Two-photon calcium imaging of neuronal responses is an increasingly accessible technology for probing population responses in cortex at single cell resolution, and with reasonable and improving temporal resolution. However, analysis of two-photon data is usually performed using ad-hoc solutions. To date, no publicly available software exists for straightforward analysis of stimulus-triggered two-photon imaging experiments. In addition, the increasing data rates of two-photon acquisition systems imply increasing cost of computing hardware required for in-memory analysis. Here we present a Matlab toolbox, FocusStack, for simple and efficient analysis of two-photon calcium imaging stacks on consumer-level hardware, with minimal memory footprint. We also present a Matlab toolbox, StimServer, for generation and sequencing of visual stimuli, designed to be triggered over a network link from a two-photon acquisition system. FocusStack is compatible out of the box with several existing two-photon acquisition systems, and is simple to adapt to arbitrary binary file formats. Analysis tools such as stack alignment for movement correction, automated cell detection and peri-stimulus time histograms are already provided, and further tools can be easily incorporated. Both packages are available as publicly-accessible source-code repositories.

How are visual scenes encoded in local neural networks of visual cortex? In rodents, visual cortex lacks a columnar organization so that processing of diverse features from a spot in visual space could be performed locally by populations of neighboring neurons. To examine how complex visual scenes are represented by local microcircuits in mouse visual cortex we measured visually evoked responses of layer 2/3 neuronal populations using 3D two-photon calcium imaging. Both natural and artificial movie scenes (10 seconds duration) evoked distributed and sparsely organized responses in local populations of 70-150 neurons within the sampled volumes. About 50% of neurons showed calcium transients during visual scene presentation, of which about half displayed reliable temporal activation patterns. The majority of the reliably responding neurons were activated primarily by one of the four visual scenes applied. Consequently, single-neurons performed poorly in decoding, which visual scene had been presented. In contrast, high levels of decoding performance (>80%) were reached when considering population responses, requiring about 80 randomly picked cells or 20 reliable responders. Furthermore, reliable responding neurons tended to have neighbors sharing the same stimulus preference. Because of this local redundancy, it was beneficial for efficient scene decoding to read out activity from spatially distributed rather than locally clustered neurons. Our results suggest a population code in layer 2/3 of visual cortex, where the visual environment is dynamically represented in the activation of distinct functional sub-networks.

Natural scenes are composed of a wide range of edge angles and spatial frequencies, with a strong overrepresentation of vertical and horizontal edges. Correspondingly, many mammalian species are much better at discriminating these cardinal orientations compared to obliques. A potential reason for this increased performance could be an increased number of neurons in the visual cortex that are tuned to cardinal orientations, which is likely to be an adaptation to the natural scene statistics. Such biased angular tuning has recently been shown in the mouse primary visual cortex. However, it is still unknown if mice also show a perceptual dominance of cardinal orientations. Here, we describe the design of a novel custom-built touchscreen chamber that allows testing natural scene perception and orientation discrimination performance by applying different task designs. Using this chamber, we applied an iterative convergence towards orientation discrimination thresholds for cardinal or oblique orientations in different cohorts of mice. Surprisingly, the expert discrimination performance was similar for both groups but showed large inter-individual differences in performance and training time. To study the discrimination of cardinal and oblique stimuli in the same mice, we, therefore, applied, a different training regime where mice learned to discriminate cardinal and oblique gratings in parallel. Parallel training revealed a higher task performance for cardinal orientations in an early phase of the training. The performance for both orientations became similar after prolonged training, suggesting that learning permits equally high perceptual tuning towards oblique stimuli. In summary, our custom-built touchscreen chamber offers a flexible tool to test natural visual perception in rodents and revealed a training-induced increase in the perception of oblique gratings. The touchscreen chamber is entirely open-source, easy to build, and freely available to the scientific community to conduct visual or multimodal behavioral studies. It is also based on the FAIR principles for data management and sharing and could therefore serve as a catalyst for testing the perception of complex and natural visual stimuli across behavioral labs.

Eye-opening is a critical point for laminar maturation of pyramidal neurons (PNs) in primary visual cortex. Knowing both the intrinsic properties and morphology of PNs from the visual cortex during development is crucial to contextualize the integration of visual inputs at different age stages. Few studies have reported changes in intrinsic excitability in these neurons but were restricted to only one layer or one stage of cortical development. Here, we used in vitro whole-cell patch-clamp to investigate the developmental impact on electrophysiological properties of layer 2/3 and layer 5 PNs in mouse visual cortex. Additionally, we evaluated the morphological changes before and after eye-opening and compared these in adult mice. Overall, we found a decrease in intrinsic excitability in both layers after eye-opening which remained stable between juvenile and adult mice. The basal dendritic length increased in layer 5 neurons, whereas spine density increased in layer 2/3 neurons after eye-opening. These data show increased number of synapses after onset of sensory input paralleled with a reduced excitability, presumably as homeostatic mechanism. Altogether, we provide a database of the properties of PNs in mouse visual cortex by considering the layer- and time-specific changes of these neurons during sensory development.

Chronically implanted neural probes are powerful tools to decode brain activity however, recording population and spiking activity over long periods remains a major challenge. Here, we designed and fabricated flexible intracortical Michigan-style arrays with a shank cross-section per electrode of 250 μm[Formula: see text] utilizing the polymer paryleneC with the goal to improve the immune acceptance. As flexible neural probes are unable to penetrate the brain due to the low buckling force threshold, a tissue-friendly insertion system was developed by reducing the effective shank length. The insertion strategy enabled the implantation of the four, bare, flexible shanks up to 2 mm into the mouse brain without increasing the implantation footprint and therefore, minimizing the acute trauma. In acute recordings from the mouse somatosensory cortex and the olfactory bulb, we demonstrated that the flexible probes were able to simultaneously detect local field potentials as well as single and multi-unit activity. Additionally, the flexible arrays outperformed stiff probes with respect to yield of single unit activity. Following the successful in vivo validation, we further improved the microfabrication towards a double-metal-layer process, and were able to double the number of electrodes per shank by keeping the shank width resulting in a cross-section per electrode of 118 μm[Formula: see text].

Retinal degeneration is one of the main causes of visual impairment and blindness. One group of retinal degenerative diseases, leading to the loss of photoreceptors, is collectively termed retinitis pigmentosa. In this group of diseases, the remaining retina is largely spared from initial cell death making retinal ganglion cells an interesting target for vision restoration methods. However, it is unknown how downstream brain areas, in particular the visual cortex, are affected by the progression of blindness. Visual deprivation studies have shown dramatic changes in the electrophysiological properties of visual cortex neurons, but changes on a cellular level in retinitis pigmentosa have not been investigated yet. Therefore, we used the rd10 mouse model to perform patch-clamp recordings of pyramidal neurons in layer 2/3 of the primary visual cortex to screen for potential changes in electrophysiological properties resulting from retinal degeneration. Compared to wild-type C57BL/6 mice, we only found an increase in intrinsic excitability around the time point of maximal retinal degeneration. In addition, we saw an increase in the current amplitude of spontaneous putative inhibitory events after a longer progression of retinal degeneration. However, we did not observe a long-lasting shift in excitability after prolonged retinal degeneration. Together, our results provide evidence of an intact visual cortex with promising potential for future therapeutic strategies to restore vision.

Local excitatory connections in mouse primary visual cortex (V1) are stronger and more prevalent between neurons that share similar functional response features. However, the details of how functional rules for local connectivity shape neuronal responses in V1 remain unknown. We hypothesised that complex responses to visual stimuli may arise as a consequence of rules for selective excitatory connectivity within the local network in the superficial layers of mouse V1. In mouse V1 many neurons respond to overlapping grating stimuli (plaid stimuli) with highly selective and facilitatory responses, which are not simply predicted by responses to single gratings presented alone. This complexity is surprising, since excitatory neurons in V1 are considered to be mainly tuned to single preferred orientations. Here we examined the consequences for visual processing of two alternative connectivity schemes: in the first case, local connections are aligned with visual properties inherited from feedforward input (a 'like-to-like' scheme specifically connecting neurons that share similar preferred orientations); in the second case, local connections group neurons into excitatory subnetworks that combine and amplify multiple feedforward visual properties (a 'feature binding' scheme). By comparing predictions from large scale computational models with in vivo recordings of visual representations in mouse V1, we found that responses to plaid stimuli were best explained by assuming feature binding connectivity. Unlike under the like-to-like scheme, selective amplification within feature-binding excitatory subnetworks replicated experimentally observed facilitatory responses to plaid stimuli; explained selective plaid responses not predicted by grating selectivity; and was consistent with broad anatomical selectivity observed in mouse V1. Our results show that visual feature binding can occur through local recurrent mechanisms without requiring feedforward convergence, and that such a mechanism is consistent with visual responses and cortical anatomy in mouse V1.

Neurons in sensory areas of neocortex exhibit responses tuned to specific features of the environment. In visual cortex, information about features such as edges or textures with particular orientations must be integrated to recognize a visual scene or object. Connectivity studies in rodent cortex have revealed that neurons make specific connections within sub-networks sharing common input tuning. In principle, this sub-network architecture enables local cortical circuits to integrate sensory information. However, whether feature integration indeed occurs locally in rodent primary sensory areas has not been examined directly. We studied local integration of sensory features in primary visual cortex (V1) of the mouse by presenting drifting grating and plaid stimuli, while recording the activity of neuronal populations with two-photon calcium imaging. Using a Bayesian model-based analysis framework, we classified single-cell responses as being selective for either individual grating components or for moving plaid patterns. Rather than relying on trial-averaged responses, our model-based framework takes into account single-trial responses and can easily be extended to consider any number of arbitrary predictive models. Our analysis method was able to successfully classify significantly more responses than traditional partial correlation (PC) analysis, and provides a rigorous statistical framework to rank any number of models and reject poorly performing models. We also found a large proportion of cells that respond strongly to only one stimulus class. In addition, a quarter of selectively responding neurons had more complex responses that could not be explained by any simple integration model. Our results show that a broad range of pattern integration processes already take place at the level of V1. This diversity of integration is consistent with processing of visual inputs by local sub-networks within V1 that are tuned to combinations of sensory features.

Pupillometry, the measure of pupil size and reactivity, has been widely used to assess cognitive processes. Changes in pupil size have been shown to correlate with various behavioral states, both externally and internally induced such as locomotion, arousal, cortical state, and decision-making processes. Besides, these pupillary responses have also been linked to the activity of neuromodulatory systems that modulate attention and perception such as the noradrenergic and cholinergic systems. Due to the extent of processes the pupil reflects, we aimed at further resolving pupillary responses in the context of behavioral state and task performance while recording pupillary transients of mice performing a vibrotactile two-alternative forced-choice task (2-AFC). We show that before the presentation of task-relevant information, pre-stimulus, pupil size differentiates between states of disengagement from task performance vs. engagement. Also, when subjects have to attend to task stimuli to attain a reward, post-stimulus, pupillary dilations exhibit a difference between correct and error responses with this difference reflecting an internal decision variable. We hypothesize that this internal decision variable relates to response confidence, the internal perception of the confidence the subject has in its choice. As opposed to this, we show that in a condition of passive performance, when the stimulus has no more task relevance due to reward being provided automatically, pupillary dilations reflect the occurrence of stimulation and reward provision but not decisional variables as under active performance. Our results provide evidence that in addition to reflecting attentiveness under task performance rather than arousal per se, pupil dilations also reflect the confidence of the subject in his ensuing response. This confidence coding is overlaid within a more pronounced pupil dilation that reflects post-decision components that are related to the response itself but not to the decision. We also provide evidence as to how different behavioral states, imposed by task demands, modulate what the pupil is reflecting, presumably showing what the underlying cognitive network is coding for.

The patch-clamp technique has revolutionized neurophysiology by allowing to study single neuronal excitability, synaptic connectivity, morphology, and the transcriptomic profile. However, the throughput in recordings is limited because of the manual replacement of patch-pipettes after each attempt which are often also unsuccessful. This has been overcome by automated cleaning the tips in detergent solutions, allowing to reuse the pipette for further recordings. Here, we developed a novel method of automated cleaning by sonicating the tips within the bath solution wherein the cells are placed, reducing the risk of contaminating the bath solution or internal solution of the recording pipette by any detergent and avoiding the necessity of a separate chamber for cleaning. We showed that the patch-pipettes can be used consecutively at least ten times and that the cleaning process does not negatively impact neither the brain slices nor other patched neurons. This method, combined with automated patch-clamp, highly improves the throughput for single and especially multiple recordings.

A general principle of sensory processing is that neurons adapt to sustained stimuli by reducing their response over time. Most of our knowledge on adaptation in single cells is based on experiments in anesthetized animals. How responses adapt in awake animals, when stimuli may be behaviorally relevant or not, remains unclear. Here we show that contrast adaptation in mouse primary visual cortex depends on the behavioral relevance of the stimulus. Cells that adapted to contrast under anesthesia maintained or even increased their activity in awake naïve mice. When engaged in a visually guided task, contrast adaptation re-occurred for stimuli that were irrelevant for solving the task. However, contrast adaptation was reversed when stimuli acquired behavioral relevance. Regulation of cortical adaptation by task demand may allow dynamic control of sensory-evoked signal flow in the neocortex.

Spike-timing-dependent synaptic plasticity (STDP) by definition requires the temporal association of pre- and postsynaptic action potentials (APs). Yet, in cortical pyramidal neurons pairing unitary EPSPs with single APs at low frequencies is ineffective at generating plasticity. Using recordings from synaptically coupled layer 5 pyramidal neurons, we show here that high-frequency (200 Hz) postsynaptic AP bursts, rather than single APs, are required for both long-term potentiation (LTP) induction and NMDA channel activation during EPSP-AP pairing at low frequencies. Furthermore, we find that AP bursts can lead to LTP induction and NMDA channel activation during EPSP-AP pairing at both positive and negative times. High-frequency AP bursts generated supralinear calcium signals in basal dendrites suggesting the generation of dendritic calcium spikes, as has been observed previously in apical dendrites during AP burst firing at frequencies greater than 100 Hz. Consistent with a role of these dendritic calcium spikes in LTP induction, pairing EPSPs with low frequency (50 Hz) AP bursts was ineffective in generating LTP. Furthermore, supralinear calcium signals in basal dendrites during AP bursts were blocked by low concentrations of the T- and R-type calcium channel antagonist nickel, which also blocked LTP and NMDA channel activation. These data suggest an important role of dendritic calcium spikes during AP bursts in determining both the efficacy and time window for STDP induction.

One of the hallmarks of neocortical circuits is the predominance of recurrent excitation between pyramidal neurons, which is balanced by recurrent inhibition from smooth GABAergic neurons. It has been previously described that in layer 2/3 of primary visual cortex (V1) of cat and monkey, pyramidal cells filled with horseradish peroxidase connect approximately in proportion to the spiny (excitatory, 95% and 81%, respectively) and smooth (GABAergic, 5% and 19%, respectively) dendrites found in the neuropil. By contrast, a recent ultrastructural study of V1 in a single mouse found that smooth neurons formed 51% of the targets of the superficial layer pyramidal cells. This suggests that either the neuropil of this particular mouse V1 had a dramatically different composition to that of V1 in cat and monkey, or that smooth neurons were specifically targeted by the pyramidal cells in that mouse. We tested these hypotheses by examining similar cells filled with biocytin in a sample of five mice. We found that the average composition of the neuropil in V1 of these mice was similar to that described for cat and monkey V1, but that the superficial layer pyramidal cells do form proportionately more synapses with smooth dendrites than the equivalent neurons in cat or monkey. These distributions may underlie the distinct differences in functional architecture of V1 between rodent and higher mammals.

Understanding how cortical circuits generate complex behavior requires investigating the cell types that comprise them. Functional differences across pyramidal neuron (PyN) types have been observed within cortical areas, but it is not known whether these local differences extend throughout the cortex, nor whether additional differences emerge when larger-scale dynamics are considered. We used genetic and retrograde labeling to target pyramidal tract, intratelencephalic and corticostriatal projection neurons and measured their cortex-wide activity. Each PyN type drove unique neural dynamics, both at the local and cortex-wide scales. Cortical activity and optogenetic inactivation during an auditory decision task revealed distinct functional roles. All PyNs in parietal cortex were recruited during perception of the auditory stimulus, but, surprisingly, pyramidal tract neurons had the largest causal role. In frontal cortex, all PyNs were required for accurate choices but showed distinct choice tuning. Our results reveal that rich, cell-type-specific cortical dynamics shape perceptual decisions.