Relationship between simultaneously recorded spiking activity and fluorescence signal in GCaMP6 transgenic mice.

Fluorescent calcium indicators are often used to investigate neural dynamics, but the relationship between fluorescence and action potentials (APs) remains unclear. Most APs can be detected when the soma almost fills the microscope's field of view, but calcium indicators are used to image populations of neurons, necessitating a large field of view, generating fewer photons per neuron, and compromising AP detection. Here, we characterized the AP-fluorescence transfer function in vivo for 48 layer 2/3 pyramidal neurons in primary visual cortex, with simultaneous calcium imaging and cell-attached recordings from transgenic mice expressing GCaMP6s or GCaMP6f. While most APs were detected under optimal conditions, under conditions typical of population imaging studies, only a minority of 1 AP and 2 AP events were detected (often <10% and ~20-30%, respectively), emphasizing the limits of AP detection under more realistic imaging conditions.

Neurons, the cells that make up the nervous system, transmit information using electrical signals known as action potentials or spikes. Studying the spiking patterns of neurons in the brain is essential to understand perception, memory, thought, and behaviour. One way to do that is by recording electrical activity with microelectrodes. Another way to study neuronal activity is by using molecules that change how they interact with light when calcium binds to them, since changes in calcium concentration can be indicative of neuronal spiking. That change can be observed with specialized microscopes know as two-photon fluorescence microscopes. Using calcium indicators, it is possible to simultaneously record hundreds or even thousands of neurons. However, calcium fluorescence and spikes do not translate one-to-one. In order to interpret fluorescence data, it is important to understand the relationship between the fluorescence signals and the spikes associated with individual neurons. The only way to directly measure this relationship is by using calcium imaging and electrical recording simultaneously to record activity from the same neuron. However, this is extremely challenging experimentally, so this type of data is rare. To shed some light on this, Huang, Ledochowitsch et al. used mice that had been genetically modified to produce a calcium indicator in neurons of the visual cortex and simultaneously obtained both fluorescence measurements and electrical recordings from these neurons. These experiments revealed that, while the majority of time periods containing multi-spike neural activity could be identified using calcium imaging microscopy, on average, less than 10% of isolated single spikes were detectable. This is an important caveat that researchers need to take into consideration when interpreting calcium imaging results. These findings are intended to serve as a guide for interpreting calcium imaging studies that look at neurons in the mammalian brain at the population level. In addition, the data provided will be useful as a reference for the development of activity sensors, and to benchmark and improve computational approaches for detecting and predicting spikes.

A large-scale standardized physiological survey reveals functional organization of the mouse visual cortex.

To understand how the brain processes sensory information to guide behavior, we must know how stimulus representations are transformed throughout the visual cortex. Here we report an open, large-scale physiological survey of activity in the awake mouse visual cortex: the Allen Brain Observatory Visual Coding dataset. This publicly available dataset includes the cortical activity of nearly 60,000 neurons from six visual areas, four layers, and 12 transgenic mouse lines in a total of 243 adult mice, in response to a systematic set of visual stimuli. We classify neurons on the basis of joint reliabilities to multiple stimuli and validate this functional classification with models of visual responses. While most classes are characterized by responses to specific subsets of the stimuli, the largest class is not reliably responsive to any of the stimuli and becomes progressively larger in higher visual areas. These classes reveal a functional organization wherein putative dorsal areas show specialization for visual motion signals.

Neuronal cell-subtype specificity of neural synchronization in mouse primary visual cortex.

Spatiotemporally synchronised neuronal activity is central to sensation, motion and cognition. Brain circuits consist of dynamically interconnected neuronal cell-types, thus elucidating how neuron types synergise within the network is key to understand the neuronal orchestra. Here we show that in neocortex neuron-network coupling is neuronal cell-subtype specific. Employing in vivo two-photon (2-p) Calcium (Ca) imaging and 2-p targeted whole-cell recordings, we cell-type specifically investigated the coupling profiles of genetically defined neuron populations in superficial layers (L) of mouse primary visual cortex (V1). Our data reveal novel subtlety of neuron-network coupling in inhibitory interneurons (INs). Parvalbumin (PV)- and Vasoactive intestinal peptide (VIP)-expressing INs exhibit skewed distributions towards strong network-coupling; in Somatostatin (SST)-expressing INs, however, two physiological subpopulations are identified with distinct neuron-network coupling profiles, providing direct evidence for subtype specificity. Our results thus add novel functional granularity to neuronal cell-typing, and provided insights critical to simplifying/understanding neural dynamics.

Autonomous patch-clamp robot for functional characterization of neurons in vivo: development and application to mouse visual cortex.

Patch clamping is the gold standard measurement technique for cell-type characterization in vivo, but it has low throughput, is difficult to scale, and requires highly skilled operation. We developed an autonomous robot that can acquire multiple consecutive patch-clamp recordings in vivo. In practice, 40 pipettes loaded into a carousel are sequentially filled and inserted into the brain, localized to a cell, used for patch clamping, and disposed. Automated visual stimulation and electrophysiology software enables functional cell-type classification of whole cell-patched cells, as we show for 37 cells in the anesthetized mouse in visual cortex (V1) layer 5. We achieved 9% yield, with 5.3 min per attempt over hundreds of trials. The highly variable and low-yield nature of in vivo patch-clamp recordings will benefit from such a standardized, automated, quantitative approach, allowing development of optimal algorithms and enabling scaling required for large-scale studies and integration with complementary techniques. NEW & NOTEWORTHY In vivo patch-clamp is the gold standard for intracellular recordings, but it is a very manual and highly skilled technique. The robot in this work demonstrates the most automated in vivo patch-clamp experiment to date, by enabling production of multiple, serial intracellular recordings without human intervention. The robot automates pipette filling, wire threading, pipette positioning, neuron hunting, break-in, delivering sensory stimulus, and recording quality control, enabling in vivo cell-type characterization.

A Suite of Transgenic Driver and Reporter Mouse Lines with Enhanced Brain-Cell-Type Targeting and Functionality.

Modern genetic approaches are powerful in providing access to diverse cell types in the brain and facilitating the study of their function. Here, we report a large set of driver and reporter transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first-generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of high-precision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.

3D Image-Guided Automatic Pipette Positioning for Single Cell Experiments in vivo.

We report a method to facilitate single cell, image-guided experiments including in vivo electrophysiology and electroporation. Our method combines 3D image data acquisition, visualization and on-line image analysis with precise control of physical probes such as electrophysiology microelectrodes in brain tissue in vivo. Adaptive pipette positioning provides a platform for future advances in automated, single cell in vivo experiments.

Arousal and locomotion make distinct contributions to cortical activity patterns and visual encoding.

Spontaneous and sensory-evoked cortical activity is highly state-dependent, yet relatively little is known about transitions between distinct waking states. Patterns of activity in mouse V1 differ dramatically between quiescence and locomotion, but this difference could be explained by either motor feedback or a change in arousal levels. We recorded single cells and local field potentials from area V1 in mice head-fixed on a running wheel and monitored pupil diameter to assay arousal. Using naturally occurring and induced state transitions, we dissociated arousal and locomotion effects in V1. Arousal suppressed spontaneous firing and strongly altered the temporal patterning of population activity. Moreover, heightened arousal increased the signal-to-noise ratio of visual responses and reduced noise correlations. In contrast, increased firing in anticipation of and during movement was attributable to locomotion effects. Our findings suggest complementary roles of arousal and locomotion in promoting functional flexibility in cortical circuits.

Influence of a subtype of inhibitory interneuron on stimulus-specific responses in visual cortex.

Inhibition modulates receptive field properties and integrative responses of neurons in cortical circuits. The contribution of specific interneuron classes to cortical circuits and emergent responses is unknown. Here, we examined neuronal responses in primary visual cortex (V1) of adult Dlx1(-/-) mice, which have a selective reduction in cortical dendrite-targeting interneurons (DTIs) that express calretinin, neuropeptide Y, and somatostatin. The V1 neurons examined in Dlx1(-/-) mice have reduced orientation selectivity and altered firing rates, with elevated late responses, suggesting that local inhibition at dendrites has a specific role in modulating neuronal computations. We did not detect overt changes in the physiological properties of thalamic relay neurons and features of thalamocortical projections, such as retinotopic maps and eye-specific inputs, in the mutant mice, suggesting that the defects are cortical in origin. These experimental results are well explained by a computational model that integrates broad tuning from dendrite-targeting and narrower tuning from soma-targeting interneuron subclasses. Our findings suggest a key role for DTIs in the fine-tuning of stimulus-specific cortical responses.

Characterization of the functional MRI response temporal linearity via optical control of neocortical pyramidal neurons.

The blood oxygenation level-dependent (BOLD) signal serves as the basis for human functional MRI (fMRI). Knowledge of the properties of the BOLD signal, such as how linear its response is to sensory stimuli, is essential for the design and interpretation of fMRI experiments. Here, we combined the cell-type and site-specific causal control provided by optogenetics and fMRI (opto-fMRI) in mice to test the linearity of BOLD signals driven by locally induced excitatory activity. We employed high-resolution mouse fMRI at 9.4 tesla to measure the BOLD response, and extracellular electrophysiological recordings to measure the effects of stimulation on single unit, multiunit, and local field potential activity. Optically driven stimulation of layer V neocortical pyramidal neurons resulted in a positive local BOLD response at the stimulated site. Consistent with a linear transform model, this locally driven BOLD response summated in response to closely spaced trains of stimulation. These properties were equivalent to responses generated through the multisynaptic method of driving neocortical activity by tactile sensory stimulation, and paralleled changes in electrophysiological measures. These results illustrate the potential of the opto-fMRI method and reinforce the critical assumption of human functional neuroimaging that--to first approximation--the BOLD response tracks local neural activity levels.

What do we gain from gamma? Local dynamic gain modulation drives enhanced efficacy and efficiency of signal transmission.

Gamma oscillations in neocortex are hypothesized to improve information transmission between groups of neurons. We recently showed that optogenetic drive of fast-spiking interneurons (FS) at 40 Hz in mouse neocortex in vivo modulates the spike count and precision of sensory evoked responses. At specific phases of alignment between stimuli and FS activation, total evoked spike count was unchanged compared to baseline, but precision was increased. In the present study, we used computational modeling to investigate the origin of these local transformations, and to make predictions about their impact on downstream signal transmission. We replicated the prior experimental findings, and found that the local gain observed can be explained by mutual inhibition of fast-spiking interneurons, leading to more robust sensory-driven spiking in a brief temporal window post-stimulus, increasing local synchrony. Enhanced spiking in a second neocortical area, without a net increase in overall driven spikes in the first area, resulted from faster depolarization of target neurons due to increased pre-synaptic synchrony. In addition, we found that the precise temporal structure of spiking in the first area impacted the gain between cortical areas. The optimal spike distribution matched the "window of opportunity" defined by the timing of inhibition in the target area: spiking beyond this window did not contribute to downstream spike generation, leading to decreased overall gain. This result predicts that efficient transmission between neocortical areas requires a mechanism to dynamically match the temporal structure of the output of one area to the timing of inhibition in the recipient zone.

Neocortical interneurons: from diversity, strength.

Interneurons in the neocortex of the brain are small, locally projecting inhibitory GABAergic cells with a broad array of anatomical and physiological properties. The diversity of interneurons is believed to be crucial for regulating myriad operations in the neocortex. Here, we describe current theories about how interneuron diversity may support distinct neocortical processes that underlie perception.

Targeted optogenetic stimulation and recording of neurons in vivo using cell-type-specific expression of Channelrhodopsin-2.

A major long-term goal of systems neuroscience is to identify the different roles of neural subtypes in brain circuit function. The ability to causally manipulate selective cell types is critical to meeting this goal. This protocol describes techniques for optically stimulating specific populations of excitatory neurons and inhibitory interneurons in vivo in combination with electrophysiology. Cell type selectivity is obtained using Cre-dependent expression of the light-activated channel Channelrhodopsin-2. We also describe approaches for minimizing optical interference with simultaneous extracellular and intracellular recording. These optogenetic techniques provide a spatially and temporally precise means of studying neural activity in the intact brain and allow a detailed examination of the effect of evoked activity on the surrounding local neural network. Injection of viral vectors requires 30-45 min, and in vivo electrophysiology with optogenetic stimulation requires 1-4 h.

Pinacidil induces vascular dilation and hyperemia in vivo and does not impact biophysical properties of neurons and astrocytes in vitro.

Vascular and neural systems are highly interdependent, as evidenced by the wealth of intrinsic modulators shared by the two systems. We tested the hypothesis that pinacidil, a selective agonist for the SUR2B receptor found on smooth muscles, could serve as an independent means of inducing vasodilation and increased local blood volume to emulate functional hyperemia. Application of pinacidil induced vasodilation and increased blood volume in the in vivo neocortex in anesthetized rats and awake mice. Direct application of this agent to the in vitro neocortical slice had no direct impact on biophysical properties of neurons or astrocytes assessed with whole-cell recording. These findings suggest that pinacidil provides an effective and selective means for inducing hyperemia in vivo, and may provide a useful tool in directly testing the impact of hemodynamics on neural activity, as recently predicted by the hemo-neural hypothesis.

Driving fast-spiking cells induces gamma rhythm and controls sensory responses.

Cortical gamma oscillations (20-80 Hz) predict increases in focused attention, and failure in gamma regulation is a hallmark of neurological and psychiatric disease. Current theory predicts that gamma oscillations are generated by synchronous activity of fast-spiking inhibitory interneurons, with the resulting rhythmic inhibition producing neural ensemble synchrony by generating a narrow window for effective excitation. We causally tested these hypotheses in barrel cortex in vivo by targeting optogenetic manipulation selectively to fast-spiking interneurons. Here we show that light-driven activation of fast-spiking interneurons at varied frequencies (8-200 Hz) selectively amplifies gamma oscillations. In contrast, pyramidal neuron activation amplifies only lower frequency oscillations, a cell-type-specific double dissociation. We found that the timing of a sensory input relative to a gamma cycle determined the amplitude and precision of evoked responses. Our data directly support the fast-spiking-gamma hypothesis and provide the first causal evidence that distinct network activity states can be induced in vivo by cell-type-specific activation.

A quantitative theory of immediate visual recognition.

Human and non-human primates excel at visual recognition tasks. The primate visual system exhibits a strong degree of selectivity while at the same time being robust to changes in the input image. We have developed a quantitative theory to account for the computations performed by the feedforward path in the ventral stream of the primate visual cortex. Here we review recent predictions by a model instantiating the theory about physiological observations in higher visual areas. We also show that the model can perform recognition tasks on datasets of complex natural images at a level comparable to psychophysical measurements on human observers during rapid categorization tasks. In sum, the evidence suggests that the theory may provide a framework to explain the first 100-150 ms of visual object recognition. The model also constitutes a vivid example of how computational models can interact with experimental observations in order to advance our understanding of a complex phenomenon. We conclude by suggesting a number of open questions, predictions, and specific experiments for visual physiology and psychophysics.