Dyrk1A influences neuronal morphogenesis through regulation of cytoskeletal dynamics in mammalian cortical neurons.

Down syndrome (DS) is the most frequent genetic cause of mental retardation. Cognitive dysfunction in these patients is correlated with reduced dendritic branching and complexity, along with fewer spines of abnormal shape that characterize the cortical neuronal profile of DS. DS phenotypes are caused by the disruptive effect of specific trisomic genes. Here, we report that overexpression of dual-specificity tyrosine phosphorylation-regulated kinase 1A, DYRK1A, is sufficient to produce the dendritic alterations observed in DS patients. Engineered changes in Dyrk1A gene dosage in vivo strongly alter the postnatal dendritic arborization processes with a similar progression than in humans. In cultured mammalian cortical neurons, we determined a reduction of neurite outgrowth and synaptogenesis. The mechanism underlying neurite dysgenesia involves changes in the dynamic reorganization of the cytoskeleton.

Attentional set-shifting in fragile X syndrome.

The ability to flexibly adapt to the changing demands of the environment is often reported as a core deficit in fragile X syndrome (FXS). However, the cognitive processes that determine this attentional set-shifting deficit remain elusive. The present study investigated attentional set-shifting ability in fragile X syndrome males with the well-validated intra/extra dimensional set-shifting paradigm (IED) which offers detailed assessment of rule learning, reversal learning, and attentional set-shifting ability within and between stimulus dimensions. A novel scoring method for IED stage errors was employed to interpret set-shifting failure in terms of repetitive decision-making, distraction to irrelevance, and set-maintenance failure. Performance of FXS males was compared to typically developing children matched on mental age, adults matched on chronological age, and individuals with Down syndrome matched on both mental and chronological age. Results revealed that a significant proportion of FXS males already failed prior to the intra-dimensional set-shift stage, whereas all control participants successfully completed the stages up to the crucial extra-dimensional set-shift. FXS males showed a specific weakness in reversal learning, which was characterized by repetitive decision-making during the reversal of newly acquired stimulus-response associations in the face of simple stimulus configurations. In contrast, when stimulus configurations became more complex, FXS males displayed increased distraction to irrelevant stimuli. These findings are interpreted in terms of the cognitive demands imposed by the stages of the IED in relation to the alleged neural deficits in FXS.

Conditional firing probabilities in cultured neuronal networks: a stable underlying structure in widely varying spontaneous activity patterns.

To properly observe induced connectivity changes after training sessions, one needs a network model that describes individual relationships in sufficient detail to enable observation of induced changes and yet reveals some kind of stability in these relationships. We analyzed spontaneous firing activity in dissociated rat cortical networks cultured on multi-electrode arrays by means of the conditional firing probability. For all pairs (i, j) of the 60 electrodes, we calculated conditional firing probability (CFP(i,j)[tau]) as the probability of an action potential at electrode j at t = tau, given that one was detected at electrode i at t = 0. If a CFP(i,j)[tau] distribution clearly deviated from a flat one, electrodes i and j were considered to be related. For all related electrode pairs, a function was fitted to the CFP-curve to obtain parameters for 'strength' and 'delay' (i.e. maximum and latency of the maximum of the curve) of each relationship. In young cultures the set of identified relationships changed rather quickly. At 16 days in vitro (DIV) 50% of the set changed within 2 days. Beyond 25 DIV this set stabilized: during a week more than 50% of the set remained intact. Most individual relationships developed rather gradually. Moreover, beyond 25 DIV relational strength appeared quite stable, with coefficients of variation (100 x SD/mean) around 25% in periods of approximately 10 h. CFP analysis provides a robust method to describe the underlying probabilistic structure of highly varying spontaneous activity in cultured cortical networks. It may offer a suitable basis for plasticity studies, in the case of changes in the probabilistic structure. CFP analysis monitors all pairs of electrodes instead of just a selected one. Still, it is likely to describe the network in sufficient detail to detect subtle changes in individual relationships.

Dendritic pathology in mental retardation: from molecular genetics to neurobiology.

Mental retardation (MR) is a developmental brain disorder characterized by impaired cognitive performance and adaptive skills that affects 1-2% of the population. During the last decade, a large number of genes have been cloned that cause MR upon mutation in humans. The causal role of these genes provides an excellent starting point to investigate the cellular, neurobiological and behavioral alterations and mechanisms responsible for the cognitive impairment in mentally retarded persons. However, studies on Down syndrome (DS) reveal that overexpression of a cluster of genes and various forms of MR that are caused by single-gene mutations, such as fragile X (FraX), Rett, Coffin-Lowry, Rubinstein-Taybi syndrome and non-syndromic forms of MR, causes similar phenotypes. In spite of the many differences in the manifestation of these forms of MR, evidence converges on the proposal that MR is primarily due to deficiencies in neuronal network connectivity in the major cognitive centers in the brain, which secondarily results in impaired information processing. Although MR has been largely regarded as a brain disorder that cannot be cured, our increased understanding of the abnormalities and mechanisms underlying MR may provide an avenue for the development of therapies for MR. In this review, we discuss the neurobiology underlying MR, with a focus on FraX and DS.

Chronic Suppression of Bioelectric Activity and Cell Survival in Primary Cultures of Rat Cerebral Cortex: Biochemical Observations.

Chronic suppression of spontaneously occurring bioelectric activity (BEA) has been shown to increase neuronal cell death in tissue culture, but may also affect astrocytes. We investigated this process in primary cultures of rat cerebral cortex by measuring the levels of NSE (neuron-specific enolase) and GFAP (glial fibrillary acidic protein) in relation to general tissue markers, including measurements for cell death and proliferation. In electrically active (control) cultures, the content of DNA, protein, and NSE became maximal between 21 and 28 days in vitro (DIV) and thereafter decreased, whereas the content of GFAP rose continuously up to 43 DIV. Chronic suppression of BEA by tetrodotoxin (TTX; from 6 DIV) decreased the content of DNA, total protein, and especially NSE. The content of GFAP was decreased in all culture series investigated, but with great temporal variations among culture series. Chronic TTX treatment (started at 6 DIV) increased the efflux of lactate dehydrogenase, a marker for cell lysis, between 12 and 21 DIV, but this efflux was mainly derived from the supporting glial cells with which the cerebral cortex cultures were cocultured. Chronic, but not acute (7 h) TTX treatment decreased total [3H]thymidine incorporation into DNA from 14 DIV; this appeared to be due to a reduced number of astrocytes. Chronic suppression of BEA with xylocaine from 6 DIV had similar effects on DNA-, protein-, and NSE-content as TTX, but led to an increased content of GFAP at 21 DIV. Chronic suppression of synaptic transmission with 10 mM Mg2+ and 0.2 mM Ca2+, starting at 6 DIV, increased the content of DNA, protein, and GFAP at 21 DIV, but NSE was still decreased. We conclude that chronic suppression of BEA in cerebral cortex cultures enhances neuronal cell death, whereas astrocytes are differentially affected, depending on the suppressing agent. As astrocytes may have a modulating effect on neuronal survival, their involvement should be regarded when studying the effects of chronic suppression of BEA on neuronal development.

Development of Neurons and Glial Cells in Cerebral Cortex, Cultured in the Presence or Absence of Bioelectric Activity: Morphological Observations.

Chronic blockade of bioelectric activity (BEA) has been shown to increase neuronal cell death in tissue culture, but the effects of this treatment on non-neuronal cells have not been investigated. To determine which cell types are affected by chronic suppression of BEA, we investigated their morphological development in primary cultures of rat cerebral cortex, grown with or without the sodium channel blocker tetrodotoxin (TTX). Morphological development was monitored by phase-contrast microscopy and by immunofluorescent staining of markers specific for neurons (NSE, MAP2, B-50, and the 200 kD neurofilament protein), astrocytes (GFAP), oligodendrocytes (galactocerebroside), macrophages (ED-1) and fibroblasts (fibronectin). Neurons in control cultures steadily increased in size and elaborated a dense network of axons and dendrites during the first 3 weeks. Astrocytes proliferated strongly and formed a 'bottom-layer' on which other cells grew. Part of the astrocytes migrated into the peripheral area of the culture, but retracted to the centre after 14 days in vitro (DIV). Oligodendrocytes and macrophages also increased in number, but oligodendrocytes were completely lost by 28 DIV. After 3 weeks, axons that had grown into the periphery of the culture gradually retracted and/or degenerated, following the retracting astrocytes. Some of the neurons died after 21 DIV, but a large part persisted until 42 DIV. Upon TTX treatment from 5/6 DIV, cultures with few macrophages showed an increase in the proportion of necrotic nuclei at 14 and 21 DIV. The retraction of peripherally located fibres was accelerated by 3 - 4 days and their degeneration was augmented. Neuronal density decreased to zero between 21 and 42 DIV. Astrocytes showed a clear decrease in density from 28 DIV. Conversely, the density of macrophages was increased about two-fold from 14 DIV. These results indicate that both neurons and glia are affected by chronic TTX treatment.

Rho proteins and the cellular mechanisms of mental retardation.

The biological basis of mental retardation is poorly understood. Mental retardation is associated with an immature morphology of synaptic spines, structures involved in neurotransmission and memory processes, suggesting that mental retardation is due to a deficiency in neuronal network formation. Recently, several genes involved in X-linked mental retardation (MRX) have been cloned. Investigation of the roles of these genes in neuronal development and function should lead to a better understanding of the cellular mechanisms underlying mental retardation. A significant number of MRX genes is directly involved in signal transduction through Rho proteins. These Rho proteins act as molecular switches which integrate extracellular and intracellular signals to regulate rearrangement of the actin cytoskeleton. Since the actin cytoskeleton mediates neuronal motility and morphogenesis, one can envision how mutations in proteins involved in Rho-dependent signaling result in mental retardation by altering neuronal network formation.

A developmental decrease in NMDA-mediated spontaneous firing in cultured rat cerebral cortex.

In primary cultures of fetal rat cerebral cortex chronic manipulation of the level and/or pattern of bioelectric activity leads to plastic changes in bioelectric activity, opposite to those seen during the manipulation. This suggests the presence of adaptive mechanisms which regulate functional development in the neuronal network. Since NMDA receptors play an important role in early postnatal bioelectric activity and have been implicated in activity-dependent plasticity in vivo, the involvement of NMDA and non-NMDA receptors in spontaneously occurring bioelectric activity was investigated in cultured rat cerebral cortex by assaying the effects of NMDA and non-NMDA antagonists on neuronal firing. In addition, the physiological consequences of chronic suppression of bioelectric activity were investigated following development in the presence of tetrodotoxin. NMDA receptors appeared at all ages to be more crucial for spontaneous bioelectric activity than non-NMDA receptors, although their relative importance decreased during the first 3 weeks. Whereas the NMDA antagonist APV strongly reduced burst firing, the non-NMDA antagonist DNQX tended to increase burst firing slightly. Following chronic suppression of bioelectric activity, non-variable burst firing was increased, thus replicating previous findings in cerebral cortex culture grown under different conditions. The prominence of NMDA receptor activation in spontaneous bioelectric activity in early cultures suggests a role for these receptors in activity-dependent functional plasticity, as found in vivo.

Activity-dependent plasticity of inhibitory and excitatory amino acid transmitter systems in cultured rat cerebral cortex.

Chronic suppression of spontaneous bioelectric activity in cultures of dissociated fetal rat cerebral cortex increases neuronal cell death and results in electrophysiological changes which indicate an altered balance between excitatory and inhibitory neurotransmission in culture. To delineate whether alterations in neurotransmitter release could underlie this imbalance, we investigated the effects of chronic tetrodotoxin (TTX) treatment on the content and release of glutamate, aspartate and gamma-aminobutyric acid (GABA) in culture. Chronic TTX treatment decreased the content of all amino acids investigated. However, only GABA was decreased relative to the neuronal marker NSE (neuron-specific enolase), indicating a disproportionate loss of GABA production following chronic silencing. Depolarization-induced release of GABA, glutamate and aspartate increased about 10-fold between 7 and 21 days in control cultures. Chronic TTX treatment significantly increased the depolarization-induced release of glutamate and aspartate at 7 days in vitro relative to control levels. At all ages it caused a two-fold increase in the ratio of evoked excitatory amino acid release to that of GABA. These observations suggest that chronic silencing of developing neocortex cell cultures increases the ratio of excitatory to inhibitory synaptic activity either by differential cell death or by reduced synaptic efficiency, on which a decrease in GABA neurotransmission appears to play a major role. Since similar mechanisms may be involved in activity-dependent plasticity in vivo, these cultures provide a useful model to analyse this phenomenon at the cell biological and molecular level.

Developmental changes in B-50 (GAP-43) in primary cultures of cerebral cortex: B-50 immunolocalization, axonal elongation rate and growth cone morphology.

Changes in neurite outgrowth parameters and in the immunolocalization of the neuronal growth-associated protein B-50 (GAP-43) were studied in cultured neocortex as a function of development. In addition, we studied the effects of chronic blockade of bioelectric activity (BEA) with tetrodotoxin (TTX) on these parameters. Axonal outgrowth rate in control cultures reached a maximum at 8 days in vitro (DIV) and declined to a low level at 21 DIV. B-50 staining shifted from the perikaryon to the axons and growth cones during the first 3 DIV. In axons the intensity of B-50 staining increased towards the growth cone. Within growth cones, the central/basal region and filopodia were intensely stained, whereas lamellipodia showed only marginal staining. Growth cone size gradually decreased after 3 DIV, due to the successive loss of lamellipodia and filopodia, and became club-shaped during the second week, until by 21 DIV growth cones were completely lost, and axons started retracting and degenerated. In the central area of the cultures, growth cones also decreased in size with time, but became stabilized as presynaptic elements onto other neurons. Acute addition of TTX did not affect the outgrowth rate at 6 DIV. Chronic TTX treatment led to an earlier retraction and degeneration of axons than in control cultures and to a loss of B-50-stained cells and varicosities during the third week, but did not affect growth cone morphology or B-50 staining. The regressive phenomena are probably due to an increased neuronal cell death shown to occur after chronic TTX treatment. The developmental changes in axonal elongation rate and growth cone morphology may be related to developmental changes in the content and/or phosphorylation of B-50 (GAP-43, which are studied in the same cultures in the following paper (Ramakers et al. (1991) Int. J. Devl Neurosci. 9, 231-241].

Developmental changes in B-50 (GAP-43) in primary cultures of cerebral cortex: content and phosphorylation of B-50.

The content and phosphorylation of the neuronal growth-associated protein B-50 (GAP-43) were studied in cultured neocortex as a function of normal development and development in the presence of tetrodotoxin (TTX), a blocker of bioelectric activity (BEA). The observations were correlated with previous morphological findings on neurite outgrowth and B-50 immunolocalization in the same cultures. In control cultures, the concentration of B-50 reached a maximum at 7 days in vitro (DIV) and decreased thereafter, whereas the concentration of neuron specific enolase (NSE), which was used as a neuronal reference marker, rose till 28 DIV and leveled off towards 42 DIV. The degree of basal phosphorylation of B-50 (relative to that of total protein) decreased after the first week in vitro. Stimulation of B-50 phosphorylation by phorbol ester also decreased with age in vitro, indicating that changes in B-50 phosphorylation were mainly due to changes in protein kinase C (PKC) activity. The chronic presence of TTX led to a reduced content of B-50 and NSE after 14 DIV. The basal phosphorylation of B-50 was neither affected by acute nor chronic TTX treatment. However, upon stimulation of PKC with phorbol esters, some alterations of B-50 phosphorylation were revealed in cultures grown in TTX. These biochemical observations are in line with the absence of effects of TTX on neurite outgrowth during the first 2 weeks in culture, and later effects of TTX on neuronal survival. The developmental changes in B-50 concentration and phosphorylation largely correlate with previous morphological observations on axonal outgrowth and growth cone shape in the same cultures. We suggest that B-50 phosphorylation plays an important role in transducing extracellular signals into directed neurite outgrowth.

Spontaneous firing as an epigenetic factor in brain development--physiological consequences of chronic tetrodotoxin and picrotoxin exposure on cultured rat neocortex neurons.

Functional consequences of either suppressing or intensifying spontaneous neuronal firing have been studied in developing rat cerebral cortex cultures using, respectively, tetrodotoxin (TTX) and picrotoxin (PTX) added chronically to the growth medium. Simple measures derived from the interspike interval histogram were able to powerfully discriminate between age and treatment groups. After return to control medium, most TTX-treated neurons spontaneously displayed stereotyped clustering of action potentials ('phasic' firing) which closely resembled the characteristic firing patterns seen acutely in the presence of PTX. The 'TTX-syndrome' thus suggests that GABAergic synaptic inhibition is ineffective in cortical networks grown under conditions which prevent the expression of bioelectric activity. In contrast, after return to control medium, neurons which had been partially disinhibited throughout development (by continuous exposure to PTX) had even less phasic firing than was measured in age-matched controls. Based upon these and previous findings, a two (main) factor model is put forth which can economically account for the major effects. The working hypothesis embodied in this model is that phasic neuronal discharges not only accelerate the maturation of excitatory connections within the neocortex but, even more important, are crucial for the development of adequate inhibitory synaptic transmission.

The role of calcium signaling in early axonal and dendritic morphogenesis of rat cerebral cortex neurons under non-stimulated growth conditions.

The effects of depolarizing stimuli on neurite outgrowth have been shown to depend on an influx of extracellular calcium. However, the role of calcium under non-stimulated growth conditions is less well established. Here we investigated the contribution of calcium signaling to early neuronal morphogenesis of rat cerebral cortex neurons at three levels by blocking L-type voltage sensitive calcium channels, by depleting intracellular calcium or by blocking myosin light chain kinase. Detailed quantitative morphological analysis of neurons treated for 1 day revealed that depletion of intracellular calcium strongly decreased the density of filopodia, arrested axonal outgrowth and strongly decreased dendritic branching. Preventing calcium influx through L-type voltage sensitive calcium channels and blocking of myosin light chain kinase activity selectively decreased dendritic branching. Our observations support an essential role for basal intracellular calcium levels in axonal elongation. Furthermore, under non-stimulated conditions calcium entry through L-type voltage sensitive calcium channels and myosin light chain kinase play an important role in dendritic branching.

Depolarization stimulates lamellipodia formation and axonal but not dendritic branching in cultured rat cerebral cortex neurons.

Electric activity is known to have profound effects on growth cone morphology and neurite outgrowth, but the nature of the response varies strongly between neurons derived from different species or brain areas. To establish the role of electric activity in neurite outgrowth and neuronal morphogenesis of rat cerebral cortex neurons, cultured neurons were depolarized for up to 72 h and quantitatively analyzed for changes in axonal and dendritic morphology. Depolarization with 25 mM potassium chloride induced a rapid increase in lamellipodia in almost all growth cones and along both axons and dendrites. Lamellipodia formation was dependent on an influx of extracellular calcium through L-type voltage-sensitive calcium channels. Prolonged depolarization for 24 h induced an increase in total axonal length, mainly due to an increase in branching. After three days of depolarization axonal outgrowth was largely the same as in control neurons, suggesting accommodation of the growth cones to chronic depolarization. Dendrites showed very little change during the first three days in culture, and dendritic length or branching were not affected by depolarization. Thus, in early cerebral cortex neurons depolarization specifically stimulates axonal outgrowth through increased branching. This increase in branching may be a consequence of the earlier increase in lamellipodia formation. In contrast, early dendrites seem to be unable to translate the increase in lamellipodia into changes in outgrowth or branching. This difference between axons and dendrites could be due to differences in the stabilization of the tubulin cytoskeleton.

Longterm stability and developmental changes in spontaneous network burst firing patterns in dissociated rat cerebral cortex cell cultures on multielectrode arrays.

Spontaneous action potentials were recorded longitudinally for 4-7 weeks from dissociated rat occipital cortex cells cultured on planar multi-electrode plates, during their development from isolated neurons into synaptically connected neuronal networks. Activity typically consisted of generalized bursts lasting up to several seconds, separated by variable epochs of sporadic firing at some of the active sites. These network bursts displayed discharge patterns with age-dependent firing rate profiles, and durations significantly increasing in the 3rd week in vitro and decreasing after about 1 month in vitro, when they evolved into short events with prompt onsets. These findings indicate that after about a month in vitro these cultured neuronal networks have developed a degree of excitability that allows almost instantaneous triggering of generalized discharges. Individual neurons tend to fire in specific and persistent temporal relationships to one another within these network bursts, suggesting that network connectivity maintains a core topology during its development.

Abnormalities in the spontaneous firing patterns of cultured rat neocortical neurons after chronic exposure to picrotoxin during development in vitro.

We have used the GABA-A antagonist picrotoxin (PTX) to investigate whether chronic disinhibition, leading to intensified neuronal firing, would induce a specific pattern of physiological alterations in cultured rat neocortex cells. Overall mean spontaneous discharge rates were little affected by 1 microM PTX but firing occurred mainly as repetitive high-frequency bursts of action potentials. This "phasic" pattern contrasted with the irregular, quasi-random, firing usually seen in control units. Neurons tested in normal growth medium after prolonged exposure to 1 microM PTX showed weaker interspike interval dependencies (Markov value) than in controls, along with reduced regularity in the occurrence of bursts. Since all physiological changes were opposite in direction to those reported earlier after chronic suppression of bioelectric activity, the results support the hypothesis that endogenous synaptic and/or action potentials are important for the maturation of neocortical networks. Since experimental alterations were found only in spike-train parameters which reflect ontogenetic changes in untreated control cultures, GABAergic inhibition (by preventing neuronal discharges from becoming too intense) presumably serves to constrain the rate of development within optimal limits.

Auditory and visual cortical activity during selective attention in fragile X syndrome: a cascade of processing deficiencies.

OBJECTIVE: This study examined whether attention deficits in fragile X syndrome (FXS) can be traced back to abnormalities in basic information processing. METHOD: Sixteen males with FXS and 22 age-matched control participants (mean age 29 years) performed a standard oddball task to examine selective attention in both auditory and visual modalities. Five FXS males were excluded from analysis because they performed below chance level on the auditory task. ERPs were recorded to investigate the N1, P2, N2b, and P3b components. RESULTS: N1 and N2b components were significantly enhanced in FXS males to both auditory and visual stimuli. Interestingly, in FXS males, the P3b to auditory stimuli was significantly reduced relative to visual stimuli. These modality differences in information processing corresponded to behavioral results, showing more errors on the auditory than on the visual task. CONCLUSIONS: The current findings suggest that attentional impairments in FXS at the behavioral level can be traced back to abnormalities in event-related cortical activity. These information processing abnormalities in FXS may hinder the allocation of attentional resources needed for optimal processing at higher-levels. SIGNIFICANCE: These findings demonstrate that auditory information processing in FXS males is critically impaired relative to visual information processing.

Auditory change detection in fragile X syndrome males: a brain potential study.

OBJECTIVE: The present study investigated involuntary change detection in a two-tone pre-attentive auditory discrimination paradigm in order to better understand the information processing mechanisms underlying attention deficits in fragile X syndrome (FXS) males. METHODS: Sixteen males with the FXS full mutation and 20 age-matched control participants (mean age 29 years) were presented with series of auditory stimuli consisting of standard and deviant tones while watching a silent movie. RESULTS: Brain potentials recorded to the tones showed that N1 and P2, sensory evoked potentials, were significantly enhanced in FXS compared to age-matched control participants. In contrast to controls, the N1 to standard tones failed to show long-term habituation to stimulus repetition in FXS. Additionally, both mismatch negativity and P3a generation, reflecting automatic change detection and the involuntary switch of attention, respectively, were significantly attenuated in FXS males. CONCLUSIONS: The current study demonstrates that auditory stimulus discrimination in the FXS brain is already compromised during the pre-attentive stages of information processing. Furthermore, the apparent pre-attentive information processing deficiencies in FXS coincide with a weakness in the involuntary engagement of attentional resources. SIGNIFICANCE: The stimulus-driven information processing deficiencies in FXS might compromise information processing in several domains and, thus, present a key-deficit in FXS neurocognition.

Profiling Fragile X Syndrome in males: strengths and weaknesses in cognitive abilities.

The present study examined the cognitive profile in Fragile X Syndrome (FXS) males, and investigated whether cognitive profiles are similar for FXS males at different levels of intellectual functioning. Cognitive abilities in non-verbal, verbal, memory and executive functioning domains were contrasted to both a non-verbal and verbal mental age reference. Model-based cluster analyses revealed three distinct subgroups which differed in level of functioning, but showed similar cognitive profiles. Results showed that cognitive performance is particularly weak on measures of reasoning- and performal abilities confined to abstract item content, but relatively strong on measures of visuo-perceptual recognition and vocabulary. Further, a significant weakness was found for verbal short-term memory. Finally, these results indicated that the choice of an appropriate reference is critically important in examining cognitive profiles. The pattern of findings that emerged from the current cognitive profiling of FXS males was interpreted to suggest a fundamental deficit in executive control.