DART.2: bidirectional synaptic pharmacology with thousandfold cellular specificity.

Precision pharmacology aims to manipulate specific cellular interactions within complex tissues. In this pursuit, we introduce DART.2 (drug acutely restricted by tethering), a second-generation cell-specific pharmacology technology. The core advance is optimized cellular specificity-up to 3,000-fold in 15 min-enabling the targeted delivery of even epileptogenic drugs without off-target effects. Additionally, we introduce brain-wide dosing methods as an alternative to local cannulation and tracer reagents for brain-wide dose quantification. We describe four pharmaceuticals-two that antagonize excitatory and inhibitory postsynaptic receptors, and two that allosterically potentiate these receptors. Their versatility is showcased across multiple mouse-brain regions, including cerebellum, striatum, visual cortex and retina. Finally, in the ventral tegmental area, we find that blocking inhibitory inputs to dopamine neurons accelerates locomotion, contrasting with previous optogenetic and pharmacological findings. Beyond enabling the bidirectional perturbation of chemical synapses, these reagents offer intersectional precision-between genetically defined postsynaptic cells and neurotransmitter-defined presynaptic partners.

Input-specific synaptic depression shapes temporal integration in mouse visual cortex.

Efficient sensory processing requires the nervous system to adjust to ongoing features of the environment. In primary visual cortex (V1), neuronal activity strongly depends on recent stimulus history. Existing models can explain effects of prolonged stimulus presentation but remain insufficient for explaining effects observed after shorter durations commonly encountered under natural conditions. We investigated the mechanisms driving adaptation in response to brief (100 ms) stimuli in L2/3 V1 neurons by performing in vivo whole-cell recordings to measure membrane potential and synaptic inputs. We find that rapid adaptation is generated by stimulus-specific suppression of excitatory and inhibitory synaptic inputs. Targeted optogenetic experiments reveal that these synaptic effects are due to input-specific short-term depression of transmission between layers 4 and 2/3. Thus, brief stimulus presentation engages a distinct adaptation mechanism from that previously reported in response to prolonged stimuli, enabling flexible control of sensory encoding across a wide range of timescales.

Input-specific synaptic depression shapes temporal integration in mouse visual cortex.

Efficient sensory processing requires the nervous system to adjust to ongoing features of the environment. In primary visual cortex (V1), neuronal activity strongly depends on recent stimulus history. Existing models can explain effects of prolonged stimulus presentation, but remain insufficient for explaining effects observed after shorter durations commonly encountered under natural conditions. We investigated the mechanisms driving adaptation in response to brief (100 ms) stimuli in L2/3 V1 neurons by performing in vivo whole-cell recordings to measure membrane potential and synaptic inputs. We find that rapid adaptation is generated by stimulus-specific suppression of excitatory and inhibitory synaptic inputs. Targeted optogenetic experiments reveal that these synaptic effects are due to input-specific short-term depression of transmission between layers 4 and 2/3. Thus, distinct mechanisms are engaged following brief and prolonged stimulus presentation and together enable flexible control of sensory encoding across a wide range of time scales.

Feedforward mechanisms of cross-orientation interactions in mouse V1.

Sensory neurons are modulated by context. For example, in mouse primary visual cortex (V1), neuronal responses to the preferred orientation are modulated by the presence of superimposed orientations ("plaids"). The effects of this modulation are diverse; some neurons are suppressed, while others have larger responses to a plaid than its components. We investigated whether this diversity could be explained by a unified circuit mechanism. We report that this masking is maintained during suppression of cortical activity, arguing against cortical mechanisms. Instead, the heterogeneity of plaid responses is explained by an interaction between stimulus geometry and orientation tuning. Highly selective neurons are uniformly suppressed by plaids, whereas the effects in weakly selective neurons depend on the spatial configuration of the stimulus, transitioning systematically between suppression and facilitation. Thus, the diverse responses emerge as a consequence of the spatial structure of feedforward inputs, with no need to invoke cortical interactions.

Distinct recruitment of feedforward and recurrent pathways across higher-order areas of mouse visual cortex.

Cortical visual processing transforms features of the external world into increasingly complex and specialized neuronal representations. These transformations arise in part through target-specific routing of information; however, within-area computations may also contribute to area-specific function. Here, we sought to determine whether higher order visual cortical areas lateromedial (LM), anterolateral (AL), posteromedial (PM), and anteromedial (AM) have specialized anatomical and physiological properties by using a combination of whole-cell recordings and optogenetic stimulation of primary visual cortex (V1) axons in vitro. We discovered area-specific differences in the strength of recruitment of interneurons through feedforward and recurrent pathways, as well as differences in cell-intrinsic properties and interneuron densities. These differences were most striking when comparing across medial and lateral areas, suggesting that these areas have distinct profiles for net excitability and integration of V1 inputs. Thus, cortical areas are not defined simply by the information they receive but also by area-specific circuit properties that enable specialized filtering of these inputs.

Mouse Higher Visual Areas Provide Both Distributed and Specialized Contributions to Visually Guided Behaviors.

Cortical parallel processing streams segregate many diverse features of a sensory scene. However, some features are distributed across streams, begging the question of whether and how such distributed representations contribute to perception. We determined the necessity of the primary visual cortex (V1) and three key higher visual areas (lateromedial [LM], anterolateral [AL], and posteromedial [PM]) for perception of orientation and contrast, two features that are robustly encoded across all four areas. Suppressing V1, LM, or AL decreased sensitivity for both orientation discrimination and contrast detection, consistent with a role for these areas in sensory perception. In comparison, suppressing PM selectively increased false alarm (FA) rates during contrast detection, without any effect on orientation discrimination. This effect was not retinotopically specific, suggesting that suppression of PM altered sensory integration or the decision-making process rather than processing of local visual features. Thus, we find that distributed representations in the visual system can nonetheless support specialized perceptual roles for higher visual cortical areas.

Magnitude, time course, and specificity of rapid adaptation across mouse visual areas.

Adaptation is a ubiquitous feature of sensory processing whereby recent experience shapes future responses. The mouse primary visual cortex (V1) is particularly sensitive to recent experience, where a brief stimulus can suppress subsequent responses for seconds. This rapid adaptation profoundly impacts perception, suggesting that its effects are propagated along the visual hierarchy. To understand how rapid adaptation influences sensory processing, we measured its effects at key nodes in the visual system: in V1, three higher visual areas (HVAs: lateromedial, anterolateral, and posteromedial), and the superior colliculus (SC) in awake mice of both sexes using single-unit recordings. Consistent with the feed-forward propagation of adaptation along the visual hierarchy, we find that neurons in layer 4 adapt less strongly than those in other layers of V1. Furthermore, neurons in the HVAs adapt more strongly, and recover more slowly, than those in V1. The magnitude and time course of adaptation was comparable in each of the HVAs and in the SC, suggesting that adaptation may not linearly accumulate along the feed-forward visual processing hierarchy. Despite the increase in adaptation in the HVAs compared with V1, the effects were similarly orientation specific across all areas. These data reveal that adaptation profoundly shapes cortical processing, with increasing impact at higher levels in the cortical hierarchy, and also strongly influencing computations in the SC. Thus, we find robust, brain-wide effects of rapid adaptation on sensory processing.NEW & NOTEWORTHY Rapid adaptation dynamically alters sensory signals to account for recent experience. To understand how adaptation affects sensory processing and perception, we must determine how it impacts the diverse set of cortical and subcortical areas along the hierarchy of the mouse visual system. We find that rapid adaptation strongly impacts neurons in primary visual cortex, the higher visual areas, and the colliculus, consistent with its profound effects on behavior.

Unique Spatial Integration in Mouse Primary Visual Cortex and Higher Visual Areas.

Neurons in the visual system integrate over a wide range of spatial scales. This diversity is thought to enable both local and global computations. To understand how spatial information is encoded across the mouse visual system, we use two-photon imaging to measure receptive fields (RFs) and size-tuning in primary visual cortex (V1) and three downstream higher visual areas (HVAs: LM (lateromedial), AL (anterolateral), and PM (posteromedial)) in mice of both sexes. Neurons in PM, compared with V1 or the other HVAs, have significantly larger RF sizes and less surround suppression, independent of stimulus eccentricity or contrast. To understand how this specialization of RFs arises in the HVAs, we measured the spatial properties of V1 inputs to each area. Spatial integration of V1 axons was remarkably similar across areas and significantly different from the tuning of neurons in their target HVAs. Thus, unlike other visual features studied in this system, specialization of spatial integration in PM cannot be explained by specific projections from V1 to the HVAs. Further, the differences in RF properties could not be explained by differences in convergence of V1 inputs to the HVAs. Instead, our data suggest that distinct inputs from other areas or connectivity within PM may support the area's unique ability to encode global features of the visual scene, whereas V1, LM, and AL may be more specialized for processing local features.SIGNIFICANCE STATEMENT Surround suppression is a common feature of visual processing whereby large stimuli are less effective at driving neuronal responses than smaller stimuli. This is thought to enhance efficiency in the population code and enable higher-order processing of visual information, such as figure-ground segregation. However, this comes at the expense of global computations. Here we find that surround suppression is not equally represented across mouse visual areas: primary visual cortex has substantially more surround suppression than higher visual areas, and one higher area has significantly less suppression than two others examined, suggesting that these areas have distinct functional roles. Thus, we have identified a novel dimension of specialization in the mouse visual cortex that may enable both local and global computations.

Contribution of Sensory Encoding to Measured Bias.

Signal detection theory (SDT) is a widely used theoretical framework that describes how variable sensory signals are integrated with a decision criterion to support perceptual decision-making. SDT provides two key measurements: sensitivity (d') and bias (c), which reflect the separability of decision variable distributions (signal and noise) and the position of the decision criterion relative to optimal, respectively. Although changes in the subject's decision criterion can be reflected in changes in bias, decision criterion placement is not the sole contributor to measured bias. Indeed, neuronal representations of bias have been observed in sensory areas, suggesting that some changes in bias are because of effects on sensory encoding. To directly test whether the sensory encoding process can influence bias, we optogenetically manipulated neuronal excitability in primary visual cortex (V1) in mice of both sexes during either an orientation discrimination or a contrast detection task. Increasing excitability in V1 significantly decreased behavioral bias, whereas decreasing excitability had the opposite effect. To determine whether this change in bias is consistent with effects on sensory encoding, we made extracellular recordings from V1 neurons in passively viewing mice. Indeed, we found that optogenetic manipulation of excitability shifted the neuronal bias in the same direction as the behavioral bias. Moreover, manipulating the quality of V1 encoding by changing stimulus contrast or interstimulus interval also resulted in consistent changes in both behavioral and neuronal bias. Thus, changes in sensory encoding are sufficient to drive changes in bias measured using SDT.SIGNIFICANCE STATEMENT Perceptual decision-making involves sensory integration followed by application of a cognitive criterion. Using signal detection theory, one can extract features of the underlying decision variables and rule: sensitivity (d') and bias (c). Because bias is measured as the difference between the optimal and actual criterion, it is sensitive to both the sensory encoding processes and the placement of the decision criterion. Here, we use behavioral and electrophysiological approaches to demonstrate that measures of bias depend on sensory processes. Optogenetic manipulations of V1 in mice bidirectionally affect both behavioral and neuronal measures of bias with little effect on sensitivity. Thus, changes in sensory encoding influence bias, and the absence of changes in sensitivity do not preclude changes in sensory encoding.

Neuronal Adaptation Reveals a Suboptimal Decoding of Orientation Tuned Populations in the Mouse Visual Cortex.

Sensory information is encoded by populations of cortical neurons. Yet, it is unknown how this information is used for even simple perceptual choices such as discriminating orientation. To determine the computation underlying this perceptual choice, we took advantage of the robust visual adaptation in mouse primary visual cortex (V1). We first designed a stimulus paradigm in which we could vary the degree of neuronal adaptation measured in V1 during an orientation discrimination task. We then determined how adaptation affects task performance for mice of both sexes and tested which neuronal computations are most consistent with the behavioral results given the adapted population responses in V1. Despite increasing the reliability of the population representation of orientation among neurons, and improving the ability of a variety of optimal decoders to discriminate target from distractor orientations, adaptation increases animals' behavioral thresholds. Decoding the animals' choice from neuronal activity revealed that this unexpected effect on behavior could be explained by an overreliance of the perceptual choice circuit on target preferring neurons and a failure to appropriately discount the activity of neurons that prefer the distractor. Consistent with this all-positive computation, we find that animals' task performance is susceptible to subtle perturbations of distractor orientation and optogenetic suppression of neuronal activity in V1. This suggests that to solve this task the circuit has adopted a suboptimal and task-specific computation that discards important task-related information.SIGNIFICANCE STATEMENT A major goal in systems neuroscience is to understand how sensory signals are used to guide behavior. This requires determining what information in sensory cortical areas is used, and how it is combined, by downstream perceptual choice circuits. Here we demonstrate that when performing a go/no-go orientation discrimination task, mice suboptimally integrate signals from orientation tuned visual cortical neurons. While they appropriately positively weight target-preferring neurons, they fail to negatively weight distractor-preferring neurons. We propose that this all-positive computation may be adopted because of its simple learning rules and faster processing, and may be a common approach to perceptual decision-making when task conditions allow.

Visual Attention: Mice Can Use Spatial Cues Too.

The use of cues to predict the location of a visual target is an important cognitive strategy for primates. While mice are generally considered to be less dependent on vision than primates, new work shows that they can also use spatial cues to direct their visual attention.

Higher-Order Areas of the Mouse Visual Cortex.

The brain has evolved to transform sensory information in the environment into neural representations that can be used for perception and action. Higher-order sensory cortical areas, with their increasingly complex receptive fields and integrative properties, are thought to be critical nodes for this function. This is especially true in the primate visual cortex, in which functionally specialized areas are engaged in parallel streams to support diverse computations. Recent anatomical and physiological studies of the mouse visual cortex have revealed a similarly complex network of specialized higher-order areas. This structure provides a useful model for determining the synaptic and circuit mechanisms through which information is transformed across distinct processing stages. In this review, we summarize the current knowledge on the layout, connectivity, and functional properties of the higher visual areas in the mouse. In addition, we speculate on the contribution of these areas to perception and action, and how knowledge of the mouse visual system can inform us about the principles that govern information processing in integrated networks.

High-fidelity optical excitation of cortico-cortical projections at physiological frequencies.

Optogenetic activation of axons is a powerful approach for determining the synaptic properties and impact of long-range projections both in vivo and in vitro. However, because of the difficulty of measuring activity in axons, our knowledge of the reliability of optogenetic axonal stimulation has relied on data from somatic recordings. Yet, there are many reasons why activation of axons may not be comparable to cell bodies. Thus we have developed an approach to more directly assess the fidelity of optogenetic activation of axonal projections. We expressed opsins (ChR2, Chronos, or oChIEF) in the mouse primary visual cortex (V1) and recorded extracellular, pharmacologically isolated presynaptic action potentials in response to axonal activation in the higher visual areas. Repetitive stimulation of axons with ChR2 resulted in a 70% reduction in the fiber volley amplitude and a 60% increase in the latency at all frequencies tested (10-40 Hz). Thus ChR2 cannot reliably recruit axons during repetitive stimulation, even at frequencies that are reliable for somatic stimulation, likely due to pronounced channel inactivation at the high light powers required to evoke action potentials. By comparison, oChIEF and Chronos evoked photocurrents that inactivated minimally and could produce reliable axon stimulation at frequencies up to 60 Hz. Our approach provides a more direct and accurate evaluation of the efficacy of new optogenetic tools and has identified Chronos and oChIEF as viable tools to interrogate the synaptic and circuit function of long-range projections.

Removable cranial windows for long-term imaging in awake mice.

Cranial window implants in head-fixed rodents are becoming a preparation of choice for stable optical access to large areas of the cortex over extended periods of time. Here we provide a highly detailed and reliable surgical protocol for a cranial window implantation procedure for chronic wide-field and cellular imaging in awake, head-fixed mice, which enables subsequent window removal and replacement in the weeks and months after the initial craniotomy. This protocol has facilitated awake, chronic imaging in adolescent and adult mice over several months from a large number of cortical brain regions; targeted virus and tracer injections from data obtained using prior awake functional mapping; and functionally targeted two-photon imaging across all cortical layers in awake mice using a microprism attachment to the cranial window. Collectively, these procedures extend the reach of chronic imaging of cortical function and dysfunction in behaving animals.

Visual circuits get the VIP treatment.

Behavioral state, specifically locomotion, has been shown to enhance sensory responses in primary visual cortex. In this issue of Cell, Fu et al. reveal the circuit elements that mediate this plasticity and suggest that these circuits may serve a general modulatory function across primary sensory areas.

A mouse model of higher visual cortical function.

During sensory experience, the retina transmits a diverse array of signals to the brain, which must be parsed to generate meaningful percepts that can guide decisions and actions. Decades of anatomical and physiological studies in primates and carnivores have revealed a complex parallel and hierarchical organization by which distinct visual features are distributed to, and processed by, different brain regions. However, these studies have been limited in their ability to dissect the circuit mechanisms involved in the transformation of sensory inputs into complex cortical representations and action patterns. Multiple groups have therefore pushed to explore the organization and function of higher visual areas in the mouse. Here we review the anatomical and physiological findings of these recent explorations in mouse visual cortex. These studies find that sensory input is processed in a diverse set of higher areas that are each interconnected with specific limbic and motor systems. This hierarchical and parallel organization is consistent with the multiple streams that have been found in the higher visual areas of primates. We therefore propose that the mouse visual system is a useful model to explore the circuits underlying the transformation of sensory inputs into goal-directed perceptions and actions.

Mouse primary visual cortex is used to detect both orientation and contrast changes.

In mammals, the lateral geniculate nucleus (LGN) and the superior colliculus (SC) are the major targets of visual inputs from the retina. The LGN projects mainly to primary visual cortex (V1) while the SC targets the thalamus and brainstem, providing two potential pathways for processing visual inputs. Indeed, cortical lesion experiments in rodents have yielded mixed results, leading to the hypothesis that performance of simple visual behaviors may involve computations performed entirely by this subcortical pathway through the SC. However, these previous experiments have been limited by both their assays of behavioral performance and their use of lesions to change cortical activity. To determine the contribution of V1 to these tasks, we trained mice to perform threshold detection tasks in which they reported changes in either the contrast or orientation of visual stimuli. We then reversibly inhibited V1 by optogenetically activating parvalbumin-expressing inhibitory neurons with channelrhodopsin-2. We found that suppressing activity in V1 substantially impaired performance in visual detection tasks. The behavioral deficit depended on the retinotopic position of the visual stimulus, confirming that the effect was due to the specific suppression of the visually driven V1 neurons. Behavioral effects were seen with only moderate changes in neuronal activity, as inactivation that raised neuronal contrast thresholds by a median of only 14% was associated with a doubling of behavioral contrast detection threshold. Thus, detection of changes in either orientation or contrast is dependent on, and highly sensitive to, the activity of neurons in V1.

Cortico-cortical projections in mouse visual cortex are functionally target specific.

Neurons in primary sensory cortex have diverse response properties, whereas higher cortical areas are specialized. Specific connectivity may be important for areal specialization, particularly in the mouse, where neighboring neurons are functionally diverse. To examine whether higher visual areas receive functionally specific input from primary visual cortex (V1), we used two-photon calcium imaging to measure responses of axons from V1 arborizing in three areas with distinct spatial and temporal frequency preferences. We found that visual preferences of presynaptic boutons in each area were distinct and matched the average preferences of recipient neurons. This specificity could not be explained by organization within V1 and instead was due to both a greater density and greater response amplitude of functionally matched boutons. Projections from a single layer (layer 5) and from secondary visual cortex were also matched to their target areas. Thus, transmission of specific information to downstream targets may be a general feature of cortico-cortical communication.

Functional specialization of mouse higher visual cortical areas.

The mouse is emerging as an important model for understanding how sensory neocortex extracts cues to guide behavior, yet little is known about how these cues are processed beyond primary cortical areas. Here, we used two-photon calcium imaging in awake mice to compare visual responses in primary visual cortex (V1) and in two downstream target areas, AL and PM. Neighboring V1 neurons had diverse stimulus preferences spanning five octaves in spatial and temporal frequency. By contrast, AL and PM neurons responded best to distinct ranges of stimulus parameters. Most strikingly, AL neurons preferred fast-moving stimuli while PM neurons preferred slow-moving stimuli. By contrast, neurons in V1, AL, and PM demonstrated similar selectivity for stimulus orientation but not for stimulus direction. Based on these findings, we predict that area AL helps guide behaviors involving fast-moving stimuli (e.g., optic flow), while area PM helps guide behaviors involving slow-moving objects.

Interneurons hyperpolarize pyramidal cells along their entire somatodendritic axis.

Although GABAergic interneurons are the main source of synaptic inhibition in the cortex, activation of GABA(A) receptors has been shown to depolarize specific neuronal compartments, resulting in excitation. By using a noninvasive approach to monitor the effect of individual interneurons on the pyramidal cell population, we found that rat hippocampal interneurons hyperpolarized pyramidal cells irrespective of the location of their synapses along the somato-dendritic axis.