Summary Paragraph Sensory, motor, and cognitive operations involve the coordinated action of large neuronal populations across multiple brain regions in both superficial and deep structures1,2. Existing extracellular probes record neural activity with excellent spatial and temporal (sub-millisecond) resolution but from only a few dozen neurons per shank. Optical Ca2+ imaging3–5 offers more coverage but lacks the temporal resolution to reliably distinguish individual spikes and does not measure local field potentials. To date, no technology compatible with unrestrained animals has combined high spatiotemporal resolution with large volume coverage. To satisfy this need, we designed, fabricated, and tested a new silicon probe called Neuropixels. Each probe has 384 recording channels that can programmably address 960 CMOS processing-compatible low-impedance TiN6 sites that tile a single 10 mm long, 70x20 µm cross section shank. The 6x9 mm probe base is fabricated with the shank on a single chip. Voltage signals are filtered, amplified, multiplexed, and digitized on the base, allowing noise-free digital data transmission directly from the probe. The combination of dense recording sites and high channel count yielded well-isolated spiking activity from hundreds of neurons per probe implanted in mice and rats. Using two probes, more than 700 well-isolated single neurons were simultaneously recorded from five brain structures in an awake mouse. The fully integrated functionality and small size of Neuropixels probes allowed recording large populations of neurons from multiple brain structures in freely moving animals. This combination of high-performance electrode technology and scalable chip fabrication methods opens the path to record brain-wide neural activity during behavior.

Circuits in the cerebral cortex consist of thousands of neurons connected by millions of synapses. A precise understanding of these local networks requires relating circuit activity with the underlying network structure. For pyramidal cells in superficial mouse visual cortex (V1), a consensus is emerging that neurons with similar visual response properties excite each other1–5, but the anatomical basis of this recurrent synaptic network is unknown. We combined physiological imaging and large-scale electron microscopy (EM) to study an excitatory network in V1. We found that layer 2/3 neurons organized into subnetworks defined by anatomical connectivity, with more connections within than between groups. More specifically, we found that pyramidal neurons with similar orientation selectivity preferentially formed synapses with each other, despite the fact that axons and dendrites of all orientation selectivities pass near (< 5 μm) each other with roughly equal probability. Therefore, we predict that mechanisms of functionally specific connectivity take place at the length scale of spines. Neurons with similar orientation tuning formed larger synapses, potentially enhancing the net effect of synaptic specificity. With the ability to study thousands of connections in a single circuit, functional connectomics is proving a powerful method to uncover the organizational logic of cortical networks.

Summary For over a century, electrical microstimulation has been the most direct method for causally linking brain function with behavior. Despite this long history, it is still unclear how the activity of neural populations is affected by stimulation. For example, there is still no consensus on where activated cells lie, or on the extent to which neural processes such as passing axons near the electrode are also activated. Past studies of this question have proven difficult because microstimulation interferes with electrophysiological recordings, which in any case provide only coarse information about the location of activated cells. We used two-photon calcium imaging, an optical method, to circumvent these hurdles. We found that microstimulation sparsely activates neurons around the electrode, sometimes as far as millimeters away, even at low currents. The pattern of activated neurons likely arises from the direct activation of axons in a volume with a diameter of tens of microns.

Summary The local field potential (LFP) is increasingly used to measure the combined activity of neurons within a region of tissue. Yet, available estimates of the size of this region are highly disparate, ranging from a few hundred microns to several millimeters. To measure the size of this region directly, we used a combination of multielectrode recordings and optical imaging. We determined the orientation selectivity of stimulus-evoked LFP signals in cat area V1 and were able to predict it on the basis of the surrounding map of orientation preference. The results show that LFP signals originate within ~250 μm of the recording electrode. This quantitative estimate of spatial scale indicates that LFPs are more local than often recognized, and provides a guide to the interpretation of the increasing number of studies that rest on LFP recordings.

The early visual system is endowed with adaptive mechanisms that rapidly adjust gain and integration time based on the local luminance (mean intensity) and contrast (standard deviation of intensity relative to the mean). Here we show that these mechanisms are matched to the statistics of the environment. First, we measured the joint distribution of luminance and contrast in patches selected from natural images and found that luminance and contrast were statistically independent of each other. This independence did not hold for artificial images with matched spectral characteristics. Second, we characterized the effects of the adaptive mechanisms in lateral geniculate nucleus (LGN), the direct recipient of retinal outputs. We found that luminance gain control had the same effect at all contrasts and that contrast gain control had the same effect at all mean luminances. Thus, the adaptive mechanisms for luminance and contrast operate independently, reflecting the very independence encountered in natural images.

Cranial window implants in head-fixed rodents are becoming a preparation of choice for stable optical access to large areas of cortex over extended periods of time. Here, we provide a highly detailed and reliable surgical protocol for a cranial window implantation procedure for chronic widefield and cellular imaging in awake, head-fixed mice, which enables subsequent window removal and replacement in the weeks and months following the initial craniotomy. This protocol has facilitated awake, chronic imaging in adolescent as well as 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.

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.

The responses of neurons in lateral geniculate nucleus (LGN) exhibit powerful suppressive phenomena such as contrast saturation, size tuning, and masking. These phenomena cannot be explained by the classical center-surround receptive field and have been ascribed to a variety of mechanisms, including feedback from cortex. We asked whether these phenomena might all be explained by a single mechanism, contrast gain control, which is inherited from retina and possibly strengthened in thalamus. We formalized an intuitive model of retinal contrast gain control that explicitly predicts gain as a function of local contrast. In the model, the output of the receptive field is divided by the output of a suppressive field, which computes the local root-mean-square contrast. The model provides good fits to LGN responses to a variety of stimuli; with a single set of parameters, it captures saturation, size tuning, and masking. It also correctly predicts that responses to small stimuli grow proportionally with contrast: were it not for the suppressive field, LGN responses would be linear. We characterized the suppressive field and found that it is similar in size to the surround of the classical receptive field (which is eight times larger than commonly estimated), it is not selective for stimulus orientation, and it responds to a wide range of frequencies, including very low spatial frequencies and high temporal frequencies. The latter property is hardly consistent with feedback from cortex. These measurements thoroughly describe the visual properties of contrast gain control in LGN and provide a parsimonious explanation for disparate suppressive phenomena.