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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.

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.

Imaging calcium signals in neurons of awake, behaving mice using two-photon microscopy makes it possible to study how learning alters coding in large neural populations. Such experiments produce extremely large datasets requiring powerful automated methods to extract responses from hundreds of neurons. Here we present a new open source toolbox for 1) segmentation of regions of interest (ROIs) representing neuronal structures, 2) inspection and manual splitting, addition or rejection of ROIs, 3) neuropil correction and signal extraction and 4) matching of ROIs in sequential recordings. Our toolbox is built around a novel method for the identification of ROIs based on temporal cross-correlations of low frequency components derived by Fourier analysis, of each pixel with its neighbors. The approach is insightful for the end user and allows for the ROI detection around neurons or irregular structures such as dendrites and axons by adjusting simple morphological constraints. This new pipeline thus provides an efficient and user-friendly approach for analyzing calcium responses in neuronal structures imaged over prolonged periods of time.

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.

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 γ-amino butyric acid (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 wild-type litter mates 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: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.

The formation, plasticity and maintenance of synaptic connections is regulated by molecular and electrical signals. β-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 β-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, β-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 β-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 β-catenin remains important for adult cortical function but through different mechanisms than during development.

© 2021 The Author(s)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.

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.

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 new insight in how lower and higher areas of the visual cortex interact to shape visual perception.

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. 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.

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.

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.

Summary Mitochondria buffer intracellular Ca 2+ and provide energy [1]. Because synaptic structures with high Ca 2+ 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.

SummaryDuring development, cortical plasticity is associated with the rearrangement of excitatory connections. While these connections become more stable with age, plasticity can still be induced in the adult cortex. Here we provide evidence that structural plasticity of inhibitory synapses onto pyramidal neurons is a major component of plasticity in the adult neocortex. In vivo two-photon imaging was used to monitor the formation and elimination of fluorescently labeled inhibitory structures on pyramidal neurons. We find that ocular dominance plasticity in the adult visual cortex is associated with rapid inhibitory synapse loss, especially of those present on dendritic spines. This occurs not only with monocular deprivation but also with subsequent restoration of binocular vision. We propose that in the adult visual cortex the experience-induced loss of inhibition may effectively strengthen specific visual inputs with limited need for rearranging the excitatory circuitry.

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.

Cognitive impairments are a major clinical feature of the common neurogenetic disease neurofibromatosis type 1 (NF1). Previous studies have demonstrated that increased neuronal inhibition underlies the learning deficits in NF1, however, the molecular mechanism underlying this cell-type specificity has remained unknown. Here, we identify an interneuron-specific attenuation of hyperpolarization-activated cyclic nucleotide-gated (HCN) current as the cause for increased inhibition in Nf1 mutants. Mechanistically, we demonstrate that HCN1 is a novel NF1-interacting protein for which loss of NF1 results in a concomitant increase of interneuron excitability. Furthermore, the HCN channel agonist lamotrigine rescued the electrophysiological and cognitive deficits in two independent Nf1 mouse models, thereby establishing the importance of HCN channel dysfunction in NF1. Together, our results provide detailed mechanistic insights into the pathophysiology of NF1-associated cognitive defects, and identify a novel target for clinical drug development.

Contains fulltext : 32422.pdf (Publisher’s version ) (Closed access) Pseudoxanthoma elasticum (PXE) is a heritable disorder of connective tissue, affecting mainly skin, eye and the cardiovascular system. PXE is characterized by dystrophic mineralization of elastic fibres. The condition is caused by loss of function mutations in ABCC6. We generated Abcc6 deficient mice (Abcc6-/-) by conventional gene targeting. As shown by light and electron microscopy Abcc6-/- mice spontaneously developed calcification of elastic fibres in blood vessel walls and in Bruch's membrane in the eye. No clear abnormalities were seen in the dermal extracellular matrix. Calcification of blood vessels was most prominent in small arteries in the cortex of the kidney, but in old mice, it occurred also in other organs and in the aorta and vena cava. Newly developed monoclonal antibodies against mouse Abcc6 localized the protein to the basolateral membranes of hepatocytes and the basal membrane in renal proximal tubules, but failed to show the protein at the pathogenic sites. Abcc6-/- mice developed a 25% reduction in plasma HDL cholesterol and an increase in plasma creatinine levels, which may be due to impaired kidney function. No changes in serum mineral balance were found. We conclude that the phenotype of the Abcc6-/- mouse shares calcification of elastic fibres with human PXE pathology, which makes this model a useful tool to further investigate the aetiology of PXE. Our data support the hypothesis that PXE is in fact a systemic disease.

Loss of Crumbs homologue 1 (CRB1) function causes either the eye disease Leber congenital amaurosis or progressive retinitis pigmentosa, depending on the amount of residual CRB1 activity and the genetic background. Crb1 localizes specifically to the sub-apical region adjacent to the adherens junction complex at the outer limiting membrane in the retina. We show that it is associated here with multiple PDZ protein 1 (Mupp1), protein associated with Lin-7 (Pals1 or Mpp5) and Mpp4. We have produced Crb1-/- mice completely lacking any functional Crb1. Although the retinas are initially normal, by 3-9 months the Crb1-/- retinas develop localized lesions where the integrity of the outer limiting membrane is lost and giant half rosettes are formed. After delamination of the photoreceptor layer, neuronal cell death occurs in the inner and outer nuclear layers of the retina. On moderate exposure to light for 3 days at 3 months of age, the number of severe focal retinal lesions significantly increases in the Crb1-/- retina. Crb2, Crb3 and Crb1 interacting proteins remain localized to the sub-apical region and therefore are not sufficient to maintain cell adhesion during light exposure in Crb1-/- retinas. Thus we propose that during light exposure Crb1 is essential to maintain, but not assemble, adherens junctions between photoreceptors and Müller glia cells and prevents retinal disorganization and dystrophy. Hence, light may be an influential factor in the development of the corresponding human diseases.

Summary 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.

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.

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. © 2014 the authors.

In this study, we compared the influence of two peptides on the selection of CD8αα and CD8αβ intraepithelial lymphocytes (IELs) of the intestine, which develop by a unique and partially thymus-independent process. Mice were used in which all T cells carried one transgenic T cell antigen receptor (TCR) (F5), and in which only well defined transgenic peptides were presented by H-2Db. The first peptide, for which the F5 TCR has a high affinity, derives from the influenza virus nucleoprotein (NP68). The second peptide, NP34, is an antagonistic variant of NP68 and is recognized by the F5 TCR with low affinity. To avoid presentation of endogenous peptides or production of T cells carrying alternative TCRs, F5 TCR transgenic mice were generated that were deficient for Tap-1 and Rag-1. In these mice, no CD3+CD8+cells were found in lymph nodes, spleen, or intestine. Introduction of transgenes encoding either NP34 or NP68 along with an endoplasmic reticulum signal sequence enabled Tap-1-independent expression of each peptide in these mice. Positive selection of F5TCR+CD8+thymocytes was not rescued by these transgenic peptides. However, the high-affinity NP68 peptide induced maturation of CD8αα IEL, whereas the low-affinity NP34 peptide stimulated development of both CD8αβ and CD8αα IEL, but in smaller numbers. When both peptides were present, CD8αβ T cells failed to develop and the number of CD8αα IELs was lower than in mice carrying the NP68 transgene alone. These data demonstrate that single ligands with a high or low affinity for TCR are capable of inducing or inhibiting the maturation of alternative subsets of IELs.

During development, thymocytes express a number of genes typical for activated peripheral T lymphocytes, including granzymes. We have now analyzed by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and cytochemistry fetal liver cells and thymocytes at various developmental stages for the expression of granzyme A-G genes. At days 13-17 of gestation, only granzyme B but none of the other granzymes is expressed in fetal liver. In the most immature, Pgp-1+IL2R alpha-, thymocyte subpopulation mRNAs for granzymes A-C but not for granzymes D-G are detectable. Upon further differentiation via Pgp-1-IL-2R alpha + into more mature Pgp-1-IL-2R alpha- thymocytes the level of expression of granzymes A, B, and C gradually declines reaching its lowest level at the CD4+ 8+ double positive stage. In fetal thymic lobes depleted of lymphoid cells by treatment with deoxyguanosine, no transcripts for granzymes A, B, and C were found indicating that the PCR signals are derived exclusively from early precursor T/natural killer (NK) lineage cells rather than from residual stromal elements. In mature CD4+CD8- and CD4-CD8+ thymocytes, granzyme B mRNA is found at similar levels in both subsets whereas granzyme A mRNA is expressed selectively in the CD4-CD8+ subset. Enzymatic activity of granzyme A was only seen in a fraction of CD4-CD8+ thymocytes negative for heat stable antigen (HSA) but not in the more immature HSA+ fraction of CD4-CD8+ thymocytes. The data suggest that (a) granzyme B is a pro-thymocyte marker for all T/NK lineage cells; (b) granzyme A transcripts are associated with thymocytes with the potential to develop into the CD8+ lineage; and (c) granzyme A enzymatic activity is only expressed in the most mature CD4-CD8+ stage, suggesting that granzyme proteins are not involved in early stages of thymocyte development.

International audience; In humans, the Crumbs homolog-1 (CRB1) gene is mutated in progressive types of autosomal recessive retinitis pigmentosa and Leber congenital amaurosis. However, there is no clear genotype-phenotype correlation for CRB1 mutations, which suggests that other components of the CRB complex may influence the severity of retinal disease. Therefore, to understand the physiological role of the Crumbs complex proteins, we generated and analysed conditional knockout mice lacking CRB2 in the developing retina. Progressive disorganization was detected during late retinal development. Progressive thinning of the photoreceptor layer and sites of cellular mislocalization was detected throughout the CRB2-deficient retina by confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography. Under scotopic conditions using electroretinography, the attenuation of the a-wave was relatively stronger than that of the b-wave, suggesting progressive degeneration of photoreceptors in adult animals. Histological analysis of newborn mice showed abnormal lamination of immature rod photoreceptors and disruption of adherens junctions between photoreceptors, Müller glia and progenitor cells. The number of late-born progenitor cells, rod photoreceptors and Müller glia cells was increased, concomitant with programmed cell death of rod photoreceptors. The data suggest an essential role for CRB2 in proper lamination of the photoreceptor layer and suppression of proliferation of late-born retinal progenitor cells.

BACKGROUND: Inhibitory innervation by parvalbumin (PV) expressing interneurons has been implicated in the onset of the sensitive period of visual plasticity. Immunohistochemical analysis of the development and plasticity of these inhibitory inputs is difficult because PV expression is low in young animals and strongly influenced by neuronal activity. Moreover, the synaptic boutons that PV neurons form onto each other cannot be distinguished from the innervated cell bodies by immunostaining for this protein because it is present throughout the cells. These problems call for the availability of a synaptic, activity-independent marker for PV+ inhibitory boutons that is expressed before sensitive period onset. We investigated whether synaptotagmin-2 (Syt2) fulfills these properties in the visual cortex. Syt2 is a synaptic vesicle protein involved in fast Ca(2+) dependent neurotransmitter release. Its mRNA expression follows a pattern similar to that of PV throughout the brain and is present in 30-40% of hippocampal PV expressing basket cells. Up to now, no quantitative analyses of Syt2 expression in the visual cortex have been carried out. METHODOLOGY/PRINCIPAL FINDINGS: We used immunohistochemistry to analyze colocalization of Syt2 with multiple interneuron markers including vesicular GABA transporter VGAT, calbindin, calretinin, somatostatin and PV in the primary visual cortex of mice during development and after dark-rearing. CONCLUSIONS/SIGNIFICANCE: We show that in the adult visual cortex Syt2 is only found in inhibitory, VGAT positive boutons. Practically all Syt2 positive boutons also contain PV and vice versa. During development, Syt2 expression can be detected in synaptic boutons prior to PV and in contrast to PV expression, Syt2 is not down-regulated by dark-rearing. These properties of Syt2 make it an excellent marker for analyzing the development and plasticity of perisomatic inhibitory innervations onto both excitatory and inhibitory neurons in the visual cortex.

During the last decade, we have gained much insight into the mechanisms that open and close a sensitive period of plasticity in the visual cortex. This brings the hope that novel treatments can be developed for brain injuries requiring renewed plasticity potential and neurodevelopmental brain disorders caused by defective synaptic plasticity. One of the central mechanisms responsible for opening the sensitive period is the maturation of inhibitory innervation. Many molecular and cellular events have been identified that drive this developmental process, including signaling through BDNF and IGF-1, transcriptional control by OTX2, maturation of the extracellular matrix, and GABA-regulated inhibitory synapse formation. The mechanisms through which the development of inhibitory innervation triggers and potentially closes the sensitive period may involve plasticity of inhibitory inputs or permissive regulation of excitatory synapse plasticity. Here, we discuss the current state of knowledge in the field and open questions to be addressed.

Recent studies have shown that maturation of CD4-8- double negative (DN) thymocytes to the CD4+8+ double positive (DP) stage is dependent on expression of the T cell receptor (TCR)-beta polypeptide. The exact mechanism by which the TCR-beta chain regulates this maturation step remains unknown. Previous experiments had suggested that in the presence of some TCR+ thymocytes, additional DN thymocytes not expressing a TCR-beta chain may be recruited to mature to the DP stage. The recent demonstration of an immature TCR-beta-CD3 complex on early thymocytes lead to the alternative hypothesis that signal transduction through an immature TCR-CD3 complex may induce maturation to the DP stage. In the latter case, maturation to the DP stage would depend on the expression of TCR-beta-CD3 in the same cell. We examined these two hypotheses by studying the expression of the intra- and extracellular CD3 epsilon, CD3 zeta, and TCR-beta polypeptides in intrathymic subpopulations during embryogenesis. CD3 epsilon and CD3 zeta were expressed intracellularly 2 and 1 d, respectively, before intracellular expression of the TCR-beta chain, potentially allowing immediate surface expression of an immature TCR-beta-CD3 complex as soon as functional rearrangement of a TCR-beta gene locus has been accomplished. Calcium mobilization could be induced by stimulation with anti-CD3 epsilon mAb as soon as intracellular TCR-beta was detectable, suggesting that a functional TCR-beta-CD3 complex is indeed expressed on the surface of early thymocytes. From day 17 on, most cells were in the DP stage, and over 95% of the DP cells expressed on the TCR-beta chain intracellularly. At day 19 of gestation, extremely low concentrations of TCR-beta chain and CD3 epsilon were detectable on the cell surface of nearly all thymocytes previously thought to be TCR-CD3 negative. These findings strongly support the hypothesis that maturation to the DP stage depends on surface expression of and subsequent signal transduction through an immature TCR-beta-CD3 complex and suggest that maturation to the DP stage by recruitment, if it occurs at all, is of minor relevance.

Several recent observations suggest that successful rearrangement of the T cell receptor (TCR) beta locus induces several important events in thymocyte maturation. Allelic exclusion is achieved by interruption of further rearrangement of the beta locus, and CD4-8- interleukin (IL)-2R+ cells enter the CD4+8+IL-2R- stage. The actual molecular events regulating this important control point are unknown, but may be related to the expression of the TCR-beta locus in immature CD4-8- thymocytes. It is not clear whether maturation is induced by intracellular appearance of TCR-beta chain or by signal transduction through an immature TCR complex on the thymocyte membrane, possibly involving TCR-beta chain homodimers and CD3. Here we show that early addition of anti-CD3 mAb to fetal thymic organ cultures induces all known events associated with the acquisition of the CD4+8+ stage. Expression of CD4 and CD8 is accelerated, IL-2R alpha is downregulated, and the cells fail to produce TCR-beta, possibly based on premature cessation of beta gene rearrangement. Upon stimulation with anti-CD3 antibodies, we see calcium mobilization in 15% of all CD4-8- thymocytes with no detectable surface TCR expression. These results suggest that functional CD3 is expressed on immature thymocytes at very low concentrations before the appearance of a complete TCR-beta chain. Ligation of CD3 at this stage may mimic the maturation signal normally generated by the immature TCR-beta homodimer-CD3 complex. The results are consistent with the notion that acquisition of the CD4+8+ stage involves signal transduction through an immature TCR complex. Later in thymocyte development, ligation of CD3 results in deletion of CD4+8+ cells. Thus, signal transduction through CD3 may result in entirely different cellular responses, depending on the stage of thymocyte differentiation. These results suggest an involvement of CD3 as a link in signal transduction for at least two different decision points in the development of a thymocyte.

How the visual cortex responds to specific stimuli is strongly influenced by visual experience during development. Monocular deprivation, for example, changes the likelihood of neurons in the visual cortex to respond to input from the deprived eye and reduces its visual acuity. Because these functional changes are accompanied by extensive reorganization of neurite morphology and dendritic spine turnover, genes regulating neuronal morphology are likely to be involved in visual plasticity. In recent years, Notch1 has been shown to mediate contact inhibition of neurite outgrowth in postmitotic neurons and implicated in the pathogenesis of various degenerative diseases of the CNS. Here, we provide the first evidence for the involvement of neuronal Notch1 signaling in synaptic morphology and plasticity in the visual cortex. By making use of the Cre/Lox system, we expressed an active form of Notch1 in cortical pyramidal neurons several weeks after birth. We show that neuronal Notch1 signals reduce dendritic spine and filopodia densities in a cell-autonomous manner and limit long-term potentiation in the visual cortex. After monocular deprivation, these effects of Notch1 activity predominantly affect responses to visual stimuli with higher spatial frequencies. This results in an enhanced effect of monocular deprivation on visual acuity. Copyright © 2008 Society for Neuroscience.

In adult primary visual cortex (V1), dendritic spines are more persistent than during development. Brain-derived neurotrophic factor (BDNF) increases synaptic strength, and its levels rise during cortical development. We therefore asked whether postsynaptic BDNF signaling through its receptor TrkB regulates spine persistence in adult V1. This question has been difficult to address because most methods used to alter TrkB signaling in vivo affect cortical development or cannot distinguish between pre- and postsynaptic mechanisms. We circumvented these problems by employing transgenic mice expressing a dominant negative TrkB–EGFP fusion protein in sparse pyramidal neurons of the adult neocortex and hippocampus, producing a Golgi-staining-like pattern. In adult V1, expression of dominant negative TrkB-EGFP resulted in reduced mushroom spine maintenance and synaptic efficacy, accompanied by an increase in long and thin spines and filopodia. In contrast, mushroom spine maintenance was unaffected in CA1, indicating that TrkB plays fundamentally different roles in structural plasticity in these brain areas.

Cell death is normally accompanied by loss of integrity of the cell membrane. Concomitant loss of osmotic pressure and permeability for DNA binding dyes like propidium iodide make it possible to distinguish viable from nonviable cells. However, after permeabilization for intracellular staining or after fixation of the cells, we find that propidium iodide leaks out of nonviable cells and is transferred to formerly viable cells. Cell size cannot be used for examining viability in permeabilized or heterogeneous cell populations. Here we show that streptavidin-tricolor enters specifically and irreversibly into dead cells, and is not transferred to formerly viable cells after fixation or permeabilization. Therefore, streptavidin-tricolor can be a useful dead-cell marker in experimental situations where conventional methods fail to distinguish between viable and nonviable cells.

BACKGROUND: Transgenic mice with mosaic, Golgi-staining-like expression of enhanced green fluorescent protein (EGFP) have been very useful in studying the dynamics of neuronal structure and function. In order to further investigate the molecular events regulating structural plasticity, it would be useful to express multiple proteins in the same sparse neurons, allowing co-expression of functional proteins or co-labeling of subcellular compartments with other fluorescent proteins. However, it has been difficult to obtain reproducible expression in the same subset of neurons for direct comparison of neurons expressing different functional proteins.PRINCIPAL FINDINGS: Here we describe a Cre-transgenic line that allows reproducible expression of transgenic proteins of choice in a small number of neurons of the adult cortex, hippocampus, striatum, olfactory bulb, subiculum, hypothalamus, superior colliculus and amygdala. We show that using these Cre-transgenic mice, multiple Cre-dependent transgenes can be expressed together in the same isolated neurons. We also describe a Cre-dependent transgenic line expressing a membrane associated EGFP (EGFP-F). Crossed with the Cre-transgenic line, EGFP-F expression starts in the adolescent forebrain, is present in dendrites, dendritic protrusions, axons and boutons and is strong enough for acute or chronic in vivo imaging.SIGNIFICANCE: This triple transgenic approach will aid the morphological and functional characterization of neurons in various Cre-dependent transgenic mice.

Inactivating genes in vivo is an important technique for establishing their function in the adult nervous system. Unfortunately, conventional knockout mice may suffer from several limitations including embryonic or perinatal lethality and the compensatory regulation of other genes. One approach to producing conditional activation or inactivation of genes involves the use of Cre recombinase to remove loxP-flanked segments of DNA. We have studied the effects of delivering Cre to the hippocampus and neocortex of adult mice by injecting replication-deficient adeno-associated virus (AAV) and lentiviral (LV) vectors into discrete regions of the forebrain. Recombinant AAV-Cre, AAV-GFP (green fluorescent protein) and LV-Cre-EGFP (enhanced GFP) were made with the transgene controlled by the cytomegalovirus promoter. Infecting 293T cells in vitro with AAV-Cre and LV-Cre-EGFP resulted in transduction of most cells as shown by GFP fluorescence and Cre immunoreactivity. Injections of submicrolitre quantities of LV-Cre-EGFP and mixtures of AAV-Cre with AAV-GFP into the neocortex and hippocampus of adult Rosa26 reporter mice resulted in strong Cre and GFP expression in the dentate gyrus and moderate to strong labelling in specific regions of the hippocampus and in the neocortex, mainly in neurons. The pattern of expression of Cre and GFP obtained with AAV and LV vectors was very similar. X-gal staining showed that Cre-mediated recombination had occurred in neurons in the same regions of the brain, starting at 3 days post-injection. No obvious toxic effects of Cre expression were detected even after four weeks post-injection. AAV and LV vectors are capable of delivering Cre to neurons in discrete regions of the adult mouse brain and producing recombination.

During differentiation, thymocytes are selected twice for expressing a functional version of the CD3-T cell receptor (TCR). A first round of selection involves the immature CD4CD6double-negative (DN) ceils that express interleukin-2 receptor-a (IL-2Ra). At this stage, thymocytes rearrange their TCRj3 genes in a random fashion (Godfrey et al., 1993) leading to a successful rearrangement in approximately 5 of 9 cells (i.e., 56%). Thymocytes that productively rearranged the TCR8 locus are selected by virtue of expressing an immature form of the CD3-TCR, termed pre-TCR, to proliferate, down-regulate IL-2Ra and mature to the CD4+CD8+ double-positive (DP) stage (Groettrup and von Boehmer, 1993; Levelt and Eichmann, 1993). At the same time, TCR8 locus rearrangement is arrested to achieve allelic exclusion. A second round of selection takes place during the DP stage. At this stage, the TCRa genes are rearranged (Petrie et al., 1993) and thymocytes are subsequently subjected to repertoire selection based on the specificity of the mature a+j3+CD3 TCR (Kisielow et al., 1988). DP thymocytes that are positively selected develop into mature CD4+ or CD8+ singlepositive (SP) thymocytes (Chan et al., 1993; Davis et al., 1993). This review deals with the first round of selection in Tcell development: selection for functional rearrangement and expression of the TCR6 chain. The important role of the TCR8 chain in thymic development has been brought out first by studies on mice carrying a functionally rearranged TCR8 transgene (von Boehmer, 1990). In normal mice, the TCR8 transgene arrested the rearrangement of the endogenous TCRj3 gene loci, reflecting the process of allelic exclusion (Uematsu et al., 1988). In Scid mice (Schuler et al., 1986) in which thymocyte development is arrested at the DN IL-2Ra+ stage (Habu et al., 1987; Shoreset al., 1990) aTCR5 transgene restored maturation of thymocytes to the DP stage (von Boehmer, 1990). The pivotal role of the TCR8 chain in early T cell development was further corroborated by the phenotypes of several mutant mouse strains: In mice that cannot produce a TCR8 chain because of a deficiency in the rearrangement machinery (RAG7or RAG2deficient mice) (Mombaerts et al., 1992a; Shinkai et al., 1992a) or because of a mutation in the TCRj3 chain itself (Mombaerts et al., 1992b), thymocyte development is blocked at the DN stage. Introduction of aTCR8 transgene in any of these mice restores T cell maturation to the DP stage (Mombaerts et al., 1992b; Shinkai et al., 1992b). In contrast with TCR8deficient mice, the thymus of TCRa-deficient mice contains normal numbers of DP thymocytes (Philpott et Review