Imaging the awake visual cortex with a genetically encoded voltage indicator.

Genetically encoded voltage indicators (GEVIs) promise to reveal the membrane potential of genetically targeted neuronal populations through noninvasive, chronic imaging of large portions of cortical space. Here we test a promising GEVI in mouse cortex during wakefulness, a challenging condition due to large hemodynamic activity, and we introduce a straightforward projection method to separate a signal dominated by membrane voltage from a signal dominated by hemodynamic activity. We expressed VSFP-Butterfly 1.2 plasmid in layer 2/3 pyramidal cells of visual cortex through electroporation in utero. We then used wide-field imaging with two cameras to measure both fluorophores of the indicator in response to visual stimuli. By taking weighted sums and differences of the two measurements, we obtained clear separation of hemodynamic and voltage signals. The hemodynamic signal showed strong heartbeat oscillations, superimposed on slow dynamics similar to blood oxygen level-dependent (BOLD) or "intrinsic" signals. The voltage signal had fast dynamics similar to neural responses measured electrically, and showed an orderly retinotopic mapping. We compared this voltage signal with calcium signals imaged in transgenic mice that express a calcium indicator (GCaMP3) throughout cortex. The voltage signal from VSFP had similar signal-to-noise ratios as the calcium signal, it was more immune to vascular artifacts, and it integrated over larger regions of visual space, which was consistent with its reporting mostly subthreshold activity rather than the spiking activity revealed by calcium signals. These results demonstrate that GEVIs provide a powerful tool to study the dynamics of neural populations at mesoscopic spatial scales in the awake cortex.

Dopamine neurons learn to encode the long-term value of multiple future rewards.

Midbrain dopamine neurons signal reward value, their prediction error, and the salience of events. If they play a critical role in achieving specific distant goals, long-term future rewards should also be encoded as suggested in reinforcement learning theories. Here, we address this experimentally untested issue. We recorded 185 dopamine neurons in three monkeys that performed a multistep choice task in which they explored a reward target among alternatives and then exploited that knowledge to receive one or two additional rewards by choosing the same target in a set of subsequent trials. An analysis of anticipatory licking for reward water indicated that the monkeys did not anticipate an immediately expected reward in individual trials; rather, they anticipated the sum of immediate and multiple future rewards. In accordance with this behavioral observation, the dopamine responses to the start cues and reinforcer beeps reflected the expected values of the multiple future rewards and their errors, respectively. More specifically, when monkeys learned the multistep choice task over the course of several weeks, the responses of dopamine neurons encoded the sum of the immediate and expected multiple future rewards. The dopamine responses were quantitatively predicted by theoretical descriptions of the value function with time discounting in reinforcement learning. These findings demonstrate that dopamine neurons learn to encode the long-term value of multiple future rewards with distant rewards discounted.

Functional Diversity of Dopamine Axons in Prefrontal Cortex During Classical Conditioning.

Midbrain dopamine neurons impact neural processing in the prefrontal cortex (PFC) through mesocortical projections. However, the signals conveyed by dopamine projections to the PFC remain unclear, particularly at the single-axon level. Here, we investigated dopaminergic axonal activity in the medial PFC (mPFC) during reward and aversive processing. By optimizing microprism-mediated two-photon calcium imaging of dopamine axon terminals, we found diverse activity in dopamine axons responsive to both reward and aversive stimuli. Some axons exhibited a preference for reward, while others favored aversive stimuli, and there was a strong bias for the latter at the population level. Long-term longitudinal imaging revealed that the preference was maintained in reward- and aversive-preferring axons throughout classical conditioning in which rewarding and aversive stimuli were paired with preceding auditory cues. However, as mice learned to discriminate reward or aversive cues, a cue activity preference gradually developed only in aversive-preferring axons. We inferred the trial-by-trial cue discrimination based on machine learning using anticipatory licking or facial expressions, and found that successful discrimination was accompanied by sharper selectivity for the aversive cue in aversive-preferring axons. Our findings indicate that a group of mesocortical dopamine axons encodes aversive-related signals, which are modulated by both classical conditioning across days and trial-by-trial discrimination within a day.

Encoding of self-initiated actions in axon terminals of the mesocortical pathway.

Significance: The initiation of goal-directed actions is a complex process involving the medial prefrontal cortex and dopaminergic inputs through the mesocortical pathway. However, it is unclear what information the mesocortical pathway conveys and how it impacts action initiation. In this study, we unveiled the indispensable role of mesocortical axon terminals in encoding the execution of movements in self-initiated actions. Aim: To investigate the role of mesocortical axon terminals in encoding the execution of movements in self-initiated actions. Approach: We designed a lever-press task in which mice internally determine the timing of the press, receiving a larger reward for longer waiting periods. Results: Our study revealed that self-initiated actions depend on dopaminergic signaling mediated by D2 receptors, whereas sensory-triggered lever-press actions do not involve D2 signaling. Microprism-mediated two-photon calcium imaging further demonstrated ramping activity in mesocortical axon terminals approximately 0.5 s before the self-initiated lever press. Remarkably, the ramping patterns remained consistent whether the mice responded to cues immediately for a smaller reward or held their response for a larger reward. Conclusions: We conclude that mesocortical dopamine axon terminals encode the timing of self-initiated actions, shedding light on a crucial aspect of the intricate neural mechanisms governing goal-directed behavior.

Long-range connections enrich cortical computations.

The cerebral cortex can perform powerful computations, including those involved in higher cognitive functions. Cortical processing for such computations is executed by local circuits and is further enriched by long-range connectivity. This connectivity is activated under specific conditions and modulates local processing, providing flexibility in the computational performance of the cortex. For instance, long-range connectivity in the primary visual cortex exerts facilitatory impacts when the cortex is silent but suppressive impacts when the cortex is strongly sensory-stimulated. These dual impacts can be captured by a divisive gain control model. Recent methodological advances such as optogenetics, anatomical tracing, and two-photon microscopy have enabled neuroscientists to probe the circuit and synaptic bases of long-range connectivity in detail. Here, I review a series of evidence indicating essential roles of long-range connectivity in visual and hierarchical processing involving numerous cortical areas. I also describe an overview of the challenges encountered in investigating underlying synaptic mechanisms and highlight recent technical approaches that may overcome these difficulties and provide new insights into synaptic mechanisms for cortical processing involving long-range connectivity.

A Canonical Scheme of Bottom-Up and Top-Down Information Flows in the Frontoparietal Network.

Goal-directed behavior often involves temporal separation and flexible context-dependent association between sensory input and motor output. The control of goal-directed behavior is proposed to lie in the frontoparietal network, but the computational architecture of this network remains elusive. Based on recent rodent studies that measured and manipulated projection neurons in the frontoparietal network together with findings from earlier primate studies, we propose a canonical scheme of information flows in this network. The parietofrontal pathway transmits the spatial information of a sensory stimulus or internal motor bias to drive motor programs in the frontal areas. This pathway might consist of multiple parallel connections, each controlling distinct motor effectors. The frontoparietal pathway sends the spatial information of cognitively processed motor plans through multiple parallel connections. Each of these connections could support distinct spatial functions that use the motor target information, including attention allocation, multi-body part coordination, and forward estimation of movement state (i.e., forward models). The parallel pathways in the frontoparietal network enable dynamic interactions between regions that are tuned for specific goal-directed behaviors. This scheme offers a promising framework within which the computational architecture of the frontoparietal network and the underlying circuit mechanisms can be delineated in a systematic way, providing a holistic understanding of information processing in this network. Clarifying this network may also improve the diagnosis and treatment of behavioral deficits associated with dysfunctional frontoparietal connectivity in various neurological disorders including Alzheimer's disease.

Interhemispherically dynamic representation of an eye movement-related activity in mouse frontal cortex.

Cortical plasticity is fundamental to motor recovery following cortical perturbation. However, it is still unclear how this plasticity is induced at a functional circuit level. Here, we investigated motor recovery and underlying neural plasticity upon optogenetic suppression of a cortical area for eye movement. Using a visually-guided eye movement task in mice, we suppressed a portion of the secondary motor cortex (MOs) that encodes contraversive eye movement. Optogenetic unilateral suppression severely impaired contraversive movement on the first day. However, on subsequent days the suppression became inefficient and capability for the movement was restored. Longitudinal two-photon calcium imaging revealed that the regained capability was accompanied by an increased number of neurons encoding for ipsiversive movement in the unsuppressed contralateral MOs. Additional suppression of the contralateral MOs impaired the recovered movement again, indicating a compensatory mechanism. Our findings demonstrate that repeated optogenetic suppression leads to functional recovery mediated by the contralateral hemisphere.

Streamlined sensory motor communication through cortical reciprocal connectivity in a visually guided eye movement task.

Cortical computation is distributed across multiple areas of the cortex by networks of reciprocal connectivity. However, how such connectivity contributes to the communication between the connected areas is not clear. In this study, we examine the communication between sensory and motor cortices. We develop an eye movement task in mice and combine it with optogenetic suppression and two-photon calcium imaging techniques. We identify a small region in the secondary motor cortex (MOs) that controls eye movements and reciprocally connects with a rostrolateral part of the higher visual areas (VRL/A/AL). These two regions encode both motor signals and visual information; however, the information flow between the regions depends on the direction of the connectivity: motor information is conveyed preferentially from the MOs to the VRL/A/AL, and sensory information is transferred primarily in the opposite direction. We propose that reciprocal connectivity streamlines information flow, enhancing the computational capacity of a distributed network.

Selective Suppression of Local Circuits during Movement Preparation in the Mouse Motor Cortex.

Prepared movements are more efficient than those that are not prepared for. Although changes in cortical activity have been observed prior to a forthcoming action, the circuits involved in motor preparation remain unclear. Here, we use in vivo two-photon calcium imaging to uncover changes in the motor cortex during variable waiting periods prior to a forepaw reaching task in mice. Consistent with previous reports, we observed a subset of neurons with increased activity during the waiting period; however, these neurons did not account for the degree of preparation as defined by reaction time (RT). Instead, the suppression of activity of distinct neurons in the same cortical area better accounts for RT. This suppression of neural activity resulted in a distinct and reproducible pattern when mice were well prepared. Thus, the selective suppression of network activity in the motor cortex may be a key feature of prepared movements.

An excitatory basis for divisive normalization in visual cortex.

Neurons in visual cortex are connected not only locally, but also through networks of distal connectivity. These distal networks recruit both excitatory and inhibitory synapses and result in divisive normalization. Normalization is traditionally thought to result from increases in synaptic inhibition. By combining optogenetic stimulation and intracellular recordings in mouse visual cortex, we found that, on the contrary, normalization is a result of a decrease in synaptic excitation.

Distal connectivity causes summation and division across mouse visual cortex.

Neurons in different locations across the cortex are connected through polysynaptic networks involving both excitation and inhibition. To probe the functional effect of such networks, we used optogenetic stimulation to trigger antidromic spikes in a local region of primary visual cortex (V1). This local activity had two effects at distal V1 locations: summation and division. The balance between the two depended on visual contrast, and a normalization model precisely captured these effects.

Traveling waves in visual cortex.

Electrode recordings and imaging studies have revealed that localized visual stimuli elicit waves of activity that travel across primary visual cortex. Traveling waves are present also during spontaneous activity, but they can be greatly reduced by widespread and intensive visual stimulation. In this Review, we summarize the evidence in favor of these traveling waves. We suggest that their substrate may lie in long-range horizontal connections and that their functional role may involve the integration of information over large regions of space.