Layer 1 of somatosensory cortex: an important site for input to a tiny cortical compartment.

Neocortical layer 1 has been proposed to be at the center for top-down and bottom-up integration. It is a locus for interactions between long-range inputs, layer 1 interneurons, and apical tuft dendrites of pyramidal neurons. While input to layer 1 has been studied intensively, the level and effect of input to this layer has still not been completely characterized. Here we examined the input to layer 1 of mouse somatosensory cortex with retrograde tracing and optogenetics. Our assays reveal that local input to layer 1 is predominantly from layers 2/3 and 5 pyramidal neurons and interneurons, and that subtypes of local layers 5 and 6b neurons project to layer 1 with different probabilities. Long-range input from sensory-motor cortices to layer 1 of somatosensory cortex arose predominantly from layers 2/3 neurons. Our optogenetic experiments showed that intra-telencephalic layer 5 pyramidal neurons drive layer 1 interneurons but have no effect locally on layer 5 apical tuft dendrites. Dual retrograde tracing revealed that a fraction of local and long-range neurons was both presynaptic to layer 5 neurons and projected to layer 1. Our work highlights the prominent role of local inputs to layer 1 and shows the potential for complex interactions between long-range and local inputs, which are both in position to modify the output of somatosensory cortex.

The impact of phosphorylated PTEN at threonine 366 on cortical connectivity and behaviour.

The lipid phosphatase PTEN (phosphatase and tensin homologue on chromosome 10) is a key tumour suppressor gene and an important regulator of neuronal signalling. PTEN mutations have been identified in patients with autism spectrum disorders, characterized by macrocephaly, impaired social interactions and communication, repetitive behaviour, intellectual disability, and epilepsy. PTEN enzymatic activity is regulated by a cluster of phosphorylation sites at the C-terminus of the protein. Here, we focused on the role of PTEN T366 phosphorylation and generated a knock-in mouse line in which Pten T366 was substituted with alanine (PtenT366A/T366A). We identify that phosphorylation of PTEN at T366 controls neuron size and connectivity of brain circuits involved in sensory processing. We show in behavioural tests that PtenT366/T366A mice exhibit cognitive deficits and selective sensory impairments, with significant differences in male individuals. We identify restricted cellular overgrowth of cortical neurons in PtenT366A/T366A brains, linked to increases in both dendritic arborization and soma size. In a combinatorial approach of anterograde and retrograde monosynaptic tracing using rabies virus, we characterize differences in connectivity to the primary somatosensory cortex of PtenT366A/T366A brains, with imbalances in long-range cortico-cortical input to neurons. We conclude that phosphorylation of PTEN at T366 controls neuron size and connectivity of brain circuits involved in sensory processing and propose that PTEN T366 signalling may account for a subset of autism-related functions of PTEN.

Drebrin controls scar formation and astrocyte reactivity upon traumatic brain injury by regulating membrane trafficking.

The brain of mammals lacks a significant ability to regenerate neurons and is thus particularly vulnerable. To protect the brain from injury and disease, damage control by astrocytes through astrogliosis and scar formation is vital. Here, we show that brain injury in mice triggers an immediate upregulation of the actin-binding protein Drebrin (DBN) in astrocytes, which is essential for scar formation and maintenance of astrocyte reactivity. In turn, DBN loss leads to defective astrocyte scar formation and excessive neurodegeneration following brain injuries. At the cellular level, we show that DBN switches actin homeostasis from ARP2/3-dependent arrays to microtubule-compatible scaffolds, facilitating the formation of RAB8-positive membrane tubules. This injury-specific RAB8 membrane compartment serves as hub for the trafficking of surface proteins involved in astrogliosis and adhesion mediators, such as ß1-integrin. Our work shows that DBN-mediated membrane trafficking in astrocytes is an important neuroprotective mechanism following traumatic brain injury in mice.

The Claustrum is Involved in Cognitive Processes Related to the Classical Conditioning of Eyelid Responses in Behaving Rabbits.

It is assumed that the claustrum (CL) is involved in sensorimotor integration and cognitive processes. We recorded the firing activity of identified CL neurons during classical eyeblink conditioning in rabbits, using a delay paradigm in which a tone was presented as conditioned stimulus (CS), followed by a corneal air puff as unconditioned stimulus (US). Neurons were identified by their activation from motor (MC), cingulate (CC), and medial prefrontal (mPFC) cortices. CL neurons were rarely activated by single stimuli of any modality. In contrast, their firing was significantly modulated during the first sessions of paired CS/US presentations, but not in well-trained animals. Neuron firing rates did not correlate with the kinematics of conditioned responses (CRs). CL local field potentials (LFPs) changed their spectral power across learning and presented well-differentiated CL-mPFC/CL-MC network dynamics, as shown by crossfrequency spectral measurements. CL electrical stimulation did not evoke eyelid responses, even in trained animals. Silencing of synaptic transmission of CL neurons by the vINSIST method delayed the acquisition of CRs but did not affect their presentation rate. The CL plays an important role in the acquisition of associative learning, mostly in relation to the novelty of CS/US association, but not in the expression of CRs.

Perirhinal input to neocortical layer 1 controls learning.

Hippocampal output influences memory formation in the neocortex, but this process is poorly understood because the precise anatomical location and the underlying cellular mechanisms remain elusive. Here, we show that perirhinal input, predominantly to sensory cortical layer 1 (L1), controls hippocampal-dependent associative learning in rodents. This process was marked by the emergence of distinct firing responses in defined subpopulations of layer 5 (L5) pyramidal neurons whose tuft dendrites receive perirhinal inputs in L1. Learning correlated with burst firing and the enhancement of dendritic excitability, and it was suppressed by disruption of dendritic activity. Furthermore, bursts, but not regular spike trains, were sufficient to retrieve learned behavior. We conclude that hippocampal information arriving at L5 tuft dendrites in neocortical L1 mediates memory formation in the neocortex.

Layer 6b Is Driven by Intracortical Long-Range Projection Neurons.

Layer 6b (L6b), the deepest neocortical layer, projects to cortical targets and higher-order thalamus and is the only layer responsive to the wake-promoting neuropeptide orexin/hypocretin. These characteristics suggest that L6b can strongly modulate brain state, but projections to L6b and their influence remain unknown. Here, we examine the inputs to L6b ex vivo in the mouse primary somatosensory cortex with rabies-based retrograde tracing and channelrhodopsin-assisted circuit mapping in brain slices. We find that L6b receives its strongest excitatory input from intracortical long-range projection neurons, including those in the contralateral hemisphere. In contrast, local intracortical input and thalamocortical input were significantly weaker. Moreover, our data suggest that L6b receives far less thalamocortical input than other cortical layers. L6b was most strongly inhibited by PV and SST interneurons. This study shows that L6b integrates long-range intracortical information and is not part of the traditional thalamocortical loop.

The Axonal Membrane Protein PRG2 Inhibits PTEN and Directs Growth to Branches.

In developing neurons, phosphoinositide 3-kinases (PI3Ks) control axon growth and branching by positively regulating PI3K/PI(3,4,5)P3, but how neurons are able to generate sufficient PI(3,4,5)P3 in the presence of high levels of the antagonizing phosphatase PTEN is difficult to reconcile. We find that normal axon morphogenesis involves homeostasis of elongation and branch growth controlled by accumulation of PI(3,4,5)P3 through PTEN inhibition. We identify a plasma membrane-localized protein-protein interaction of PTEN with plasticity-related gene 2 (PRG2). PRG2 stabilizes membrane PI(3,4,5)P3 by inhibiting PTEN and localizes in nanoclusters along axon membranes when neurons initiate their complex branching behavior. We demonstrate that PRG2 is both sufficient and necessary to account for the ability of neurons to generate axon filopodia and branches in dependence on PI3K/PI(3,4,5)P3 and PTEN. Our data indicate that PRG2 is part of a neuronal growth program that induces collateral branch growth in axons by conferring local inhibition of PTEN.

Optically Induced Calcium-Dependent Gene Activation and Labeling of Active Neurons Using CaMPARI and Cal-Light.

The advent of optogenetic methods has made it possible to use endogeneously produced molecules to image and manipulate cellular, subcellular, and synaptic activity. It has also led to the development of photoactivatable calcium-dependent indicators that mark active synapses, neurons, and circuits. Furthermore, calcium-dependent photoactivation can be used to trigger gene expression in active neurons. Here we describe two sets of protocols, one using CaMPARI and a second one using Cal-Light. CaMPARI, a calcium-modulated photoactivatable ratiometric integrator, enables rapid network-wide, tunable, all-optical functional circuit mapping. Cal-Light, a photoactivatable calcium sensor, while slower to respond than CaMPARI, has the capacity to trigger the expression of genes, including effectors, activators, indicators, or other constructs. Here we describe the rationale and provide procedures for using these two calcium-dependent constructs (1) in vitro in dissociated primary neuronal cell cultures (CaMPARI & Cal-Light); (2) in vitro in acute brain slices for circuit mapping (CaMPARI); (3) in vivo for triggering photoconversion or gene expression (CaMPARI & Cal-Light); and finally, (4) for recovering photoconverted neurons post-fixation with immunocytochemistry (CaMPARI). The approaches and protocols we describe are examples of the potential uses of both CaMPARI & Cal-Light. The ability to mark and manipulate neurons that are active during specific epochs of behavior has a vast unexplored experimental potential.

Improved methods for marking active neuron populations.

Marking functionally distinct neuronal ensembles with high spatiotemporal resolution is a key challenge in systems neuroscience. We recently introduced CaMPARI, an engineered fluorescent protein whose green-to-red photoconversion depends on simultaneous light exposure and elevated calcium, which enabled marking active neuronal populations with single-cell and subsecond resolution. However, CaMPARI (CaMPARI1) has several drawbacks, including background photoconversion in low calcium, slow kinetics and reduced fluorescence after chemical fixation. In this work, we develop CaMPARI2, an improved sensor with brighter green and red fluorescence, faster calcium unbinding kinetics and decreased photoconversion in low calcium conditions. We demonstrate the improved performance of CaMPARI2 in mammalian neurons and in vivo in larval zebrafish brain and mouse visual cortex. Additionally, we herein develop an immunohistochemical detection method for specific labeling of the photoconverted red form of CaMPARI. The anti-CaMPARI-red antibody provides strong labeling that is selective for photoconverted CaMPARI in activated neurons in rodent brain tissue.

The emergence of mesencephalic trigeminal neurons.

BACKGROUND: The cells of the mesencephalic trigeminal nucleus (MTN) are the proprioceptive sensory neurons that innervate the jaw closing muscles. These cells differentiate close to the two key signalling centres that influence the dorsal midbrain, the isthmus, which mediates its effects via FGF and WNT signalling and the roof plate, which is a major source of BMP signalling as well as WNT signalling. METHODS: In this study, we have set out to analyse the importance of FGF, WNT and BMP signalling for the development of the MTN. We have employed pharmacological inhibitors of these pathways in explant cultures as well as utilising the electroporation of inhibitory constructs in vivo in the chick embryo. RESULTS: We find that interfering with either FGF or WNT signalling has pronounced effects on MTN development whilst abrogation of BMP signalling has no effect. We show that treatment of explants with either FGF or WNT antagonists results in the generation of fewer MTN neurons and affects MTN axon extension and that inhibition of both these pathways has an additive effect. To complement these studies, we have used in vivo electroporation to inhibit BMP, FGF and WNT signalling within dorsal midbrain cells prior to, and during, their differentiation as MTN neurons. Again, we find that inhibition of BMP signalling has no effect on the development of MTN neurons. We additionally find that cells electroporated with inhibitory constructs for either FGF or WNT signalling can differentiate as MTN neurons suggesting that these pathways are not required cell intrinsically for the emergence of these neurons. Indeed, we also show that explants of dorsal mesencephalon lacking both the isthmus and roof plate can generate MTN neurons. However, we did find that inhibiting FGF or WNT signalling had consequences for MTN differentiation. CONCLUSIONS: Our results suggest that the emergence of MTN neurons is an intrinsic property of the dorsal mesencephalon of gnathostomes, and that this population undergoes expansion, and maturation, along with the rest of the dorsal midbrain under the influence of FGF and WNT signalling.

Investigation of hippocampal synaptic transmission and plasticity in mice deficient in the actin-binding protein Drebrin.

The dynamic regulation of the actin cytoskeleton plays a key role in controlling the structure and function of synapses. It is vital for activity-dependent modulation of synaptic transmission and long-term changes in synaptic morphology associated with memory consolidation. Several regulators of actin dynamics at the synapse have been identified, of which a salient one is the postsynaptic actin stabilising protein Drebrin (DBN). It has been suggested that DBN modulates neurotransmission and changes in dendritic spine morphology associated with synaptic plasticity. Given that a decrease in DBN levels is correlated with cognitive deficits associated with ageing and dementia, it was hypothesised that DBN protein abundance instructs the integrity and function of synapses. We created a novel DBN deficient mouse line. Analysis of gross brain and neuronal morphology revealed no phenotype in the absence of DBN. Electrophysiological recordings in acute hippocampal slices and primary hippocampal neuronal cultures showed that basal synaptic transmission, and both long-term and homeostatic synaptic plasticity were unchanged, suggesting that loss of DBN is not sufficient in inducing synapse dysfunction. We propose that the overall lack of changes in synaptic function and plasticity in DBN deficient mice may indicate robust compensatory mechanisms that safeguard cytoskeleton dynamics at the synapse.

A software tool for the analysis of neuronal morphology data.

Anatomy plays a fundamental role in supporting and shaping nervous system activity. The remarkable progress of computer processing power within the last two decades has enabled the generation of electronic databases of complete three-dimensional (3D) dendritic and axonal morphology for neuroanatomical studies. Several laboratories are freely posting their reconstructions online after result publication v.gr. NeuroMorpho.Org (Nat Rev Neurosci7:318-324, 2006). These neuroanatomical archives represent a crucial resource to explore the relationship between structure and function in the brain (Front Neurosci6:49, 2012). However, such 'Cartesian' descriptions bear little intuitive information for neuroscientists. Here, we developed a simple prototype of a MATLAB-based software tool to quantitatively describe the 3D neuronal structures from public repositories. The program imports neuronal reconstructions and quantifies statistical distributions of basic morphological parameters such as branch length, tortuosity, branch's genealogy and bifurcation angles. Using these morphological distributions, our algorithm can generate a set of virtual neurons readily usable for network simulations.

Maturation of postnatally generated olfactory bulb granule cells depends on functional γ-protocadherin expression.

γ-protocadherins (γ-pcdhs) are transmembrane receptor proteins ubiquitously expressed in the postnatal and adult mouse brain. γ-pcdhs are required for normal neuronal development as shown for spinal cord interneurons, retinal ganglion cells and cortical neurons. To test the role of γ-pcdhs during development of subventricular zone progenitor cells and their subsequent differentiation into olfactory granule cells we generated a conditional γ-pcdh(lox/lox) allele (γ-pcdh(lox/lox)) allowing for functional γ-pcdh inactivation upon lentivirus-mediated Cre-recombinase expression selectively in subventricular zone progenitor cells. While γ-pcdh loss did not alter the proliferation of subventricular zone progenitors, γ-pcdh ko progenitors that reached the main olfactory bulb showed a significant reduction in dendritic arborization and failed to develop dendritic spines. Our results suggest that olfactory bulb granule cell maturation necessitates functional γ-pcdh expression.

Charting monosynaptic connectivity maps by two-color light-sheet fluorescence microscopy.

Cellular resolution three-dimensional (3D) visualization of defined, fluorescently labeled long-range neuronal networks in the uncut adult mouse brain has been elusive. Here, a virus-based strategy is described that allowed fluorescent labeling of centrifugally projecting neuronal populations in the ventral forebrain and their directly, monosynaptically connected bulbar interneurons upon a single stereotaxic injection into select neuronal populations. Implementation of improved tissue clearing combined with light-sheet fluorescence microscopy permitted imaging of the resulting connectivity maps in a single whole-brain scan. Subsequent 3D reconstructions revealed the exact distribution of the diverse neuronal ensembles monosynaptically connected with distinct bulbar interneuron populations. Moreover, rehydratation of brains after light-sheet fluorescence imaging enabled the immunohistochemical identification of synaptically connected neurons. Thus, this study describes a method for identifying monosynaptic connectivity maps from distinct, virally labeled neuronal populations that helps in better understanding of information flow in neural systems.

Stochastic choice of allelic expression in human neural stem cells.

Monoallelic gene expression, such as genomic imprinting, is well described. Less well-characterized are genes undergoing stochastic monoallelic expression (MA), where specific clones of cells express just one allele at a given locus. We performed genome-wide allelic expression assessment of human clonal neural stem cells derived from cerebral cortex, striatum, and spinal cord, each with differing genotypes. We assayed three separate clonal lines from each donor, distinguishing stochastic MA from genotypic effects. Roughly 2% of genes showed evidence for autosomal MA, and in about half of these, allelic expression was stochastic between different clones. Many of these loci were known neurodevelopmental genes, such as OTX2 and OLIG2. Monoallelic genes also showed increased levels of DNA methylation compared to hypomethylated biallelic loci. Identified monoallelic gene loci showed altered chromatin signatures in fetal brain, suggesting an in vivo correlate of this phenomenon. We conclude that stochastic allelic expression is prevalent in neural stem cells, providing clonal diversity to developing tissues such as the human brain.