Inhibitory Units: An Organizing Nidus for Feature-Selective SubNetworks in Area V1.

Neuronal circuits often display small-world network architecture characterized by neuronal cliques of dense local connectivity communicating with each other through a limited number of cells that participate in multiple cliques. The principles by which such cliques organize to encode information remain poorly understood. Similarly tuned pyramidal cells that preferentially target each other may form multicellular encoding units performing distinct computational tasks. The existence of such units can reflect upon both spontaneous and stimulus-driven population events.We applied two-photon calcium imaging to study spontaneous population bursts in layer 2/3 of area V1 in male C57BL/6 mice. To identify potential small-world cliques, we searched for pyramidal cells whose calcium events had a consistent temporal relationship with the events of local inhibitory interneurons. This was guided by the intuition that groups of neurons whose synchronous firing represents a temporally coherent computational unit should be inhibited together. Pyramidal members of these interneuron-centered clusters on average displayed stronger functional connectivity between each other than with nonmember pyramidal neurons. The structure of the clusters evolved during postnatal development: cluster size and overlap between clusters decreased with developmental maturation. Pyramidal neurons in a cluster showed higher than chance tuning function similarity between each other and with the linked interneuron. Thus, spontaneous population events in V1 are shaped by small-world subnetworks of pyramidal neurons that share functional properties and work as a coherent unit with a local interneuron. These interneuron-pyramidal cell partnerships may represent a fundamental neocortical unit of computation at the population level.SIGNIFICANCE STATEMENT Neuronal circuit in layer 2/3 of mouse area V1 possesses small-world network architecture, where cliques of densely interconnected neurons ("small worlds") communicate via restricted number of hub cells. We show that: (1) in mouse V1 individual small-world cliques preferably incorporate pyramidal neurons with similar visual feature tuning, and (2) ongoing population activity of such pyramidal neuron clique is temporally linked to the activity of the local interneuron sharing its feature tuning with the clique members. Functional grouping of similarly tuned interneurons and pyramidal cells into cliques may ensure that ensembles of functionally alike pyramidal cells recruited during perceptual tasks and spontaneous activity are also turned off together as a unit, with interneurons serving as organizers of linked pyramidal ensemble activity.

Complex Visual Motion Representation in Mouse Area V1.

Rodent visual cortex has a hierarchical architecture similar to that of higher mammals (Coogan and Burkhalter, 1993; Marshel et al., 2011; Wang et al., 2012). Although notable differences exist between the species in terms or receptive field sizes and orientation map organization (Dräger, 1975; Gattass et al., 1987; Van den Bergh et al., 2010), mouse V1 is thought to respond to local orientation and visual motion elements rather than to global patterns of motion, similar to V1 in higher mammals (Niell and Stryker, 2008; Bonin et al., 2011). However, recent results are inconclusive: some argue mouse V1 is analogous to monkey V1 (Juavinett and Callaway, 2015); others argue that it displays complex motion responses (Muir et al., 2015). We used type I plaids formed by the additive superposition of moving gratings (Adelson and Movshon, 1982; Movshon et al., 1985; Albright and Stoner, 1995) to investigate this question. We show that mouse V1 contains a considerably smaller fraction of component-motion-selective neurons (∼17% vs ∼84%), and a larger fraction of pattern-motion-selective neurons (∼10% vs <1.3%) compared with primate/cat V1. The direction of optokinetic nystagmus correlates with visual perception in higher mammals (Fox et al., 1975; Logothetis and Schall, 1990; Wei and Sun, 1998; Watanabe, 1999; Naber et al., 2011). Measurement of optokinetic responses to plaid stimuli revealed that mice demonstrate bistable perception, sometimes tracking individual stimulus components and others the global pattern of motion. Moreover, bistable optokinetic responses cannot be entirely attributed to subcortical circuitry as V1 lesions alter the fraction of responses occurring along pattern versus component motion. These observations suggest that area V1 input contributes to complex motion perception in the mouse. SIGNIFICANCE STATEMENT: Area V1 in the mouse is hierarchically similar but not necessarily identical to area V1 in cats and primates. Here we demonstrate that area V1 neurons process complex motion plaid stimuli differently in mice versus in cats or primates. Specifically, a smaller proportion of mouse V1 cells are sensitive to component motion, and a larger proportion to pattern motion than are found in area V1 of cats/primates. Furthermore, we demonstrate for the first time that mice exhibit bistable visual perception of plaid stimuli, and that this depends, at least in part, on area V1 input. Finally, we suggest that the relative proportion of component-motion-selective responses to pattern-motion-selective responses in mouse V1 may bias visual perception, as evidenced by changes in the direction of elicited optokinetic responses.

The Effect of Single Pyramidal Neuron Firing Within Layer 2/3 and Layer 4 in Mouse V1.

The influence of cortical cell spiking activity on nearby cells has been studied extensively in vitro. Less is known, however, about the impact of single cell firing on local cortical networks in vivo. In a pioneering study, Kwan and Dan (Kwan and Dan, 2012) reported that in mouse layer 2/3 (L2/3), under anesthesia, stimulating a single pyramidal cell recruits ~2.1% of neighboring units. Here we employ two-photon calcium imaging in layer 2/3 of mouse V1, in conjunction with single-cell patch clamp stimulation in layer 2/3 or layer 4, to probe, in both the awake and lightly anesthetized states, how (i) activating single L2/3 pyramidal neurons recruits neighboring units within L2/3 and from layer 4 (L4) to L2/3, and whether (ii) activating single pyramidal neurons changes population activity in local circuit. To do this, it was essential to develop an algorithm capable of quantifying how sensitive the calcium signal is at detecting effectively recruited units ("followers"). This algorithm allowed us to estimate the chance of detecting a follower as a function of the probability that an epoch of stimulation elicits one extra action potential (AP) in the follower cell. Using this approach, we found only a small fraction (<0.75%) of L2/3 cells to be significantly activated within a radius of ~200 µm from a stimulated neighboring L2/3 pyramidal cell. This fraction did not change significantly in the awake vs. the lightly anesthetized state, nor when stimulating L2/3 vs. underlying L4 pyramidal neurons. These numbers are in general agreement with, though lower than, the percentage of neighboring cells (2.1% pyramidal cells and interneurons combined) reported by Kwan and Dan to be activated upon stimulating single L2/3 pyramidal neurons under anesthesia (Kwan and Dan, 2012). Interestingly, despite the small number of individual units found to be reliably driven, we did observe a modest but significant elevation in aggregate population responses compared to sham stimulation. This underscores the distributed impact that single cell stimulation has on neighboring microcircuit responses, revealing only a small minority of relatively strongly connected partners. One sentence summary: Patch-clamp stimulation in conjunction with 2-photon imaging shows that activating single layer-2/3 or layer-4 pyramidal neurons produces few (<1% of local units) reliable single-cell followers in L2/3 of mouse area V1, either under light anesthesia or in quiet wakefulness: instead, single cell stimulation was found to elevate aggregate population activity in a weak but highly distributed fashion.

Visually Driven Neuropil Activity and Information Encoding in Mouse Primary Visual Cortex.

Cortical neuropil modulations recorded by calcium imaging reflect the activity of large aggregates of axo-dendritic processes and synaptic compartments from a large number of neurons. The organization of this activity impacts neuronal firing but is not well understood. Here we used in vivo 2-photon imaging with Oregon Green Bapta (OGB) and GCaMP6s to study neuropil visual responses to moving gratings in layer 2/3 of mouse area V1. We found neuropil responses to be strongly modulated and more reliable than neighboring somatic activity. Furthermore, stimulus independent modulations in neuropil activity, i.e., noise correlations, were highly coherent across the cortical surface, up to distances of at least 200 µm. Pairwise neuropil-to-neuropil-patch noise correlation strength was much higher than cell-to-cell noise correlation strength and depended strongly on brain state, decreasing in quiet wakefulness relative to light anesthesia. The profile of neuropil noise correlation strength decreased gently with distance, dropping by ~11% at a distance of 200 µm. This was comparatively slower than the profile of cell-to-cell noise correlations, which dropped by ~23% at 200 µm. Interestingly, in spite of the "salt & pepper" organization of orientation and direction encoding across mouse V1 neurons, populations of neuropil patches, even of moderately large size (radius ~100 µm), showed high accuracy for discriminating perpendicularly moving gratings. This was commensurate to the accuracy of corresponding cell populations. The dynamic, stimulus dependent, nature of neuropil activity further underscores the need to carefully separate neuropil from cell soma activity in contemporary imaging studies.

Magnetic stimulation and depression of mammalian networks in primary neuronal cell cultures.

For transcranial magnetic stimulation (TMS), the coupling of induced electric fields with neurons in gray matter is not well understood. There is little information on optimal stimulation parameters and on basic cellular mechanisms. For this reason, magnetic stimulation of spontaneously active neuronal networks, grown on microelectrode arrays in culture, was employed as a test environment. This allowed use of smaller coils and the continual monitoring of network action potential (AP) activity before, during, and for long periods after stimulation. Biphasic, rectangular, and 500 micros long pulses were used at mean pulse frequencies (MPFs) ranging from 3 to 100 Hz on both spinal cord (SC) and frontal cortex (FC) cultures. Contrary to stimulation of organized fiber bundles, APs were not elicited directly. Responses were predominantly inhibitory, dose dependent, with onset times between 10 s and several minutes. Spinal networks showed a greater sensitivity to activity suppression. Under pharmacological disinhibition, some excitation was seen at low pulse frequencies. FC cultures showed greater excitatory responses than SC networks. The observed primary inhibitory responses imply interference with synaptic exocytosis mechanisms. With 20,000 pulses at 10 Hz, 40% inhibition was maintained for over 30 min with full recovery, suggesting possible application to nonchemical, noninvasive pain management.