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Using optogenetics to silence the cortex, the authors show that thalamic inputs to layer 4 V1 neurons in anesthetized mice only contribute a third of the total excitation to these cells during presentation of visual stimuli. Moreover, they find that a small offset in the center of ON and OFF receptive subfields accounts for the orientation tuning of thalamic excitation to these cells. Cortical neurons in thalamic recipient layers receive excitation from the thalamus and the cortex. The relative contribution of these two sources of excitation to sensory tuning is poorly understood. We optogenetically silenced the visual cortex of mice to isolate thalamic excitation onto layer 4 neurons during visual stimulation. Thalamic excitation contributed to a third of the total excitation and was organized in spatially offset, yet overlapping, ON and OFF receptive fields. This receptive field structure predicted the orientation tuning of thalamic excitation. Finally, both thalamic and total excitation were similarly tuned to orientation and direction and had the same temporal phase relationship to the visual stimulus. Our results indicate that tuning of thalamic excitation is unlikely to be imparted by direction- or orientation-selective thalamic neurons and that a principal role of cortical circuits is to amplify tuned thalamic excitation.

Detecting the direction of motion of an object is essential for our representation of the visual environment. The visual cortex is one of the main stages in the mammalian nervous system in which the direction of motion may be computed de novo. Experiments and theories indicate that cortical neurons respond selectively to motion direction by combining inputs that provide information about distinct spatial locations with distinct time delays. Despite the importance of this spatiotemporal offset for direction selectivity, its origin and cellular mechanisms are not fully understood. We show that approximately 80 ± 10 thalamic neurons, which respond with distinct time courses to stimuli in distinct locations, excite mouse visual cortical neurons during visual stimulation. The integration of thalamic inputs with the appropriate spatiotemporal offset provides cortical neurons with a primordial bias for direction selectivity. These data show how cortical neurons selectively combine the spatiotemporal response diversity of thalamic neurons to extract fundamental features of the visual world. Direction selectivity emerges de novo in layer 4 neurons of primary visual cortex through the convergence of synaptic inputs from thalamic neurons that respond with distinct time courses to visual stimuli in distinct locations.

How intracortical recurrent circuits in mammalian sensory cortex influence dynamics of sensory representation is not understood. Previous methods could not distinguish the relative contributions of recurrent circuits and thalamic afferents to cortical dynamics. We accomplish this by optogenetically manipulating thalamus and cortex. Over the initial 40 ms of visual stimulation, excitation from recurrent circuits in visual cortex progressively increased to exceed direct thalamocortical excitation. Even when recurrent excitation exceeded thalamic excitation, upon silencing thalamus, sensory-evoked activity in cortex decayed rapidly, with a time constant of 10 ms, which is similar to a neuron's integration time window. In awake mice, this cortical decay function predicted the time-locking of cortical activity to thalamic input at frequencies <15 Hz and attenuation of the cortical response to higher frequencies. Under anesthesia, depression at thalamocortical synapses disrupted the fidelity of sensory transmission. Thus, we determine dynamics intrinsic to cortical recurrent circuits that transform afferent input in time. By optogenetically silencing thalamus, the authors show that visual cortex does not sustain a response without thalamus for more than a few tens of milliseconds. This rapid cortical activity decay predicts the temporal dynamics of sensory activity transmission between thalamus and cortex in awake animals, whereas under anesthesia, the fidelity of thalamo-cortical connection is dominated by the effect of synaptic depression.

Reinforcement has long been thought to require striatal synaptic plasticity. Indeed, direct striatal manipulations such as self-stimulation of direct-pathway projection neurons (dMSNs) are sufficient to induce reinforcement within minutes. However, it’s unclear what role, if any, is played by downstream circuitry. Here, we used dMSN self-stimulation in mice as a model for striatum-driven reinforcement and mapped the underlying circuitry across multiple basal ganglia nuclei and output targets. We found that mimicking the effects of dMSN activation on downstream circuitry, through optogenetic suppression of basal ganglia output nucleus substantia nigra reticulata (SNr) or activation of SNr targets in the brainstem or thalamus, was also sufficient to drive rapid reinforcement. Remarkably, silencing motor thalamus—but not other selected targets of SNr—was the only manipulation that reduced dMSN-driven reinforcement. Together, these results point to an unexpected role for basal ganglia output to motor thalamus in striatum-driven reinforcement.

Relating the functional properties of neurons in an intact organism with their cellular and synaptic characteristics is necessary for a mechanistic understanding of brain function. However, while the functional properties of cortical neurons (e.g., tuning to sensory stimuli) are necessarily determined in vivo, detailed cellular and synaptic analysis relies on in vitro techniques. Here we describe an approach that combines in vivo calcium imaging (for functional characterization) with photo-activation of fluorescent proteins (for neuron labeling), thereby allowing targeted in vitro recording of multiple neurons with known functional properties. We expressed photo-activatable GFP rendered non-diffusible through fusion with a histone protein (H2B-PAGFP) in the mouse visual cortex to rapidly photo-label constellations of neurons in vivo at cellular and sub-cellular resolution using two-photon excitation. This photo-labeling method was compatible with two-photon calcium imaging of neuronal responses to visual stimuli, allowing us to label constellations of neurons with specific functional properties. Photo-labeled neurons were easily identified in vitro in acute brain slices and could be targeted for whole-cell recording. We also demonstrate that in vitro and in vivo image stacks of the same photo-labeled neurons could be registered to one another, allowing the exact in vivo response properties of individual neurons recorded in vitro to be known. The ability to perform in vitro recordings from neurons with known functional properties opens up exciting new possibilities for dissecting the cellular, synaptic, and circuit mechanisms that underlie neuronal function in vivo.

In the published version of this article, a data point is missing from Fig. 4f, and the y-axis label reads “RFpre”; it should read “RFpref.” The original article has not been corrected. The original and corrected figures are shown in the accompanying Publisher Correction.

A hallmark of the mammalian visual system is spatial information processing. This relies on feedforward activity spanning multiple brain areas, and on interconnected neurons with spatial receptive fields (RFs) aligned across these areas. This organization allows neurons to iteratively analyze information from the same point of the visual field. It remains unclear if this framework extends beyond the visual system, especially into cognitive areas of frontal cortex that exert feedback control over early sensory areas. Here, we surveyed the mouse frontal cortex (anterior cingulate and secondary motor areas), and discovered neurons with low latency, highly localized visual RFs. Just like in visual cortex, responses were often highly selective for bright or dark stimuli. The responses lagged visual cortical areas by only ∼25 ms, and their RFs were comparable in size. Further, the representation of visual space in frontal cortex showed a strong bias for the central (binocular) visual field, but there was no evidence of a topographically organized retinotopic map. Importantly, these visual responses were abolished by optogenetic silencing of visual cortex, revealing a causal role for feedforward hierarchical connectivity that extends visual spatial processing directly into motor and cognitive regions of mouse frontal cortex.

The mammalian brain contains many regions in which feedforward excitatory afferents impinge on neurons that are interconnected by recurrent excitatory circuits. The functional in vivo properties of feedforward versus recurrent excitation are poorly understood due to the technical difficulty of distinguishing between these two sources of excitation in individual neurons. To address these issues I developed novel techniques for measuring the in vivo functional properties of the specific neuronal circuits that impinge onto individual neurons. Chapters 1 and 2 describe how feedforward and recurrent excitation onto individual neurons in the mouse primary visual cortex respond to visual stimuli. In the primary visual cortex, individual neurons are tuned to specific features of visual stimuli namely their orientation, direction of movement, and retinal location of bright and dark regions. I examined the extent to which these sensory tuning properties are present in the thalamic versus intracortical excitation onto single cortical neurons using in vivo intracellular recording techniques during optogenetic silencing of cortical excitatory neurons to isolate thalamic excitation. These results reveal that the main tuning properties observed in the primary visual cortex, namely receptive field structure, orientation selectivity, and direction selectivity, are already present in the thalamic excitation onto individual neurons. Estimation of the intracortical excitation from the total excitation recorded in the absence of cortical silencing revealed that thalamic and intracortical excitation share similar tuning properties demonstrating that tuned feedforward thalamic excitation is amplified by recurrent intracortical excitation. Chapter 3 describes a novel photolabeling method that allows neurons with known in vivo functional properties to be studied in in vitro brain slice preparations in order to reveal their cellular, synaptic, and circuit properties. This technique helps bridge the gap between systems and cellular neuroscience and opens up new possibilities for understanding how neuronal circuits underlie neuronal function in the intact brain. Together, these novel experimental approaches allow us to understand a fundamental aspect of neuronal circuit operation: the transfer and integration of information via synaptic connections. Such knowledge will aid in our understanding of the neuronal mechanisms underlying sensation, learning, and behavior.

Detecting the direction of an object's motion is essential for our representation of the visual environment. Visual cortex is one of the main stages in the mammalian nervous system where motion direction may be computed de novo. Experiments and theories indicate that cortical neurons respond selectively to motion direction by combining inputs that provide information about distinct spatial locations with distinct time-delays. Despite the importance of this spatiotemporal offset for direction selectivity its origin and cellular mechanisms are not fully understood. We show that ~80+/-10 thalamic neurons responding with distinct time-courses to stimuli in distinct locations contribute to the excitation of mouse visual cortical neurons during visual stimulation. Integration of thalamic inputs with the appropriate spatiotemporal offset provides cortical neurons with the primordial bias for direction selectivity. These data show how cortical neurons selectively combine the spatiotemporal response diversity of thalamic neurons to extract fundamental features of the visual world.

Primary visual cortex (V1) exhibits two types of gamma rhythm: broadband activity in the 30-90 Hz range, and a narrowband oscillation seen in mice at frequencies close to 60 Hz. We investigated the sources of the narrowband gamma oscillation, the factors modulating its strength, and its relationship to broadband gamma activity. Narrowband and broadband gamma power were uncorrelated. Increasing visual contrast had opposite effects on the two rhythms: it increased broadband activity, but suppressed the narrowband oscillation. The narrowband oscillation was strongest in layer 4, and was mediated primarily by excitatory currents entrained by the synchronous, rhythmic firing of neurons in the lateral geniculate nucleus (LGN). The power and peak frequency of the narrowband gamma oscillation increased with light intensity. Silencing the cortex optogenetically did not affect narrowband oscillation in either LGN firing or cortical excitatory currents, suggesting that this oscillation reflects unidirectional flow of signals from thalamus to cortex.