The influence of cortical activity on perception depends on behavioral state and sensory context.

The mechanistic link between neural circuit activity and behavior remains unclear. While manipulating cortical activity can bias certain behaviors and elicit artificial percepts, some tasks can still be solved when cortex is silenced or removed. Here, mice were trained to perform a visual detection task during which we selectively targeted groups of visually responsive and co-tuned neurons in L2/3 of primary visual cortex (V1) for two-photon photostimulation. The influence of photostimulation was conditional on two key factors: the behavioral state of the animal and the contrast of the visual stimulus. The detection of low-contrast stimuli was enhanced by photostimulation, while the detection of high-contrast stimuli was suppressed, but crucially, only when mice were highly engaged in the task. When mice were less engaged, our manipulations of cortical activity had no effect on behavior. The behavioral changes were linked to specific changes in neuronal activity. The responses of non-photostimulated neurons in the local network were also conditional on two factors: their functional similarity to the photostimulated neurons and the contrast of the visual stimulus. Functionally similar neurons were increasingly suppressed by photostimulation with increasing visual stimulus contrast, correlating with the change in behavior. Our results show that the influence of cortical activity on perception is not fixed, but dynamically and contextually modulated by behavioral state, ongoing activity and the routing of information through specific circuits.

Cortico-cortical feedback engages active dendrites in visual cortex.

Sensory processing in the neocortex requires both feedforward and feedback information flow between cortical areas1. In feedback processing, higher-level representations provide contextual information to lower levels, and facilitate perceptual functions such as contour integration and figure-ground segmentation2,3. However, we have limited understanding of the circuit and cellular mechanisms that mediate feedback influence. Here we use long-range all-optical connectivity mapping in mice to show that feedback influence from the lateromedial higher visual area (LM) to the primary visual cortex (V1) is spatially organized. When the source and target of feedback represent the same area of visual space, feedback is relatively suppressive. By contrast, when the source is offset from the target in visual space, feedback is relatively facilitating. Two-photon calcium imaging data show that this facilitating feedback is nonlinearly integrated in the apical tuft dendrites of V1 pyramidal neurons: retinotopically offset (surround) visual stimuli drive local dendritic calcium signals indicative of regenerative events, and two-photon optogenetic activation of LM neurons projecting to identified feedback-recipient spines in V1 can drive similar branch-specific local calcium signals. Our results show how neocortical feedback connectivity and nonlinear dendritic integration can together form a substrate to support both predictive and cooperative contextual interactions.

All-optical interrogation of neural circuits in behaving mice.

Recent advances combining two-photon calcium imaging and two-photon optogenetics with computer-generated holography now allow us to read and write the activity of large populations of neurons in vivo at cellular resolution and with high temporal resolution. Such 'all-optical' techniques enable experimenters to probe the effects of functionally defined neurons on neural circuit function and behavioral output with new levels of precision. This greatly increases flexibility, resolution, targeting specificity and throughput compared with alternative approaches based on electrophysiology and/or one-photon optogenetics and can interrogate larger and more densely labeled populations of neurons than current voltage imaging-based implementations. This protocol describes the experimental workflow for all-optical interrogation experiments in awake, behaving head-fixed mice. We describe modular procedures for the setup and calibration of an all-optical system (~3 h), the preparation of an indicator and opsin-expressing and task-performing animal (~3-6 weeks), the characterization of functional and photostimulation responses (~2 h per field of view) and the design and implementation of an all-optical experiment (achievable within the timescale of a normal behavioral experiment; ~3-5 h per field of view). We discuss optimizations for efficiently selecting and targeting neuronal ensembles for photostimulation sequences, as well as generating photostimulation response maps from the imaging data that can be used to examine the impact of photostimulation on the local circuit. We demonstrate the utility of this strategy in three brain areas by using different experimental setups. This approach can in principle be adapted to any brain area to probe functional connectivity in neural circuits and investigate the relationship between neural circuit activity and behavior.

Two-Photon Optogenetic Stimulation of Drosophila Neurons.

Optogenetics enables experimental control over neural activity using light. Channelrhodopsin and its variants are typically activated using visible light excitation but can also be activated using infrared two-photon excitation. Two-photon excitation can improve the spatial precision of stimulation in scattering tissue but has several practical limitations that need to be considered before use. Here we describe the methodology and best practices for using two-photon optogenetic stimulation of neurons within the brain of the fruit fly, Drosophila melanogaster, with an emphasis on projection neurons of the antennal lobe.

Are Human Dendrites Different?

The first patch-clamp recordings from the dendrites of human neocortical neurons have recently been reported by Beaulieu-Laroche et al. and Gidon et al. These studies have shown that human dendrites are electrically excitable, exhibiting backpropagating action potentials and fast dendritic calcium spikes. This new frontier highlights the potential for interspecies differences in the biophysics of dendritic computation.

The Organization of Projections from Olfactory Glomeruli onto Higher-Order Neurons.

Each odorant receptor corresponds to a unique glomerulus in the brain. Projections from different glomeruli then converge in higher brain regions, but we do not understand the logic governing which glomeruli converge and which do not. Here, we use two-photon optogenetics to map glomerular connections onto neurons in the lateral horn, the region of the Drosophila brain that receives the majority of olfactory projections. We identify 39 morphological types of lateral horn neurons (LHNs) and show that different types receive input from different combinations of glomeruli. We find that different LHN types do not have independent inputs; rather, certain combinations of glomeruli converge onto many of the same LHNs and so are over-represented. Notably, many over-represented combinations are composed of glomeruli that prefer chemically dissimilar ligands whose co-occurrence indicates a behaviorally relevant "odor scene." The pattern of glomerulus-LHN connections thus represents a prediction of what ligand combinations will be most salient.

Stereotyped connectivity and computations in higher-order olfactory neurons.

In the first brain relay of the olfactory system, odors are encoded by combinations of glomeruli, but it is not known how glomerular signals are ultimately integrated. In Drosophila melanogaster, the majority of glomerular projections target the lateral horn. Here we show that lateral horn neurons (LHNs) receive input from sparse and stereotyped combinations of glomeruli that are coactivated by odors, and certain combinations of glomeruli are over-represented. One morphological LHN type is broadly tuned and sums input from multiple glomeruli. These neurons have a broader dynamic range than their individual glomerular inputs do. By contrast, a second morphological type is narrowly tuned and receives prominent odor-selective inhibition through both direct and indirect pathways. We show that this wiring scheme confers increased selectivity. The biased stereotyped connectivity of the lateral horn contrasts with the probabilistic wiring of the mushroom body, reflecting the distinct roles of these regions in innate as compared to learned behaviors.

Precise temperature compensation of phase in a rhythmic motor pattern.

Most animal species are cold-blooded, and their neuronal circuits must maintain function despite environmental temperature fluctuations. The central pattern generating circuits that produce rhythmic motor patterns depend on the orderly activation of circuit neurons. We describe the effects of temperature on the pyloric rhythm of the stomatogastric ganglion of the crab, Cancer borealis. The pyloric rhythm is a triphasic motor pattern in which the Pyloric Dilator (PD), Lateral Pyloric (LP), and Pyloric (PY) neurons fire in a repeating sequence. While the frequency of the pyloric rhythm increased about 4-fold (Q(10) approximately 2.3) as the temperature was shifted from 7 degrees C to 23 degrees C, the phase relationships of the PD, LP, and PY neurons showed almost perfect temperature compensation. The Q(10)'s of the input conductance, synaptic currents, transient outward current (I(A)), and the hyperpolarization-activated inward current (I(h)), all of which help determine the phase of LP neuron activity, ranged from 1.8 to 4. We studied the effects of temperature in >1,000 computational models (with different sets of maximal conductances) of a bursting neuron and the LP neuron. Many bursting models failed to monotonically increase in frequency as temperature increased. Temperature compensation of LP neuron phase was facilitated when model neurons' currents had Q(10)'s close to 2. Together, these data indicate that although diverse sets of maximal conductances may be found in identified neurons across animals, there may be strong evolutionary pressure to restrict the Q(10)'s of the processes that contribute to temperature compensation of neuronal circuits.