes et al. (2020a, 2020b).

A Head-Fixation System for Continuous Monitoring of Force Generated During Behavior.

Many studies in neuroscience use head-fixed behavioral preparations, which confer a number of advantages, including the ability to limit the behavioral repertoire and use techniques for large-scale monitoring of neural activity. But traditional studies using this approach use extremely limited behavioral measures, in part because it is difficult to detect the subtle movements and postural adjustments that animals naturally exhibit during head fixation. Here we report a new head-fixed setup with analog load cells capable of precisely monitoring the continuous forces exerted by mice. The load cells reveal the dynamic nature of movements generated not only around the time of task-relevant events, such as presentation of stimuli and rewards, but also during periods in between these events, when there is no apparent overt behavior. It generates a new and rich set of behavioral measures that have been neglected in previous experiments. We detail the construction of the system, which can be 3D-printed and assembled at low cost, show behavioral results collected from head-fixed mice, and demonstrate that neural activity can be highly correlated with the subtle, whole-body movements continuously produced during head restraint.

Opponent regulation of action performance and timing by striatonigral and striatopallidal pathways.

The basal ganglia have been implicated in action selection and timing, but the relative contributions of the striatonigral (direct) and striatopallidal (indirect) pathways to these functions remain unclear. We investigated the effects of optogenetic stimulation of D1+ (direct) and A2A+ (indirect) neurons in the ventrolateral striatum in head-fixed mice on a fixed time reinforcement schedule. Direct pathway stimulation initiates licking, whereas indirect pathway stimulation suppresses licking and results in rebound licking after stimulation. Moreover, direct and indirect pathways also play distinct roles in timing. Direct pathway stimulation produced a resetting of the internal timing process, whereas indirect pathway stimulation transiently paused timing, and proportionally delayed the next bout of licking. Our results provide evidence for the continuous and opposing contributions of the direct and indirect pathways in the production and timing of reward-guided behavior.

Ventral Tegmental Dopamine Neurons Control the Impulse Vector during Motivated Behavior.

The ventral tegmental area (VTA) is a major source of dopamine, especially to the limbic brain regions. Despite decades of research, the function of VTA dopamine neurons remains controversial. Here, using a novel head-fixed behavioral system with five orthogonal force sensors, we show for the first time that the activity of dopamine neurons precisely represents the impulse vector (force exerted over time) generated by the animal. Distinct populations of VTA dopamine neurons contribute to components of the impulse vector in different directions. Optogenetic excitation of these neurons shows a linear relationship between signal injected and impulse generated. Optogenetic inhibition paused force generation or produced force in the backward direction. At the same time, these neurons also regulate the initiation and execution of anticipatory licking. Our results indicate that VTA dopamine controls the magnitude, direction, and duration of force used to move toward or away from any motivationally relevant stimuli.

Temporally restricted dopaminergic control of reward-conditioned movements.

Midbrain dopamine (DA) neurons encode both reward- and movement-related events and are implicated in disorders of reward processing as well as movement. Consequently, disentangling the contribution of DA neurons in reinforcing versus generating movements is challenging and has led to lasting controversy. In this study, we dissociated these functions by parametrically varying the timing of optogenetic manipulations in a Pavlovian conditioning task and examining the influence on anticipatory licking before reward delivery. Inhibiting both ventral tegmental area and substantia nigra pars compacta DA neurons in the post-reward period had a significantly greater behavioral effect than inhibition in the pre-reward period of the task. Furthermore, the contribution of DA neurons to behavior decreased linearly as a function of elapsed time after reward. Together, the results indicate a temporally restricted role of DA neurons primarily related to reinforcing stimulus-reward associations and suggest that directly generating movements is a comparatively less important function.

Breakdown of spatial coding and interneuron synchronization in epileptic mice.

Temporal lobe epilepsy causes severe cognitive deficits, but the circuit mechanisms remain unknown. Interneuron death and reorganization during epileptogenesis may disrupt the synchrony of hippocampal inhibition. To test this, we simultaneously recorded from the CA1 and dentate gyrus in pilocarpine-treated epileptic mice with silicon probes during head-fixed virtual navigation. We found desynchronized interneuron firing between the CA1 and dentate gyrus in epileptic mice. Since hippocampal interneurons control information processing, we tested whether CA1 spatial coding was altered in this desynchronized circuit, using a novel wire-free miniscope. We found that CA1 place cells in epileptic mice were unstable and completely remapped across a week. This spatial instability emerged around 6 weeks after status epilepticus, well after the onset of chronic seizures and interneuron death. Finally, CA1 network modeling showed that desynchronized inputs can impair the precision and stability of CA1 place cells. Together, these results demonstrate that temporally precise intrahippocampal communication is critical for spatial processing.

Precise Coordination of Three-Dimensional Rotational Kinematics by Ventral Tegmental Area GABAergic Neurons.

The ventral tegmental area (VTA) is a midbrain region implicated in a variety of motivated behaviors. However, the function of VTA GABAergic (Vgat+) neurons remains poorly understood. Here, using three-dimensional motion capture, in vivo electrophysiology, calcium imaging, and optogenetics, we demonstrate a novel function of VTAVgat+ neurons. We found three distinct populations of neurons, each representing head angle about a principal axis of rotation: yaw, roll, and pitch. For each axis, opponent cell groups were found that increase firing when the head moves in one direction and decrease firing in the opposite direction. Selective excitation and inhibition of VTAVgat+ neurons generate opposite rotational movements. Thus, VTAVgat+ neurons serve a critical role in the control of rotational kinematics while pursuing a moving target. This general-purpose steering function can guide animals toward desired spatial targets in any motivated behavior.

Gain Modulation by Corticostriatal and Thalamostriatal Input Signals during Reward-Conditioned Behavior.

The cortex and thalamus send excitatory projections to the striatum, but little is known about how these inputs, either individually or collectively, regulate striatal dynamics during behavior. The lateral striatum receives overlapping input from the secondary motor cortex (M2), an area involved in licking, and the parafascicular thalamic nucleus (PF). Using neural recordings, together with optogenetic terminal inhibition, we examine the contribution of M2 and PF projections on medium spiny projection neuron (MSN) activity as mice performed an anticipatory licking task. Each input has a similar contribution to striatal activity. By comparing how suppressing single or multiple projections altered striatal activity, we find that cortical and thalamic input signals modulate MSN gain and that this effect is more pronounced in a temporally specific period of the task following the cue presentation. These results demonstrate that cortical and thalamic inputs synergistically regulate striatal output during reward-conditioned behavior.

Haploinsufficiency of myostatin protects against aging-related declines in muscle function and enhances the longevity of mice.

The molecular mechanisms behind aging-related declines in muscle function are not well understood, but the growth factor myostatin (MSTN) appears to play an important role in this process. Additionally, epidemiological studies have identified a positive correlation between skeletal muscle mass and longevity. Given the role of myostatin in regulating muscle size, and the correlation between muscle mass and longevity, we tested the hypotheses that the deficiency of myostatin would protect oldest-old mice (28-30 months old) from an aging-related loss in muscle size and contractility, and would extend the maximum lifespan of mice. We found that MSTN(+/-) and MSTN(-/-) mice were protected from aging-related declines in muscle mass and contractility. While no differences were detected between MSTN(+/+) and MSTN(-/-) mice, MSTN(+/-) mice had an approximately 15% increase in maximal lifespan. These results suggest that targeting myostatin may protect against aging-related changes in skeletal muscle and contribute to enhanced longevity.

Neural basis of benzodiazepine reward: requirement for α2 containing GABAA receptors in the nucleus accumbens.

Despite long-standing concerns regarding the abuse liability of benzodiazepines, the mechanisms underlying properties of benzodiazepines that may be relevant to abuse are still poorly understood. Earlier studies showed that compounds selective for α1-containing GABAA receptors (α1GABAARs) are abused by humans and self-administered by animals, and that these receptors may underlie a preference for benzodiazepines as well as neuroplastic changes observed in the ventral tegmental area following benzodiazepine administration. There is some evidence, however, that even L-838, 417, a compound with antagonistic properties at α1GABAARs and agonistic properties at the other three benzodiazepine-sensitive GABAA receptor subtypes, is self-administered, and that the α2GABAARs may have a role in benzodiazepine-induced reward enhancement. Using a two-bottle choice drinking paradigm to evaluate midazolam preference and an intracranial self-stimulation (ICSS) paradigm to evaluate the impact of midazolam on reward enhancement, we demonstrated that mice carrying a histidine-to-arginine point mutation in the α2 subunit which renders it insensitive to benzodiazepines (α2(H101R) mice) did not prefer midazolam and did not show midazolam-induced reward enhancement in ICSS, in contrast to wild-type controls, suggesting that α2GABAARs are necessary for the reward enhancing effects and preference for oral benzodiazepines. Through a viral-mediated knockdown of α2GABAARs in the nucleus accumbens (NAc), we demonstrated that α2 in the NAc is necessary for the preference for midazolam. Findings imply that α2GABAARs in the NAc are involved in at least some reward-related properties of benzodiazepines, which might partially underlie repeated drug-taking behavior.

Physiological loading of tendons induces scleraxis expression in epitenon fibroblasts.

Scleraxis is a basic helix-loop-helix transcription factor that plays a central role in promoting fibroblast proliferation and matrix synthesis during the embryonic development of tendons. Mice with a targeted inactivation of scleraxis (Scx(-/-)) fail to properly form limb tendons, but the role that scleraxis has in regulating the growth and adaptation of tendons of adult organisms is unknown. To determine if scleraxis expression changes in response to a physiological growth stimulus to tendons, we subjected adult mice that express green fluorescent protein (GFP) under the control of the scleraxis promoter (ScxGFP) to a 6-week-treadmill training program designed to induce adaptive growth in Achilles tendons. Age matched sedentary ScxGFP mice were used as controls. Scleraxis expression was sparsely observed in the epitenon region of sedentary mice, but in response to treadmill training, scleraxis was robustly expressed in fibroblasts that appeared to be emerging from the epitenon and migrating into the superficial regions of tendon fascicles. Treadmill training also led to an increase in scleraxis, tenomodulin, and type I collagen gene expression as measured by qPCR. These results suggest that in addition to regulating the embryonic formation of limb tendons, scleraxis also appears to play an important role in the adaptation of adult tendons to physiological loading.