Combined cellomics and proteomics analysis reveals shared neuronal morphology and molecular pathway phenotypes for multiple schizophrenia risk genes.

An enigma in studies of neuropsychiatric disorders is how to translate polygenic risk into disease biology. For schizophrenia, where > 145 significant GWAS loci have been identified and only a few genes directly implicated, addressing this issue is a particular challenge. We used a combined cellomics and proteomics approach to show that polygenic risk can be disentangled by searching for shared neuronal morphology and cellular pathway phenotypes of candidate schizophrenia risk genes. We first performed an automated high-content cellular screen to characterize neuronal morphology phenotypes of 41 candidate schizophrenia risk genes. The transcription factors Tcf4 and Tbr1 and the RNA topoisomerase Top3b shared a neuronal phenotype marked by an early and progressive reduction in synapse numbers upon knockdown in mouse primary neuronal cultures. Proteomics analysis subsequently showed that these three genes converge onto the syntaxin-mediated neurotransmitter release pathway, which was previously implicated in schizophrenia, but for which genetic evidence was weak. We show that dysregulation of multiple proteins in this pathway may be due to the combined effects of schizophrenia risk genes Tcf4, Tbr1, and Top3b. Together, our data provide new biological functions for schizophrenia risk genes and support the idea that polygenic risk is the result of multiple small impacts on common neuronal signaling pathways.

Learning the association between a context and a target location in infancy.

Extracting the statistical regularities present in the environment is a central learning mechanism in infancy. For instance, infants are able to learn the associations between simultaneously or successively presented visual objects (Fiser & Aslin, ; Kirkham, Slemmer & Johnson, ). The present study extends these results by investigating whether infants can learn the association between a target location and the context in which it is presented. With this aim, we used a visual associative learning procedure inspired by the contextual cuing paradigm, with infants from 8 to 12 months of age. In two experiments, in which we varied the complexity of the stimuli, we first habituated infants to several scenes where the location of a target (a cartoon character) was consistently associated with a context, namely a specific configuration of geometrical shapes. Second, we examined whether infants learned the covariation between the target location and the context by measuring looking times at scenes that either respected or violated the association. In both experiments, results showed that infants learned the target-context associations, as they looked longer at the familiar scenes than at the novel ones. In particular, infants selected clusters of co-occurring contextual shapes and learned the covariation between the target location and this subset. These results support the existence of a powerful and versatile statistical learning mechanism that may influence the orientation of infants' visual attention toward areas of interest in their environment during early developmental stages. A video abstract of this article can be viewed at: https://www.youtube.com/watch?v=9Hm1unyLBn0.

Why try saving the ANS? An alternative proposal.

Leibovich et al. propose that continuous magnitudes and a number sense are used holistically to judge numerosity. We point out that their proposal is incomplete and implausible: incomplete, as it does not explain how continuous magnitudes are calculated; implausible, as it cannot explain performance in estimation tasks. We propose that we do not possess a number sense. Instead, we assume that numerosity judgments are accomplished by weighing the different continuous magnitudes constituting numerosity.

Image Informatics Strategies for Deciphering Neuronal Network Connectivity.

Brain function relies on an intricate network of highly dynamic neuronal connections that rewires dramatically under the impulse of various external cues and pathological conditions. Amongst the neuronal structures that show morphological plasticity are neurites, synapses, dendritic spines and even nuclei. This structural remodelling is directly connected with functional changes such as intercellular communication and the associated calcium bursting behaviour. In vitro cultured neuronal networks are valuable models for studying these morpho-functional changes. Owing to the automation and standardization of both image acquisition and image analysis, it has become possible to extract statistically relevant readouts from such networks. Here, we focus on the current state-of-the-art in image informatics that enables quantitative microscopic interrogation of neuronal networks. We describe the major correlates of neuronal connectivity and present workflows for analysing them. Finally, we provide an outlook on the challenges that remain to be addressed, and discuss how imaging algorithms can be extended beyond in vitro imaging studies.

The neural mechanism underlying ordinal numerosity processing.

Changes in the sensory properties of numerosity stimuli have a direct effect on the outcomes of nonsymbolic number tasks. This suggests a prominent role of sensory properties in numerosity processing. However, the current consensus holds that numerosity is processed independent of its sensory properties. To investigate the role of sensory cues in ordinal number processes, we manipulated both dimensions orthogonally. Participants passively viewed the stimuli while their brain activity was measured using EEG. The results revealed an interaction between numerosity and its sensory properties in the absence of main effects. Different neural responses were present for trials where numerosity and sensory cues changed in the same direction compared with trials where they changed in opposite directions. These results show that the sensory cues are expected to change in concert with numerosity and support the notion that the visual cues are taken into account when judging numerosity.

The neural mechanisms underlying passive and active processing of numerosity.

To investigate the difference in passive viewing and active processing of numerosity, we presented participants arrays of dots and concurrently measured their EEG. In the first condition, participants naïve to the subject under study passively viewed the dot-arrays. In the second condition, the participants were informed about the changes in numerosity and had to actively process numerosity. The visual properties of the dot-arrays were controlled and could therefore not explain possible numerosity related effects. The results revealed no numerosity related effects in the passive and active conditions. Instead, when the data was reorganised according to visual cue size (surface or diameter, etc.), strong effects of the visual cues were present at lateral occipital and parietal electrode sites. These electrode sites and time windows correspond to the ERP components often suggested to support numerosity processes. Furthermore, a larger central-parietal P3 amplitude effect was present for active versus passive numerosity processing. This result was not influenced by numerosity itself and could not be explained by response processing. It therefore appears to reflect general cognitive processes. Together, our results show that we do not (automatically) extract numerosity from a visual scene during passive or active processing of numerosity. Instead, these results are consistent with the notion that we rely on the continuous sensory properties of numerosity stimuli to make numerosity judgments.

Task- and age-dependent effects of visual stimulus properties on children's explicit numerosity judgments.

Researchers investigating numerosity processing manipulate the visual stimulus properties (e.g., surface). This is done to control for the confound between numerosity and its visual properties and should allow the examination of pure number processes. Nevertheless, several studies have shown that, despite different visual controls, visual cues remained to exert their influence on numerosity judgments. This study, therefore, investigated whether the impact of the visual stimulus manipulations on numerosity judgments is dependent on the task at hand (comparison task vs. same-different task) and whether this impact changes throughout development. In addition, we examined whether the influence of visual stimulus manipulations on numerosity judgments plays a role in the relation between performance on numerosity tasks and mathematics achievement. Our findings confirmed that the visual stimulus manipulations affect numerosity judgments; more important, we found that these influences changed with increasing age and differed between the comparison and the same-different tasks. Consequently, direct comparisons between numerosity studies using different tasks and age groups are difficult. No meaningful relationship between the performance on the comparison and same-different tasks and mathematics achievement was found in typically developing children, nor did we find consistent differences between children with and without mathematical learning disability (MLD).

Thinking about touch facilitates tactile but not auditory processing.

Mental imagery is considered to be important for normal conscious experience. It is most frequently investigated in the visual, auditory and motor domain (imagination of movement), while the studies on tactile imagery (imagination of touch) are scarce. The current study investigated the effect of tactile and auditory imagery on the left/right discriminations of tactile and auditory stimuli. In line with our hypothesis, we observed that after tactile imagery, tactile stimuli were responded to faster as compared to auditory stimuli and vice versa. On average, tactile stimuli were responded to faster as compared to auditory stimuli, and stimuli in the imagery condition were on average responded to slower as compared to baseline performance (left/right discrimination without imagery assignment). The former is probably due to the spatial and somatotopic proximity of the fingers receiving the taps and the thumbs performing the response (button press), the latter to a dual task cost. Together, these results provide the first evidence of a behavioural effect of a tactile imagery assignment on the perception of real tactile stimuli.

Children's representation of symbolic and nonsymbolic magnitude examined with the priming paradigm.

How people process and represent magnitude has often been studied using number comparison tasks. From the results of these tasks, a comparison distance effect (CDE) is generated, showing that it is easier to discriminate two numbers that are numerically further apart (e.g., 2 and 8) compared with numerically closer numbers (e.g., 6 and 8). However, it has been suggested that the CDE reflects decisional processes rather than magnitude representation. In this study, therefore, we investigated the development of symbolic and nonsymbolic number processes in kindergartners and first, second, and sixth graders using the priming paradigm. This task has been shown to measure magnitude and not decisional processes. Our findings revealed that a priming distance effect (PDE) is already present in kindergartners and that it remains stable across development. This suggests that formal schooling does not affect magnitude representation. No differences were found between the symbolic and nonsymbolic PDE, indicating that both notations are processed with comparable precision. Finally, a poorer performance on a standardized mathematics test seemed to be associated with a smaller PDE for both notations, possibly suggesting that children with lower mathematics scores have a less precise coding of magnitude. This supports the defective number module hypothesis, which assumes an impairment of number sense.

Generating nonsymbolic number stimuli.

Studies investigating nonsymbolic numbers (e.g., dot arrays) are confronted with the problem that changes in numerosity are always accompanied by changes in the visual properties of the stimulus. It is therefore debated whether the visual properties of the stimulus rather than number can explain the results obtained in studies investigating nonsymbolic number processing. In this report, we present a program (available at http://titiagebuis.eu/Materials.html ; note that the program is designed to work with the Psychophysics Toolbox in MATLAB) that exports information about the visual properties of stimuli that co-vary with number (area extended, item size, total surface, density, and circumference). Consequently, insight into the relation between the visual properties of the stimulus and numerical distance can be achieved, and post hoc analyses can be conducted to directly reveal whether numerical distance or (some combinations of) the visual properties of a stimulus could be the most likely candidate underlying the results. Here, we report data that demonstrate the program's usefulness for research on nonsymbolic number stimuli.

Multiple dimensions in bi-directional synesthesia.

Grapheme-color synesthetes report seeing a specific color when a number is perceived. The reverse, the synesthetic experience of a specific grapheme after the percept of a color is extremely rare. However, recent studies have revealed these interactions at both behavioral and neurophysiological levels. We investigated whether similar neuronal processes (i.e. perceptual and/or attentional) may underlie this bi-directional interaction by measuring event-related potentials (ERPs) during both a number-color and color-number priming task. In addition, we investigated the unitarity of synesthesia by comparing two distinct subtypes of synesthetes, projectors and associators, and assessed whether consistencies between measures (i.e. behavioral and electrophysiological) were present across synesthetes. Our results show longer reaction times for incongruent compared with congruent trials in both tasks. This priming effect is also present in the P3b latency (parietal electrode site) and P3a amplitude (frontal electrode site) of the ERP data. Interestingly, projector and associator synesthetes did not reveal distinct behavioral or electrophysiological patterns. Instead, a dissociation was found when synesthetes were divided in two groups on the basis of their behavioral data. Synesthetes with a large behavioral priming effect revealed ERP modulation at the frontal and parietal electrode sites, whereas synesthetes with a small priming effect revealed a frontal effect only. Together, these results show, for the first time, that similar neural mechanisms underlie bi-directional synesthesia in synesthetes that do not report a synesthetic experience of a grapheme when a color is perceived. In addition, they add support for the notion of the existence of both 'lower' and 'higher' synesthetes.

The development of automated access to symbolic and non-symbolic number knowledge in children: an ERP study.

Infants can visually detect changes in numerosity, which suggests that a (non-symbolic) numerosity system is already present early in life. This non-symbolic system is hypothesized to serve as the basis for the later acquired symbolic system. Little is known about the processes underlying the transition from the non-symbolic to symbolic code. In the current study we investigated the development of automatization of symbolic number processing in children from second (6.0 years) and fourth grade (8.0 years) and adults using a symbolic and non-symbolic size congruency task and event-related potentials (ERPs) as a measure. The comparison between symbolic and non-symbolic size congruency effects (SCEs) allowed us to disentangle processes necessary to perform the task from processes specific to numerosity notation. In contrast to previous studies, second graders already revealed a behavioral symbolic SCE similar to that of adults. In addition, the behavioral SCE increased for symbolic and decreased for non-symbolic notation with increasing age. For all age groups, the ERP data showed that the two magnitudes interfered at a level before selective activation of the response system, for both notations. However, only for the second graders distinct processes were recruited to perform the symbolic size comparison task. This shift in recruited processes for the symbolic task only might reflect the functional specialization of the parietal cortex.

Automatic quantity processing in 5-year olds and adults.

In this study adults performed numerical and physical size judgments on a symbolic (Arabic numerals) and non-symbolic (groups of dots) size congruity task. The outcomes would reveal whether a size congruity effect (SCE) can be obtained irrespective of notation. Subsequently, 5-year-old children performed a physical size judgment on both tasks. The outcomes will give a better insight in the ability of 5-year-olds to automatically process symbolic and non-symbolic numerosities. Adult performance on the symbolic and non-symbolic size congruity tasks revealed a SCE for numerical and physical size judgments, indicating that the non-symbolic size congruity task is a valid indicator for automatic processing of non-symbolic numerosities. Physical size judgments on both tasks by children revealed a SCE only for non-symbolic notation, indicating that the lack of a symbolic SCE is not related to the mathematical or cognitive abilities required for the task but instead to an immature association between the number symbol and its meaning.

Memory strength gates the involvement of a CREB-dependent cortical fear engram in remote memory.

Encoding and retrieval of contextual memories is initially mediated by sparsely activated neurons, so-called engram cells, in the hippocampus. Subsequent memory persistence is thought to depend on network-wide changes involving progressive contribution of cortical regions, a process referred to as systems consolidation. Using a viral-based TRAP (targeted recombination in activated populations) approach, we studied whether consolidation of contextual fear memory by neurons in the medial prefrontal cortex (mPFC) is modulated by memory strength and CREB function. We demonstrate that activity of a small subset of mPFC neurons is sufficient and necessary for remote memory expression, but their involvement depends on the strength of conditioning. Furthermore, selective disruption of CREB function in mPFC engram cells after mild conditioning impairs remote memory expression. Together, our data demonstrate that memory consolidation by mPFC engram cells requires CREB-mediated transcription, with the functionality of this network hub being gated by memory strength.

Gpr158 Deficiency Impacts Hippocampal CA1 Neuronal Excitability, Dendritic Architecture, and Affects Spatial Learning.

G-protein-coupled receptor 158 (Gpr158) is highly expressed in striatum, hippocampus and prefrontal cortex. It gained attention as it was implicated in physiological responses to stress and depression. Recently, Gpr158 has been shown to act as a pathway-specific synaptic organizer in the hippocampus, required for proper mossy fiber-CA3 neurocircuitry establishment, structure, and function. Although rodent Gpr158 expression is highest in CA3, considerable expression occurs in CA1 especially after the first postnatal month. Here, we combined hippocampal-dependent behavioral paradigms with subsequent electrophysiological and morphological analyses from the same group of mice to assess the effects of Gpr158 deficiency on CA1 physiology and function. We demonstrate deficits in spatial memory acquisition and retrieval in the Morris water maze paradigm, along with deficits in the acquisition of extinction memory in the passive avoidance test in Gpr158 KO mice. Electrophysiological recordings from CA1 pyramidal neurons revealed normal basal excitatory and inhibitory synaptic transmission, however, Schaffer collateral stimulation yielded dramatically reduced post-synaptic currents. Interestingly, intrinsic excitability of CA1 pyramidals was found increased, potentially acting as a compensatory mechanism to the reductions in Schaffer collateral-mediated drive. Both ex vivo and in vitro, neurons deficient for or with lowered levels of Gpr158 exhibited robust reductions in dendritic architecture and complexity, i.e., reduced length, surface, bifurcations, and branching. This effect was localized in the apical but not basal dendrites of adult CA1 pyramidals, indicative of compartment-specific alterations. A significant positive correlation between spatial memory acquisition and extent of complexity of CA1 pyramidals was found. Taken together, we provide first evidence of significant disruptions in hippocampal CA1 neuronal dendritic architecture and physiology, driven by Gpr158 deficiency. Importantly, the hippocampal neuronal morphology deficits appear to support the impairments in spatial memory acquisition observed in Gpr158 KO mice.

Order information in verbal working memory shifts the subjective midpoint in both the line bisection and the landmark tasks.

A largely substantiated view in the domain of working memory is that the maintenance of serial order is achieved by generating associations of each item with an independent representation of its position, so-called position markers. Recent studies reported that the ordinal position of an item in verbal working memory interacts with spatial processing. This suggests that position markers might be spatial in nature. However, these interactions were so far observed in tasks implying a clear binary categorization of space (i.e., with left and right responses or targets). Such binary categorizations leave room for alternative interpretations, such as congruency between non-spatial categorical codes for ordinal position (e.g., begin and end) and spatial categorical codes for response (e.g., left and right). Here we discard this interpretation by providing evidence that this interaction can also be observed in a task that draws upon a continuous processing of space, the line bisection task. Specifically, bisections are modulated by ordinal position in verbal working memory, with lines bisected more towards the right after retrieving items from the end compared to the beginning of the memorized sequence. This supports the idea that position markers are intrinsically spatial in nature.

Sensory-integration system rather than approximate number system underlies numerosity processing: A critical review.

It is widely accepted that human and nonhuman species possess a specialized system to process large approximate numerosities. The theory of an evolutionarily ancient approximate number system (ANS) has received converging support from developmental studies, comparative experiments, neuroimaging, and computational modelling, and it is one of the most dominant and influential theories in numerical cognition. The existence of an ANS system is significant, as it is believed to be the building block of numerical development in general. The acuity of the ANS is related to future arithmetic achievements, and intervention strategies therefore aim to improve the ANS. Here we critically review current evidence supporting the existence of an ANS. We show that important shortcomings and confounds exist in the empirical studies on human and non-human animals as well as the logic used to build computational models that support the ANS theory. We conclude that rather than taking the ANS theory for granted, a more comprehensive explanation might be provided by a sensory-integration system that compares or estimates large approximate numerosities by integrating the different sensory cues comprising number stimuli.

Dynamics of the mouse brain cortical synaptic proteome during postnatal brain development.

Development of the brain involves the formation and maturation of numerous synapses. This process requires prominent changes of the synaptic proteome and potentially involves thousands of different proteins at every synapse. To date the proteome analysis of synapse development has been studied sparsely. Here, we analyzed the cortical synaptic membrane proteome of juvenile postnatal days 9 (P9), P15, P21, P27, adolescent (P35) and different adult ages P70, P140 and P280 of C57Bl6/J mice. Using a quantitative proteomics workflow we quantified 1560 proteins of which 696 showed statistically significant differences over time. Synaptic proteins generally showed increased levels during maturation, whereas proteins involved in protein synthesis generally decreased in abundance. In several cases, proteins from a single functional molecular entity, e.g., subunits of the NMDA receptor, showed differences in their temporal regulation, which may reflect specific synaptic development features of connectivity, strength and plasticity. SNARE proteins, Snap 29/47 and Stx 7/8/12, showed higher expression in immature animals. Finally, we evaluated the function of Cxadr that showed high expression levels at P9 and a fast decline in expression during neuronal development. Knock down of the expression of Cxadr in cultured primary mouse neurons revealed a significant decrease in synapse density.

Topographic representation of high-level cognition: numerosity or sensory processing?

A recent study showed that topographic field maps of complex cognitive functions, such as numerosity, exist in the human brain. This is an exciting, novel approach for studying the neural representation of high-level cognition. However, the results can also be explained on the basis of the confounding non-numerical sensory cues of numerosity.

Concurrent validity of approximate number sense tasks in adults and children.

Reasoning with non-symbolic numerosities is suggested to be rooted in the Approximate Number System (ANS) and evidence pointing to a relationship between the acuity of this system and mathematics is available. In order to use the acuity of this ANS as a screening instrument to detect future math problems, it is important to model ANS acuity over development. However, whether ANS acuity and its development have been described accurately can be questioned. Namely, different tasks were used to examine the developmental trajectory of ANS acuity and studies comparing performances on these different tasks are scarce. In the present study, we examined whether different tasks designed to measure the acuity of the ANS are comparable and lead to related ANS acuity measures (i.e., the concurrent validity of these tasks). We contrasted the change detection task, which is used in infants, with tasks that are more commonly used in older children and adults (i.e., comparison and same-different tasks). Together, our results suggest that ANS acuity measures obtained with different tasks are not related. This poses serious problems for the comparison of ANS acuity measures derived from different tasks and thus for the establishment of the developmental trajectory of ANS acuity.