Altered integration of excitatory inputs onto the basal dendrites of layer 5 pyramidal neurons in a mouse model of Fragile X syndrome.

Layer 5 (L5) pyramidal neurons receive predictive and sensory inputs in a compartmentalized manner at their apical and basal dendrites, respectively. To uncover how integration of sensory inputs is affected in autism spectrum disorders (ASD), we used two-photon glutamate uncaging to activate spines in the basal dendrites of L5 pyramidal neurons from a mouse model of Fragile X syndrome (FXS), the most common genetic cause of ASD. While subthreshold excitatory inputs integrate linearly in wild-type animals, surprisingly those with FXS summate sublinearly, contradicting what would be expected of sensory hypersensitivity classically associated with ASD. We next investigated the mechanism underlying this sublinearity by performing knockdown of the regulatory ß4 subunit of BK channels, which rescued the synaptic integration, a result that was corroborated with numerical simulations. Taken together, these findings suggest that there is a differential impairment in the integration of feedforward sensory and feedback predictive inputs in L5 pyramidal neurons in FXS and potentially other forms of ASD, as a result of specifically localized subcellular channelopathies. These results challenge the traditional view that FXS and other ASD are characterized by sensory hypersensitivity, proposing instead a hyposensitivity of sensory inputs and hypersensitivity of predictive inputs onto cortical neurons.

Do Written Responses to Open-Ended Questions on Fourth-Grade Online Formative Assessments in Mathematics Help Predict Scores on End-of-Year Standardized Tests?

Predicting long-term student achievement is a critical task for teachers and for educational data mining. However, most of the models do not consider two typical situations in real-life classrooms. The first is that teachers develop their own questions for online formative assessment. Therefore, there are a huge number of possible questions, each of which is answered by only a few students. Second, online formative assessment often involves open-ended questions that students answer in writing. These types of questions in online formative assessment are highly valuable. However, analyzing the responses automatically can be a complex process. In this paper, we address these two challenges. We analyzed 621,575 answers to closed-ended questions and 16,618 answers to open-ended questions by 464 fourth-graders from 24 low socioeconomic status (SES) schools. Using regressors obtained from linguistic features of the answers and an automatic incoherent response classifier, we built a linear model that predicts the score on an end-of-year national standardized test. We found that despite answering 36.4 times fewer open-ended questions than closed questions, including features of the students' open responses in our model improved our prediction of their end-of-year test scores. To the best of our knowledge, this is the first time that a predictor of end-of-year test scores has been improved by using automatically detected features of answers to open-ended questions on online formative assessments.

Selective activation of BK channels in small-headed dendritic spines suppresses excitatory postsynaptic potentials.

Dendritic spines are the main receptacles of excitatory information in the brain. Their particular morphology, with a small head connected to the dendrite by a slender neck, has inspired theoretical and experimental work to understand how these structural features affect the processing, storage and integration of synaptic inputs in pyramidal neurons (PNs). The activation of glutamate receptors in spines triggers a large voltage change as well as calcium signals at the spine head. Thus, voltage-gated and calcium-activated potassium channels located in the spine head likely play a key role in synaptic transmission. Here we study the presence and function of large conductance calcium-activated potassium (BK) channels in spines from layer 5 PNs. We found that BK channels are localized to dendrites and spines regardless of their size, but their activity can only be detected in spines with small head volumes (≤0.09 µm3 ), which reduces the amplitude of two-photon uncaging excitatory postsynaptic potentials recorded at the soma. In addition, we found that calcium signals in spines with small head volumes are significantly larger than those observed in spines with larger head volumes. In accordance with our experimental data, numerical simulations predict that synaptic inputs impinging onto spines with small head volumes generate voltage responses and calcium signals within the spine head itself that are significantly larger than those observed in spines with larger head volumes, which are sufficient to activate spine BK channels. These results show that BK channels are selectively activated in small-headed spines, suggesting a new level of dendritic spine-mediated regulation of synaptic processing, integration and plasticity in cortical PNs. KEY POINTS: BK channels are expressed in the visual cortex and layer 5 pyramidal neuron somata, dendrites and spines regardless of their size. BK channels are selectively activated in small-headed spines (≤0.09 µm3 ), which reduces the amplitude of two-photon (2P) uncaging excitatory postsynaptic potentials (EPSPs) recorded at the soma. Two-photon imaging revealed that intracellular calcium responses in the head of 2P-activated spines are significantly larger in small-headed spines (≤0.09 µm3 ) than in spines with larger head volumes. In accordance with our experimental data, numerical simulations showed that synaptic inputs impinging onto spines with small head volumes (≤0.09 µm3 ) generate voltage responses and calcium signals within the spine head itself that are significantly larger than those observed in spines with larger head volumes, sufficient to activate spine BK channels and suppress EPSPs.

Developing Computational Thinking Teaching Strategies to Model Pandemics and Containment Measures.

COVID-19 has been extremely difficult to control. The lack of understanding of key aspects of pandemics has affected virus transmission. On the other hand, there is a demand to incorporate computational thinking (CT) in the curricula with applications in STEM. However, there are still no exemplars in the curriculum that apply CT to real-world problems such as controlling a pandemic or other similar global crises. In this paper, we fill this gap by proposing exemplars of CT for modeling the pandemic. We designed exemplars following the three pillars of the framework for CT from the Inclusive Mathematics for Sustainability in a Digital Economy (InMside) project by Asia-Pacific Economic Cooperation (APEC): algorithmic thinking, computational modeling, and machine learning. For each pillar, we designed a progressive sequence of activities that covers from elementary to high school. In an experimental study with elementary and middle school students from 2 schools of high vulnerability, we found that the computational modeling exemplar can be implemented by teachers and correctly understood by students. We conclude that it is feasible to introduce the exemplars at all grade levels and that this is a powerful example of Science Technology, Engineering, and Mathematics (STEM) integration that helps reflect and tackle real-world and challenging public health problems of great impact for students and their families.

Automatic Detection of Gaze and Body Orientation in Elementary School Classrooms.

Detecting the direction of the gaze and orientation of the body of both teacher and students is essential to estimate who is paying attention to whom. It also provides vital clues for understanding their unconscious, non-verbal behavior. These are called "honest signals" since they are unconscious subtle patterns in our interaction with other people that help reveal the focus of our attention. Inside the classroom, they provide important clues about teaching practices and students' responses to different conscious and unconscious teaching strategies. Scanning this non-verbal behavior in the classroom can provide important feedback to the teacher in order for them to improve their teaching practices. This type of analysis usually requires sophisticated eye-tracking equipment, motion sensors, or multiple cameras. However, for this to be a useful tool in the teacher's daily practice, an alternative must be found using only a smartphone. A smartphone is the only instrument that a teacher always has at their disposal and is nowadays considered truly ubiquitous. Our study looks at data from a group of first-grade classrooms. We show how video recordings on a teacher's smartphone can be used in order to estimate the direction of the teacher and students' gaze, as well as their body orientation. Using the output from the OpenPose software, we run Machine Learning (ML) algorithms to train an estimator to recognize the direction of the students' gaze and body orientation. We found that the level of accuracy achieved is comparable to that of human observers watching frames from the videos. The mean square errors (RMSE) of the predicted pitch and yaw angles for head and body directions are on average 11% lower than the RMSE between human annotators. However, our solution is much faster, avoids the tedium of doing it manually, and makes it possible to design solutions that give the teacher feedback as soon as they finish the class.

S100ß-mediated astroglial control of firing and input processing in layer 5 pyramidal neurons of the mouse visual cortex.

KEY POINTS: Inputs impinging on layer 5 pyramidal neurons perform essential operations as these cells represent one of the most important output carriers of the cerebral cortex. However, the contribution of astrocytes, a type of glial cell, to these operations is poorly documented. Here we found that optogenetic activation of astrocytes in the vicinity of layer 5 in the mouse primary visual cortex induces spiking in local pyramidal neurons through Nav1.6 ion channels and prolongs the responses elicited in these neurons by stimulation of their distal inputs in cortical layer 1. This effect partially involved glutamatergic signalling but relied mostly on the astrocytic calcium-binding protein S100ß, which regulates the concentration of calcium in the extracellular space around neurons. These findings show that astrocytes contribute to the fundamental computational operations of the cortex by acting on the ionic environment of neurons. ABSTRACT: The most complex cerebral functions are performed by the cortex, whose most important output is carried out by its layer 5 pyramidal neurons. Their firing reflects integration of the sensory and contextual information that they receive. There is evidence that astrocytes influence cortical neuron firing through the release of gliotransmitters such as ATP, glutamate or GABA. These effects have been described at the network and at the synaptic levels, but it is still unclear how astrocytes influence neuron input-output transfer function at the cellular level. Here, we used optogenetic tools coupled with electrophysiological, imaging and anatomical approaches to test whether and how astrocytic activation affected processing of distal inputs to layer 5 pyramidal neurons (L5PNs). We show that optogenetic activation of astrocytes near L5PN cell body prolonged firing induced by distal inputs to L5PNs and potentiated their ability to trigger spikes. The observed astrocytic effects on L5PN firing involved glutamatergic transmission to some extent but relied mostly on release of S100ß, an astrocytic Ca2+ -binding protein that decreases extracellular Ca2+ once released. This astrocyte-evoked decrease in extracellular Ca2+ elicited firing mediated by activation of Nav1.6 channels. Our findings suggest that astrocytes contribute to the cortical fundamental computational operations by controlling the extracellular ionic environment.

A spike-timing-dependent plasticity rule for dendritic spines.

The structural organization of excitatory inputs supporting spike-timing-dependent plasticity (STDP) remains unknown. We performed a spine STDP protocol using two-photon (2P) glutamate uncaging (pre) paired with postsynaptic spikes (post) in layer 5 pyramidal neurons from juvenile mice. Here we report that pre-post pairings that trigger timing-dependent LTP (t-LTP) produce shrinkage of the activated spine neck and increase in synaptic strength; and post-pre pairings that trigger timing-dependent LTD (t-LTD) decrease synaptic strength without affecting spine shape. Furthermore, the induction of t-LTP with 2P glutamate uncaging in clustered spines (<5 µm apart) enhances LTP through a NMDA receptor-mediated spine calcium accumulation and actin polymerization-dependent neck shrinkage, whereas t-LTD was dependent on NMDA receptors and disrupted by the activation of clustered spines but recovered when separated by >40 µm. These results indicate that synaptic cooperativity disrupts t-LTD and extends the temporal window for the induction of t-LTP, leading to STDP only encompassing LTP.

Probing Single Synapses via the Photolytic Release of Neurotransmitters.

The development of two-photon microscopy has revolutionized our understanding of how synapses are formed and how they transform synaptic inputs in dendritic spines-tiny protrusions that cover the dendrites of pyramidal neurons that receive most excitatory synaptic information in the brain. These discoveries have led us to better comprehend the neuronal computations that take place at the level of dendritic spines as well as within neuronal circuits with unprecedented resolution. Here, we describe a method that uses a two-photon (2P) microscope and 2P uncaging of caged neurotransmitters for the activation of single and multiple spines in the dendrites of cortical pyramidal neurons. In addition, we propose a cost-effective description of the components necessary for the construction of a one laser source-2P microscope capable of nearly simultaneous 2P uncaging of neurotransmitters and 2P calcium imaging of the activated spines and nearby dendrites. We provide a brief overview on how the use of these techniques have helped researchers in the last 15 years unravel the function of spines in: (a) information processing; (b) storage; and (c) integration of excitatory synaptic inputs.

Remodeled cortical inhibition prevents motor seizures in generalized epilepsy.

OBJECTIVE: Deletions of CACNA1A, encoding the α1 subunit of CaV 2.1 channels, cause epilepsy with ataxia in humans. Whereas the deletion of Cacna1a in γ-aminobutyric acidergic (GABAergic) interneurons (INs) derived from the medial ganglionic eminence (MGE) impairs cortical inhibition and causes generalized seizures in Nkx2.1Cre ;Cacna1ac/c mice, the targeted deletion of Cacna1a in somatostatin-expressing INs (SOM-INs), a subset of MGE-derived INs, does not result in seizures, indicating a crucial role of parvalbumin-expressing (PV) INs. Here we identify the cellular and network consequences of Cacna1a deletion specifically in PV-INs. METHODS: We generated PVCre ;Cacna1ac/c mutant mice carrying a conditional Cacna1a deletion in PV neurons and evaluated the cortical cellular and network outcomes of this mutation by combining immunohistochemical assays, in vitro electrophysiology, 2-photon imaging, and in vivo video-electroencephalographic recordings. RESULTS: PVCre ;Cacna1ac/c mice display reduced cortical perisomatic inhibition and frequent absences but only rare motor seizures. Compared to Nkx2.1Cre ;Cacna1ac/c mice, PVCre ;Cacna1ac/c mice have a net increase in cortical inhibition, with a gain of dendritic inhibition through sprouting of SOM-IN axons, largely preventing motor seizures. This beneficial compensatory remodeling of cortical GABAergic innervation is mTORC1-dependent and its inhibition with rapamycin leads to a striking increase in motor seizures. Furthermore, we show that a direct chemogenic activation of cortical SOM-INs prevents motor seizures in a model of kainate-induced seizures. INTERPRETATION: Our findings provide novel evidence suggesting that the remodeling of cortical inhibition, with an mTOR-dependent gain of dendritic inhibition, determines the seizure phenotype in generalized epilepsy and that mTOR inhibition can be detrimental in epilepsies not primarily due to mTOR hyperactivation. Ann Neurol 2018;84:436-451.

Evolution of dopamine receptors: phylogenetic evidence suggests a later origin of the DRD2l and DRD4rs dopamine receptor gene lineages.

Dopamine receptors are integral membrane proteins whose endogenous ligand is dopamine. They play a fundamental role in the central nervous system and dysfunction of dopaminergic neurotransmission is responsible for the generation of a variety of neuropsychiatric disorders. From an evolutionary standpoint, phylogenetic relationships among the DRD1 class of dopamine receptors are still a matter of debate as in the literature different tree topologies have been proposed. In contrast, phylogenetic relationships among the DRD 2 group of receptors are well understood. Understanding the time of origin of the different dopamine receptors is also an issue that needs further study, especially for the genes that have restricted phyletic distributions (e.g., DRD2l and DRD4rs). Thus, the goal of this study was to investigate the evolution of dopamine receptors, with emphasis on shedding light on the phylogenetic relationships among the D1 class of dopamine receptors and the time of origin of the DRD2l and DRD4rs gene lineages. Our results recovered the monophyly of the two groups of dopamine receptors. Within the DRD1 group the monophyly of each paralog was recovered with strong support, and phylogenetic relationships among them were well resolved. Within the DRD1 class of dopamine receptors we recovered the sister group relationship between the DRD1C and DRD1E, and this clade was recovered sister to a cyclostome sequence. The DRD1 clade was recovered sister to the aforementioned clade, and the group containing DRD5 receptors was sister to all other DRD1 paralogs. In agreement with the literature, among the DRD2 class of receptors, DRD2 was recovered sister to DRD3, whereas DRD4 was sister to the DRD2/DRD3 clade. According to our phylogenetic tree, the DRD2l and DRD4rs gene lineages would have originated in the ancestor of gnathostomes between 615 and 473 mya. Conservation of sequences required for dopaminergic neurotransmission and small changes in regulatory regions suggest a functional refinement of the dopaminergic pathways along evolution.

NOVA2-mediated RNA regulation is required for axonal pathfinding during development.

The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators. NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2. Transcriptome-wide searches for isoform-specific functions, using NOVA1 and NOVA2 specific HITS-CLIP and RNA-seq data from mouse cortex lacking either NOVA isoform, reveals that NOVA2 uniquely regulates alternative splicing events of a series of axon guidance related genes during cortical development. Corresponding axonal pathfinding defects were specific to NOVA2 deficiency: Nova2-/- but not Nova1-/- mice had agenesis of the corpus callosum, and axonal outgrowth defects specific to ventral motoneuron axons and efferent innervation of the cochlea. Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo.

Input transformation by dendritic spines of pyramidal neurons.

In the mammalian brain, most inputs received by a neuron are formed on the dendritic tree. In the neocortex, the dendrites of pyramidal neurons are covered by thousands of tiny protrusions known as dendritic spines, which are the major recipient sites for excitatory synaptic information in the brain. Their peculiar morphology, with a small head connected to the dendritic shaft by a slender neck, has inspired decades of theoretical and more recently experimental work in an attempt to understand how excitatory synaptic inputs are processed, stored and integrated in pyramidal neurons. Advances in electrophysiological, optical and genetic tools are now enabling us to unravel the biophysical and molecular mechanisms controlling spine function in health and disease. Here I highlight relevant findings, challenges and hypotheses on spine function, with an emphasis on the electrical properties of spines and on how these affect the storage and integration of excitatory synaptic inputs in pyramidal neurons. In an attempt to make sense of the published data, I propose that the raison d'etre for dendritic spines lies in their ability to undergo activity-dependent structural and molecular changes that can modify synaptic strength, and hence alter the gain of the linearly integrated sub-threshold depolarizations in pyramidal neuron dendrites before the generation of a dendritic spike.

Activity-dependent dendritic spine neck changes are correlated with synaptic strength.

Most excitatory inputs in the mammalian brain are made on dendritic spines, rather than on dendritic shafts. Spines compartmentalize calcium, and this biochemical isolation can underlie input-specific synaptic plasticity, providing a raison d'etre for spines. However, recent results indicate that the spine can experience a membrane potential different from that in the parent dendrite, as though the spine neck electrically isolated the spine. Here we use two-photon calcium imaging of mouse neocortical pyramidal neurons to analyze the correlation between the morphologies of spines activated under minimal synaptic stimulation and the excitatory postsynaptic potentials they generate. We find that excitatory postsynaptic potential amplitudes are inversely correlated with spine neck lengths. Furthermore, a spike timing-dependent plasticity protocol, in which two-photon glutamate uncaging over a spine is paired with postsynaptic spikes, produces rapid shrinkage of the spine neck and concomitant increases in the amplitude of the evoked spine potentials. Using numerical simulations, we explore the parameter regimes for the spine neck resistance and synaptic conductance changes necessary to explain our observations. Our data, directly correlating synaptic and morphological plasticity, imply that long-necked spines have small or negligible somatic voltage contributions, but that, upon synaptic stimulation paired with postsynaptic activity, they can shorten their necks and increase synaptic efficacy, thus changing the input/output gain of pyramidal neurons.

Spatial light modulator microscopy.

The use of spatial light modulators (SLMs) for two-photon laser microscopy is described. SLM phase modulation can be used to generate nearly any spatiotemporal pattern of light, enabling simultaneous illumination of any number of selected regions of interest. We take advantage of this flexibility to perform fast two-photon imaging or uncaging experiments on dendritic spines and neocortical neurons. By operating in the spatial Fourier plane, an SLM can effectively mimic any arbitrary optical transfer function and thus replace, in software, many of the functions provided by hardware in standard microscopes, such as focusing, magnification, and aberration correction.

Two-photon optical interrogation of individual dendritic spines with caged dopamine.

We introduce a novel caged dopamine compound (RuBi-Dopa) based on ruthenium photochemistry. RuBi-Dopa has a high uncaging efficiency and can be released with visible (blue-green) and IR light in a two-photon regime. We combine two-photon photorelease of RuBi-Dopa with two-photon calcium imaging for an optical imaging and manipulation of dendritic spines in living brain slices, demonstrating that spines can express functional dopamine receptors. This novel compound allows mapping of functional dopamine receptors in living brain tissue with exquisite spatial resolution.

Two-photon microscopy with diffractive optical elements and spatial light modulators.

Two-photon microscopy is often performed at slow frame rates due to the need to serially scan all points in a field of view with a single laser beam. To overcome this problem, we have developed two optical methods that split and multiplex a laser beam across the sample. In the first method a diffractive optical element (DOE) generates a fixed number of beamlets that are scanned in parallel resulting in a corresponding increase in speed or in signal-to-noise ratio in time-lapse measurements. The second method uses a computer-controlled spatial light modulator (SLM) to generate any arbitrary spatio-temporal light pattern. With an SLM one can image or photostimulate any predefined region of the image such as neurons or dendritic spines. In addition, SLMs can be used to mimic a large number of optical transfer functions including light path corrections as adaptive optics.

RuBi-Glutamate: Two-Photon and Visible-Light Photoactivation of Neurons and Dendritic spines.

We describe neurobiological applications of RuBi-Glutamate, a novel caged-glutamate compound based on ruthenium photochemistry. RuBi-Glutamate can be excited with visible wavelengths and releases glutamate after one- or two-photon excitation. It has high quantum efficiency and can be used at low concentrations, partly avoiding the blockade of GABAergic transmission present with other caged compounds. Two-photon uncaging of RuBi-Glutamate has a high spatial resolution and generates excitatory responses in individual dendritic spines with physiological kinetics. With laser beam multiplexing, two-photon RuBi-Glutamate uncaging can also be used to depolarize and fire pyramidal neurons with single-cell resolution. RuBi-Glutamate therefore enables the photoactivation of neuronal dendrites and circuits with visible or two-photon light sources, achieving single cell, or even single spine, precision.

SLM Microscopy: Scanless Two-Photon Imaging and Photostimulation with Spatial Light Modulators.

Laser microscopy has generally poor temporal resolution, caused by the serial scanning of each pixel. This is a significant problem for imaging or optically manipulating neural circuits, since neuronal activity is fast. To help surmount this limitation, we have developed a "scanless" microscope that does not contain mechanically moving parts. This microscope uses a diffractive spatial light modulator (SLM) to shape an incoming two-photon laser beam into any arbitrary light pattern. This allows the simultaneous imaging or photostimulation of different regions of a sample with three-dimensional precision. To demonstrate the usefulness of this microscope, we perform two-photon uncaging of glutamate to activate dendritic spines and cortical neurons in brain slices. We also use it to carry out fast (60 Hz) two-photon calcium imaging of action potentials in neuronal populations. Thus, SLM microscopy appears to be a powerful tool for imaging and optically manipulating neurons and neuronal circuits. Moreover, the use of SLMs expands the flexibility of laser microscopy, as it can substitute traditional simple fixed lenses with any calculated lens function.

Sodium channels amplify spine potentials.

Dendritic spines mediate most excitatory synapses in the brain. Past theoretical work and recent experimental evidence have suggested that spines could contain sodium channels. We tested this by measuring the effect of the sodium channel blocker tetrodotoxin (TTX) on depolarizations generated by two-photon uncaging of glutamate on spines from mouse neocortical pyramidal neurons. In practically all spines examined, uncaging potentials were significantly reduced by TTX. This effect was postsynaptic and spatially localized to the spine and occurred with uncaging potentials of different amplitudes and in spines of different neck lengths. Our data confirm that spines from neocortical pyramidal neurons are electrically isolated from the dendrite and indicate that they have sodium channels and are therefore excitable structures. Spine sodium channels could boost synaptic potentials and facilitate action potential backpropagation.

Injury of skeletal muscle and specific cytokines induce the expression of gap junction channels in mouse dendritic cells.

Dendritic cells (DCs) in culture express at least connexin43, a protein subunit of gap junctions, and form gap junction channels, which could be important for T-cells activation. Here, we evaluated whether DCs express connexins in vivo and also to identify components of their microenvironment that regulate the functional expression of gap junctions. In vivo studies were performed in lymph nodes of mice under control conditions or after skeletal muscle damage. In double immunolabeling studies, connexin45 was frequently detected in DEC205(+) DCs in lymph nodes of control animals, whereas connexin43 was rarely found in DCs. However, connexin43 was upregulated in DCs after skeletal muscle damage. Upregulation of connexin43 gene expression by tissue damage was also confirmed in mice carrying a beta-galactosidase reporter gene in a connexin43 allele. The effect of several cytokines on the expression of functional gap junctions between cultured DCs was also tested. Under control conditions, cultured DCs did not communicate via gap junctions. However, after treatment with keratinocyte-conditioned medium or cytokine mixtures containing at least TNF-alpha and IL-1beta, they became transiently coupled through a pathway sensitive to octanol, a gap junction blocker. Cellular coupling induced by effective cytokine mixtures was prevented by IL-6. Single cytokines (TNF-alpha, IL-1beta, IFN-gamma, or IL-6) or other mixtures than the described above did not induce coupling via gap junctions. Increased levels of connexin43 and connexin45 protein and mRNA accompanied the appearance of cellular coupling. These studies provide demonstration of connexin expression and regulation by specific danger signals in DCs.