Kenichi Ohki

Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex

Neurons in the cerebral cortex are organized into anatomical columns, with ensembles of cells arranged from the surface to the white matter. Within a column, neurons often share functional properties, such as selectivity for stimulus orientation; columns with distinct properties, such as different preferred orientations, tile the cortical surface in orderly patterns. This functional architecture was discovered with the relatively sparse sampling of microelectrode recordings. Optical imaging of membrane voltage or metabolic activity elucidated the overall geometry of functional maps, but is averaged over many cells (resolution >100 µm). Consequently, the purity of functional domains and the precision of the borders between them could not be resolved. Here, we labelled thousands of neurons of the visual cortex with a calcium-sensitive indicator in vivo. We then imaged the activity of neuronal populations at single-cell resolution with two-photon microscopy up to a depth of 400 µm. In rat primary visual cortex, neurons had robust orientation selectivity but there was no discernible local structure; neighbouring neurons often responded to different orientations. In area 18 of cat visual cortex, functional maps were organized at a fine scale. Neurons with opposite preferences for stimulus direction were segregated with extraordinary spatial precision in three dimensions, with columnar borders one to two cells wide. These results indicate that cortical maps can be built with single-cell precision.

Highly ordered arrangement of single neurons in orientation pinwheels.

In the visual cortex of higher mammals, neurons are arranged across the cortical surface in an orderly map of preferred stimulus orientations. This map contains ‘orientation pinwheels’, structures that are arranged like the spokes of a wheel such that orientation changes continuously around a centre. Conventional optical imaging first demonstrated these pinwheels, but the technique lacked the spatial resolution to determine the response properties and arrangement of cells near pinwheel centres. Electrophysiological recordings later demonstrated sharply selective neurons near pinwheel centres, but it remained unclear whether they were arranged randomly or in an orderly fashion. Here we use two-photon calcium imaging in vivo to determine the microstructure of pinwheel centres in cat visual cortex with single-cell resolution. We find that pinwheel centres are highly ordered: neurons selective to different orientations are clearly segregated even in the very centre. Thus, pinwheel centres truly represent singularities in the cortical map. This highly ordered arrangement at the level of single cells suggests great precision in the development of cortical circuits underlying orientation selectivity.

Similarity of Visual Selectivity among Clonally Related Neurons in Visual Cortex

Neurons in rodent visual cortex are organized in a salt-and-pepper fashion for orientation selectivity, but it is still unknown how this functional architecture develops. A recent study reported that the progeny of single cortical progenitor cells are preferentially connected in the postnatal cortex. If these neurons acquire similar selectivity through their connections, a salt-and-pepper organization may be generated, because neurons derived from different progenitors are intermingled in rodents. Here we investigated whether clonally related cells have similar preferred orientation by using a transgenic mouse, which labels all the progeny of single cortical progenitor cells. We found that preferred orientations of clonally related cells are similar to each other, suggesting that cell lineage is involved in the development of response selectivity of neurons in the cortex. However, not all clonally related cells share response selectivity, suggesting that cell lineage is not the only determinant of response selectivity.

Functional labeling of neurons and their projections using the synthetic activity–dependent promoter E-SARE

Identifying the neuronal ensembles that respond to specific stimuli and mapping their projection patterns in living animals are fundamental challenges in neuroscience. To this end, we engineered a synthetic promoter, the enhanced synaptic activity–responsive element (E-SARE), that drives neuronal activity–dependent gene expression more potently than other existing immediate-early gene promoters. Expression of a drug-inducible Cre recombinase downstream of E-SARE enabled imaging of neuronal populations that respond to monocular visual stimulation and tracking of their long-distance thalamocortical projections in living mice. Targeted cell-attached recordings and calcium imaging of neurons in sensory cortices revealed that E-SARE reporter expression correlates with sensory-evoked neuronal activity at the single-cell level and is highly specific to the type of stimuli presented to the animals. This activity-dependent promoter can expand the repertoire of genetic approaches for high-resolution anatomical and functional analysis of neural circuits.

Geometrical and topological relationships between multiple functional maps in cat primary visual cortex

The mammalian striate cortex is organized such that the receptive field properties of neighboring neurons change gradually across the cortical surface, forming so-called cortical maps. The presence of such maps has been demonstrated in different species of mammals for several parameters characterizing the visual space: retinotopy, ocular dominance, orientation, direction of motion and spatial frequency. In this study we used the optical imaging of intrinsic signals to simultaneously record the multiple functional maps in the same animal in order to obtain a comprehensive set of rules that govern mutual dependencies among the functional maps. Our results indicate that while orientation, direction and ocular dominance are represented on the cortex in a mutually dependent manner, the representation of spatial frequency is independent of the other types of cortical representations. The presence and/or absence of mutual dependence among the multiple functional maps are suggested to provide an important clue for the understanding of the development of visual cortical information representation in neonatal animals.

Transient neuronal coactivations embedded in globally propagating waves underlie resting-state functional connectivity.

Significance Resting-state functional connectivity (FC) is a commonly used method in neuroimaging to noninvasively study network organization of brains in humans and other animals. FC is reproducible across different institutions and sensitive enough to detect network changes due to psychiatric disorders. FC is thus a core tool for projects such as the Human Connectome Project. However, because hemodynamic signals are an indirect measure of neuronal activity, actual spatiotemporal neuronal activity underlying FC is still unknown. This study used simultaneous wide-field optical imaging of neuronal calcium signals and hemodynamic signals in transgenic mice to understand the spatiotemporal neuronal dynamics underlying FC. Transient spatial patterns of neuronal coactivations embedded within waves of activity propagating across neocortex were found to be particularly important for FC. Resting-state functional connectivity (FC), which measures the correlation of spontaneous hemodynamic signals (HemoS) between brain areas, is widely used to study brain networks noninvasively. It is commonly assumed that spatial patterns of HemoS-based FC (Hemo-FC) reflect large-scale dynamics of underlying neuronal activity. To date, studies of spontaneous neuronal activity cataloged heterogeneous types of events ranging from waves of activity spanning the entire neocortex to flash-like activations of a set of anatomically connected cortical areas. However, it remains unclear how these various types of large-scale dynamics are interrelated. More importantly, whether each type of large-scale dynamics contributes to Hemo-FC has not been explored. Here, we addressed these questions by simultaneously monitoring neuronal calcium signals (CaS) and HemoS in the entire neocortex of mice at high spatiotemporal resolution. We found a significant relationship between two seemingly different types of large-scale spontaneous neuronal activity—namely, global waves propagating across the neocortex and transient coactivations among cortical areas sharing high FC. Different sets of cortical areas, sharing high FC within each set, were coactivated at different timings of the propagating global waves, suggesting that spatial information of cortical network characterized by FC was embedded in the phase of the global waves. Furthermore, we confirmed that such transient coactivations in CaS were indeed converted into spatially similar coactivations in HemoS and were necessary to sustain the spatial structure of Hemo-FC. These results explain how global waves of spontaneous neuronal activity propagating across large-scale cortical network contribute to Hemo-FC in the resting state.

The role of the parahippocampal gyrus in source memory for external and internal events.

We can discriminate between the memories of real and imagined events. In this study, the traces of the perceived external events and the imagined internal events were investigated in the established paradigm of reality monitoring using event-related fMRI. In the retrieval phase, we found that the left parahippocampal gyrus represented the traces of visually encoded memory. The right inferior parietal cortex was activated when subjects judged that the original event was imagined. We suggest that these traces are used to distinguish what is seen from what is thought during reality monitoring. Furthermore, we found that the incorrect judgments were associated with signal increases in the left frontal operculum, suggesting that this area is a candidate for the monitoring system of contextual information or failure in the retrieval phase.

Temporal characterization of memory retrieval processes: an fMRI study of the ‘tip of the tongue’ phenomenon

‘Tip of the tongue’ (TOT) is a natural phenomenon in which people cannot retrieve a target word immediately, even though they feel confident that they know the target. This provides us an opportunity to understand the human memory system, because cognitive components of memory retrieval such as retrieval effort and successful retrieval are temporally dissociated from each other during the TOT states. The purpose of the present study was to reveal the neural correlates of the cognitive components of the retrieval process by separating cognitive phases of the TOT phenomenon using event‐related functional magnetic resonance imaging with multiple regression analysis. We demonstrated that the left dorsolateral prefrontal cortex (DLPFC) and anterior cingulate cortex were activated at the time of successful retrieval, and the left DLPFC also showed activation when the subjects successfully retrieved the target names as compared to when they gave up. This result suggests that the left DLPFC is specific to the successful retrieval process. During the TOT state, a number of regions were activated, and this suggests that widely distributed brain regions are engaged when people make a hard effort to retrieve a proper name in the TOT state. Our new approach employing temporal resolution of the TOT phenomenon may contribute to the understanding of the mechanisms of the human memory system.

Laminar differences in the orientation selectivity of geniculate afferents in mouse primary visual cortex

It has been debated whether orientation selectivity in mouse primary visual cortex (V1) is derived from tuned lateral geniculate nucleus (LGN) inputs or computed from untuned LGN inputs. However, few studies have measured orientation tuning of LGN axons projecting to V1. We measured the response properties of mouse LGN axons terminating in V1 and found that LGN axons projecting to layer 4 were generally less tuned for orientation than axons projecting to more superficial layers of V1. We also found several differences in response properties between LGN axons and V1 neurons in layer 4. These results suggest that orientation selectivity of mouse V1 may not simply be inherited from LGN inputs, but could also depend on thalamocortical or V1 circuits.

Target dependence of orientation and direction selectivity of corticocortical projection neurons in the mouse V1

Higher order visual areas that receive input from the primary visual cortex (V1) are specialized for the processing of distinct features of visual information. However, it is still incompletely understood how this functional specialization is acquired. Here we used in vivo two photon calcium imaging in the mouse visual cortex to investigate whether this functional distinction exists at as early as the level of projections from V1 to two higher order visual areas, AL and LM. Specifically, we examined whether sharpness of orientation and direction selectivity and optimal spatial and temporal frequency of projection neurons from V1 to higher order visual areas match with that of target areas. We found that the V1 input to higher order visual areas were indeed functionally distinct: AL preferentially received inputs from V1 that were more orientation and direction selective and tuned for lower spatial frequency compared to projection of V1 to LM, consistent with functional differences between AL and LM. The present findings suggest that selective projections from V1 to higher order visual areas initiates parallel processing of sensory information in the visual cortical network.