Experience shapes chandelier cell function and structure in the visual cortex.

Detailed characterization of interneuron types in primary visual cortex (V1) has greatly contributed to understanding visual perception, yet the role of chandelier cells (ChCs) in visual processing remains poorly characterized. Using viral tracing we found that V1 ChCs predominantly receive monosynaptic input from local layer 5 pyramidal cells and higher-order cortical regions. Two-photon calcium imaging and convolutional neural network modeling revealed that ChCs are visually responsive but weakly selective for stimulus content. In mice running in a virtual tunnel, ChCs respond strongly to events known to elicit arousal, including locomotion and visuomotor mismatch. Repeated exposure of the mice to the virtual tunnel was accompanied by reduced visual responses of ChCs and structural plasticity of ChC boutons and axon initial segment length. Finally, ChCs only weakly inhibited pyramidal cells. These findings suggest that ChCs provide an arousal-related signal to layer 2/3 pyramidal cells that may modulate their activity and/or gate plasticity of their axon initial segments during behaviorally relevant events.

Thalamic regulation of ocular dominance plasticity in adult visual cortex.

Experience-dependent plasticity in the adult visual system is generally thought of as a cortical process. However, several recent studies have shown that perceptual learning or monocular deprivation can also induce plasticity in the adult dorsolateral geniculate nucleus (dLGN) of the thalamus. How plasticity in the thalamus and cortex interact in the adult visual system is ill-understood. To assess the influence of thalamic plasticity on plasticity in primary visual cortex (V1), we made use of our previous finding that during the critical period ocular dominance (OD) plasticity occurs in dLGN and requires thalamic synaptic inhibition. Using multielectrode recordings we find that this is also true in adult mice, and that in the absence of thalamic inhibition and plasticity, OD plasticity in adult V1 is absent. To study the influence of V1 on thalamic plasticity, we silenced V1 and show that during the critical period, but not in adulthood, the OD shift in dLGN is partially caused by feedback from V1. We conclude that during adulthood the thalamus plays an unexpectedly dominant role in experience-dependent plasticity in V1. Our findings highlight the importance of considering the thalamus as a potential source of plasticity in learning events that are typically thought of as cortical processes.

Impaired Direction Selectivity in the Nucleus of the Optic Tract of Albino Mice.

Purpose: Human albinos have a low visual acuity. This is partially due to the presence of spontaneous erroneous eye movements called pendular nystagmus. This nystagmus is present in other albino vertebrates and has been hypothesized to be caused by aberrant wiring of retinal ganglion axons to the nucleus of the optic tract (NOT), a part of the accessory optic system involved in the optokinetic response to visual motion. The NOT in pigmented rodents is preferentially responsive to ipsiversive motion (i.e., motion in the contralateral visual field in the temporonasal direction). We compared the response to visual motion in the NOT of albino and pigmented mice to understand if motion coding and preference are impaired in the NOT of albino mice. Methods: We recorded neuronal spiking activity with Neuropixels probes in the visual cortex and NOT in C57BL/6JRj mice (pigmented) and DBA/1JRj mice with oculocutaneous albinism (albino). Results: We found that in pigmented mice, NOT is retinotopically organized, and neurons are direction tuned, whereas in albino mice, neuronal tuning is severely impaired. Neurons in the NOT of albino mice do not have a preference for ipsiversive movement. In contrast, neuronal tuning in visual cortex was preserved in albino mice and did not differ significantly from the tuning in pigmented mice. Conclusions: We propose that excessive interhemispheric crossing of retinal projections in albinos may cause the disrupted left/right direction encoding we found in NOT. This, in turn, impairs the normal horizontal optokinetic reflex and leads to pendular albino nystagmus.

SpecSeg is a versatile toolbox that segments neurons and neurites in chronic calcium imaging datasets based on low-frequency cross-spectral power.

Imaging calcium signals in neurons of animals using single- or multi-photon microscopy facilitates the study of coding in large neural populations. Such experiments produce massive datasets requiring powerful methods to extract responses from hundreds of neurons. We present SpecSeg, an open-source toolbox for (1) segmentation of regions of interest (ROIs) representing neuronal structures, (2) inspection and manual editing of ROIs, (3) neuropil correction and signal extraction, and (4) matching of ROIs in sequential recordings. ROI segmentation in SpecSeg is based on temporal cross-correlations of low-frequency components derived by Fourier analysis of each pixel with its neighbors. The approach is user-friendly, intuitive, and insightful and enables ROI detection around neurons or neurites. It works for single- (miniscope) and multi-photon microscopy data, eliminating the need for separate toolboxes. SpecSeg thus provides an efficient and versatile approach for analyzing calcium responses in neuronal structures imaged over prolonged periods of time.

A parameter-free statistical test for neuronal responsiveness.

Neurophysiological studies depend on a reliable quantification of whether and when a neuron responds to stimulation. Simple methods to determine responsiveness require arbitrary parameter choices, such as binning size, while more advanced model-based methods require fitting and hyperparameter tuning. These parameter choices can change the results, which invites bad statistical practice and reduces the replicability. New recording techniques that yield increasingly large numbers of cells would benefit from a test for cell-inclusion that requires no manual curation. Here, we present the parameter-free ZETA-test, which outperforms t-tests, ANOVAs, and renewal-process-based methods by including more cells at a similar false-positive rate. We show that our procedure works across brain regions and recording techniques, including calcium imaging and Neuropixels data. Furthermore, in illustration of the method, we show in mouse visual cortex that (1) visuomotor-mismatch and spatial location are encoded by different neuronal subpopulations and (2) optogenetic stimulation of VIP cells leads to early inhibition and subsequent disinhibition.

The essential role of recurrent processing for figure-ground perception in mice.

The segregation of figures from the background is an important step in visual perception. In primary visual cortex, figures evoke stronger activity than backgrounds during a delayed phase of the neuronal responses, but it is unknown how this figure-ground modulation (FGM) arises and whether it is necessary for perception. Here, we show, using optogenetic silencing in mice, that the delayed V1 response phase is necessary for figure-ground segregation. Neurons in higher visual areas also exhibit FGM and optogenetic silencing of higher areas reduced FGM in V1. In V1, figures elicited higher activity of vasoactive intestinal peptide-expressing (VIP) interneurons than the background, whereas figures suppressed somatostatin-positive interneurons, resulting in an increased activation of pyramidal cells. Optogenetic silencing of VIP neurons reduced FGM in V1, indicating that disinhibitory circuits contribute to FGM. Our results provide insight into how lower and higher areas of the visual cortex interact to shape visual perception.

Somatostatin interneurons restrict cell recruitment to retinally driven spontaneous activity in the developing cortex.

During early development, before the eyes open, synaptic refinement of sensory networks depends on activity generated by developing neurons themselves. In the mouse visual system, retinal cells spontaneously depolarize and recruit downstream neurons to bursts of activity, where the number of recruited cells determines the resolution of synaptic retinotopic refinement. Here we show that during the second post-natal week in mouse visual cortex, somatostatin (SST)-expressing interneurons control the recruitment of cells to retinally driven spontaneous activity. Suppressing SST interneurons increases cell participation and allows events to spread farther along the cortex. During the same developmental period, a second type of high-participation, retina-independent event occurs. During these events, cells receive such large excitatory charge that inhibition is overwhelmed and large parts of the cortex participate in each burst. These results reveal a role of SST interneurons in restricting retinally driven activity in the visual cortex, which may contribute to the refinement of retinotopy.

Disruption of Critical Period Plasticity in a Mouse Model of Neurofibromatosis Type 1.

Neurofibromatosis type 1 (NF1) is a common monogenic neurodevelopmental disorder associated with physical and cognitive problems. The cognitive issues are thought to arise from increased release of the neurotransmitter GABA. Modulating the signaling pathways causing increased GABA release in a mouse model of NF1 reverts deficits in hippocampal learning. However, clinical trials based on these approaches have so far been unsuccessful. We therefore used a combination of slice electrophysiology, in vivo two-photon calcium imaging, and optical imaging of intrinsic signal in a mouse model of NF1 to investigate whether cortical development is affected in NF1, possibly causing lifelong consequences that cannot be rescued by reducing inhibition later in life. We find that, in NF1 mice of both sexes, inhibition increases strongly during the development of the visual cortex and remains high. While this increase in cortical inhibition does not affect spontaneous cortical activity patterns during early cortical development, the critical period for ocular dominance plasticity is shortened in NF1 mice due to its early closure but unaltered onset. Notably, after environmental enrichment, differences in inhibitory innervation and ocular dominance plasticity between NF1 mice and WT littermates disappear. These results provide the first evidence for critical period dysregulation in NF1 and suggest that treatments aimed at normalizing levels of inhibition will need to start at early stages of development.SIGNIFICANCE STATEMENT Neurofibromatosis type 1 is associated with cognitive problems for which no treatment is currently available. This study shows that, in a mouse model of neurofibromatosis type 1, cortical inhibition is increased during development and critical period regulation is disturbed. Rearing the mice in an environment that stimulates cognitive function overcomes these deficits. These results uncover critical period dysregulation as a novel mechanism in the pathogenesis of neurofibromatosis type 1. This suggests that targeting the affected signaling pathways in neurofibromatosis type 1 for the treatment of cognitive disabilities may have to start at a much younger age than has so far been tested in clinical trials.

Activity in Lateral Visual Areas Contributes to Surround Suppression in Awake Mouse V1.

Neuronal response to sensory stimuli depends on the context. The response in primary visual cortex (V1), for instance, is reduced when a stimulus is surrounded by a similar stimulus [1-3]. The source of this surround suppression is partially known. In mouse, local horizontal integration by somatostatin-expressing interneurons contributes to surround suppression [4]. In primates, however, surround suppression arises too quickly to come from local horizontal integration alone, and myelinated axons from higher visual areas, where cells have larger receptive fields, are thought to provide additional surround suppression [5, 6]. Silencing higher visual areas indeed decreased surround suppression in the awake primate by increasing responses to large stimuli [7, 8], although not under anesthesia [9, 10]. In smaller mammals, like mice, fast surround suppression could be possible without feedback. Recent studies revealed a small reduction in V1 responses when silencing higher areas [11, 12] but have not investigated surround suppression. To determine whether higher visual areas contribute to V1 surround suppression, even when this is not necessary for fast processing, we inhibited the areas lateral to V1, particularly the lateromedial area (LM), a possible homolog of primate V2 [13], while recording in V1 of awake and anesthetized mice. We found that part of the surround suppression depends on activity from lateral visual areas in the awake, but not anesthetized, mouse. Inhibiting the lateral visual areas specifically increased responses in V1 to large stimuli. We present a model explaining how excitatory feedback to V1 can have these suppressive effects for large stimuli.

NMNAT Proteins that Limit Wallerian Degeneration Also Regulate Critical Period Plasticity in the Visual Cortex.

Many brain regions go through critical periods of development during which plasticity is enhanced. These critical periods are associated with extensive growth and retraction of thalamocortical and intracortical axons. Here, we investigated whether a signaling pathway that is central in Wallerian axon degeneration also regulates critical period plasticity in the primary visual cortex (V1). Wallerian degeneration is characterized by rapid disintegration of axons once they are separated from the cell body. This degenerative process is initiated by reduced presence of cytoplasmic nicotinamide mononucleotide adenylyltransferases (NMNATs) and is strongly delayed in mice overexpressing cytoplasmic NMNAT proteins, such as WldS mutant mice producing a UBE4b-NMNAT1 fusion protein or NMNAT3 transgenic mice. Here, we provide evidence that in WldS mice and NMNAT3 transgenic mice, ocular dominance (OD) plasticity in the developing visual cortex is reduced. This deficit is only observed during the second half of the critical period. Additionally, we detect an early increase of visual acuity in the V1 of WldS mice. We do not find evidence for Wallerian degeneration occurring during OD plasticity. Our findings suggest that NMNATs do not only regulate Wallerian degeneration during pathological conditions but also control cellular events that mediate critical period plasticity during the physiological development of the cortex.

Visual Processing by Calretinin Expressing Inhibitory Neurons in Mouse Primary Visual Cortex.

Inhibition in the cerebral cortex is delivered by a variety of GABAergic interneurons. These cells have been categorized by their morphology, physiology, gene expression and connectivity. Many of these classes appear to be conserved across species, suggesting that the classes play specific functional roles in cortical processing. What these functions are, is still largely unknown. The largest group of interneurons in the upper layers of mouse primary visual cortex (V1) is formed by cells expressing the calcium-binding protein calretinin (CR). This heterogeneous class contains subsets of vasoactive intestinal polypeptide (VIP) interneurons and somatostatin (SOM) interneurons. Here we show, using in vivo two-photon calcium imaging in mice, that CR neurons can be sensitive to stimulus orientation, but that they are less selective on average than the overall neuronal population. Responses of CR neurons are suppressed by a surrounding stimulus, but less so than the overall population. In rats and primates, CR interneurons have been suggested to provide disinhibition, but we found that in mice their in vivo activation by optogenetics causes a net inhibition of cortical activity. Our results show that the average functional properties of CR interneurons are distinct from the averages of the parvalbumin, SOM and VIP interneuron populations.

Amblyopia: The Thalamus Is a No-Go Area for Visual Acuity.

When one eye does not function well during development, the visual cortex becomes less responsive to it and visual acuity declines. New research suggests that reduced response strength and deteriorating acuity occur in separate circuits.

ß-Catenin in the Adult Visual Cortex Regulates NMDA-Receptor Function and Visual Responses.

The formation, plasticity and maintenance of synaptic connections is regulated by molecular and electrical signals. ß-Catenin is an important protein in these events and regulates cadherin-mediated cell adhesion and the recruitment of pre- and postsynaptic proteins in an activity-dependent fashion. Mutations in the ß-catenin gene can cause cognitive disability and autism, with life-long consequences. Understanding its synaptic function may thus be relevant for the treatment of these disorders. So far, ß-catenin's function has been studied predominantly in cell culture and during development but knowledge on its function in adulthood is limited. Here, we show that ablating ß-catenin in excitatory neurons of the adult visual cortex does not cause the same synaptic deficits previously observed during development. Instead, it reduces NMDA-receptor currents and impairs visual processing. We conclude that ß-catenin remains important for adult cortical function but through different mechanisms than during development.

Thalamic inhibition regulates critical-period plasticity in visual cortex and thalamus.

During critical periods of development, experience shapes cortical circuits, resulting in the acquisition of functions used throughout life. The classic example of critical-period plasticity is ocular dominance (OD) plasticity, which optimizes binocular vision but can reduce the responsiveness of the primary visual cortex (V1) to an eye providing low-grade visual input. The onset of the critical period of OD plasticity involves the maturation of inhibitory synapses within V1, specifically those containing the GABAA receptor α1 subunit. Here we show that thalamic relay neurons in mouse dorsolateral geniculate nucleus (dLGN) also undergo OD plasticity. This process depends on thalamic α1-containing synapses and is required for consolidation of the OD shift in V1 during long-term deprivation. Our findings demonstrate that thalamic inhibitory circuits play a central role in the regulation of the critical period. This has far-reaching consequences for the interpretation of studies investigating the molecular and cellular mechanisms regulating critical periods of brain development.

Mitochondrial Dynamics in Visual Cortex Are Limited In Vivo and Not Affected by Axonal Structural Plasticity.

Mitochondria buffer intracellular Ca2+ and provide energy [1]. Because synaptic structures with high Ca2+ buffering [2-4] or energy demand [5] are often localized far away from the soma, mitochondria are actively transported to these sites [6-11]. Also, the removal and degradation of mitochondria are tightly regulated [9, 12, 13], because dysfunctional mitochondria are a source of reactive oxygen species, which can damage the cell [14]. Deficits in mitochondrial trafficking have been proposed to contribute to the pathogenesis of Parkinson's disease, schizophrenia, amyotrophic lateral sclerosis, optic atrophy, and Alzheimer's disease [13, 15-19]. In neuronal cultures, about a third of mitochondria are motile, whereas the majority remains stationary for several days [8, 20]. Activity-dependent mechanisms cause mitochondria to stop at synaptic sites [7, 8, 20, 21], which affects synapse function and maintenance. Reducing mitochondrial content in dendrites decreases spine density [22, 23], whereas increasing mitochondrial content or activity increases it [7]. These bidirectional interactions between synaptic activity and mitochondrial trafficking suggest that mitochondria may regulate synaptic plasticity. Here we investigated the dynamics of mitochondria in relation to axonal boutons of neocortical pyramidal neurons for the first time in vivo. We find that under these circumstances practically all mitochondria are stationary, both during development and in adulthood. In adult visual cortex, mitochondria are preferentially localized at putative boutons, where they remain for several days. Retinal-lesion-induced cortical plasticity increases turnover of putative boutons but leaves mitochondrial turnover unaffected. We conclude that in visual cortex in vivo, mitochondria are less dynamic than in vitro, and that structural plasticity does not affect mitochondrial dynamics.

Inhibitory interneurons in visual cortical plasticity.

For proper maturation of the neocortex and acquisition of specific functions and skills, exposure to sensory stimuli is vital during critical periods of development when synaptic connectivity is highly malleable. To preserve reliable cortical processing, it is essential that these critical periods end after which learning becomes more conditional and active interaction with the environment becomes more important. How these age-dependent forms of plasticity are regulated has been studied extensively in the primary visual cortex. This has revealed that inhibitory innervation plays a crucial role and that a temporary decrease in inhibition is essential for plasticity to take place. Here, we discuss how different interneuron subsets regulate plasticity during different stages of cortical maturation. We propose a theory in which different interneuron subsets select the sources of neuronal input that undergo plasticity.

Ocular dominance plasticity disrupts binocular inhibition-excitation matching in visual cortex.

BACKGROUND: To ensure that neuronal networks function in a stable fashion, neurons receive balanced inhibitory and excitatory inputs. In various brain regions, this balance has been found to change temporarily during plasticity. Whether changes in inhibition have an instructive or permissive role in plasticity remains unclear. Several studies have addressed this question using ocular dominance plasticity in the visual cortex as a model, but so far, it remains controversial whether changes in inhibition drive this form of plasticity by directly affecting eye-specific responses or through increasing the plasticity potential of excitatory connections. RESULTS: We tested how three major classes of interneurons affect eye-specific responses in normally reared or monocularly deprived mice by optogenetically suppressing their activity. We find that in contrast to somatostatin-expressing or vasoactive intestinal polypeptide-expressing interneurons, parvalbumin (PV)-expressing interneurons strongly inhibit visual responses. In individual neurons of normal mice, inhibition and excitation driven by either eye are balanced, and suppressing PV interneurons does not alter ocular preference. Monocular deprivation disrupts the binocular balance of inhibition and excitation in individual neurons, causing suppression of PV interneurons to change their ocular preference. Importantly, however, these changes do not consistently favor responses to one of the eyes at the population level. CONCLUSIONS: Monocular deprivation disrupts the binocular balance of inhibition and excitation of individual cells. This disbalance does not affect the overall expression of ocular dominance. Our data therefore support a permissive rather than an instructive role of inhibition in ocular dominance plasticity.

Competition and Homeostasis of Excitatory and Inhibitory Connectivity in the Adult Mouse Visual Cortex.

During cortical development, synaptic competition regulates the formation and adjustment of neuronal connectivity. It is unknown whether synaptic competition remains active in the adult brain and how inhibitory neurons participate in this process. Using morphological and electrophysiological measurements, we show that expressing a dominant-negative form of the TrkB receptor (TrkB.T1) in the majority of pyramidal neurons in the adult visual cortex does not affect excitatory synapse densities. This is in stark contrast to the previously reported loss of excitatory input which occurs if the exact same transgene is expressed in sparse neurons at the same age. This indicates that synaptic competition remains active in adulthood. Additionally, we show that interneurons not expressing the TrkB.T1 transgene may have a competitive advantage and obtain more excitatory synapses when most neighboring pyramidal neurons do express the transgene. Finally, we demonstrate that inhibitory synapses onto pyramidal neurons are reduced when TrkB signaling is interfered with in most pyramidal neurons but not when few pyramidal neurons have this deficit. This adjustment of inhibitory innervation is therefore not a cell-autonomous consequence of decreased TrkB signaling but more likely a homeostatic mechanism compensating for activity changes at the population level.

Lack of functional specialization of neurons in the mouse primary visual cortex that have expressed calretinin.

Calretinin is a calcium-binding protein often used as a marker for a subset of inhibitory interneurons in the mammalian neocortex. We studied the labeled cells in offspring from a cross of a Cre-dependent reporter line with the CR-ires-Cre mice, which express Cre-recombinase in the same pattern as calretinin. We found that in the mature visual cortex, only a minority of the cells that have expressed calretinin and Cre-recombinase during their lifetime is GABAergic and only about 20% are immunoreactive for calretinin. The reason behind this is that calretinin is transiently expressed in many cortical pyramidal neurons during development. To determine whether neurons that express or have expressed calretinin share any distinct functional characteristics, we recorded their visual response properties using GCaMP6s calcium imaging. The average orientation selectivity, size tuning, and temporal and spatial frequency tuning of this group of cells, however, match the response profile of the general neuronal population, revealing the lack of functional specialization for the features studied.

Orientation-tuned surround suppression in mouse visual cortex.

The firing rates of neurons in primary visual cortex (V1) are suppressed by large stimuli, an effect known as surround suppression. In cats and monkeys, the strength of suppression is sensitive to orientation; responses to regions containing uniform orientations are more suppressed than those containing orientation contrast. This effect is thought to be important for scene segmentation, but the underlying neural mechanisms are poorly understood. We asked whether it is possible to study these mechanisms in the visual cortex of mice, because of recent advances in technology for studying the cortical circuitry in mice. It is unknown whether neurons in mouse V1 are sensitive to orientation contrast. We measured the orientation selectivity of surround suppression in the different layers of mouse V1. We found strong surround suppression in layer 4 and the superficial layers, part of which was orientation tuned: iso-oriented surrounds caused more suppression than cross-oriented surrounds. Surround suppression was delayed relative to the visual response and orientation-tuned suppression was delayed further, suggesting two separate suppressive mechanisms. Previous studies proposed that surround suppression depends on the activity of inhibitory somatostatin-positive interneurons in the superficial layers. To test the involvement of the superficial layers we topically applied lidocaine. Silencing of the superficial layers did not prevent orientation-tuned suppression in layer 4. These results show that neurons in mouse V1, which lacks orientation columns, show orientation-dependent surround suppression in layer 4 and the superficial layers and that surround suppression in layer 4 does not require contributions from neurons in the superficial layers.