Maturation, Refinement, and Serotonergic Modulation of Cerebellar Cortical Circuits in Normal Development and in Murine Models of Autism.

The formation of the complex cerebellar cortical circuits follows different phases, with initial synaptogenesis and subsequent processes of refinement guided by a variety of mechanisms. The regularity of the cellular and synaptic organization of the cerebellar cortex allowed detailed studies of the structural plasticity mechanisms underlying the formation of new synapses and retraction of redundant ones. For the attainment of the monoinnervation of the Purkinje cell by a single climbing fiber, several signals are involved, including electrical activity, contact signals, homosynaptic and heterosynaptic interaction, calcium transients, postsynaptic receptors, and transduction pathways. An important role in this developmental program is played by serotonergic projections that, acting on temporally and spatially regulated postsynaptic receptors, induce and modulate the phases of synaptic formation and maturation. In the adult cerebellar cortex, many developmental mechanisms persist but play different roles, such as supporting synaptic plasticity during learning and formation of cerebellar memory traces. A dysfunction at any stage of this process can lead to disorders of cerebellar origin, which include autism spectrum disorders but are not limited to motor deficits. Recent evidence in animal models links impairment of Purkinje cell function with autism-like symptoms including sociability deficits, stereotyped movements, and interspecific communication by vocalization.

Development of a pluripotent stem cell derived neuronal model to identify chemically induced pathway perturbations in relation to neurotoxicity: effects of CREB pathway inhibition.

According to the advocated paradigm shift in toxicology, acquisition of knowledge on the mechanisms underlying the toxicity of chemicals, such as perturbations of biological pathways, is of primary interest. Pluripotent stem cells (PSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), offer a unique opportunity to derive physiologically relevant human cell types to measure molecular and cellular effects of such pathway modulations. Here we compared the neuronal differentiation propensity of hESCs and hiPSCs with the aim to develop novel hiPSC-based tools for measuring pathway perturbation in relation to molecular and cellular effects in vitro. Among other fundamental pathways, also, the cAMP responsive element binding protein (CREB) pathway was activated in our neuronal models and gave us the opportunity to study time-dependent effects elicited by chemical perturbations of the CREB pathway in relation to cellular effects. We show that the inhibition of the CREB pathway, using 2-naphthol-AS-E-phosphate (KG-501), induced an inhibition of neurite outgrowth and synaptogenesis, as well as a decrease of MAP2(+) neuronal cells. These data indicate that a CREB pathway inhibition can be related to molecular and cellular effects that may be relevant for neurotoxicity testing, and, thus, qualify the use of our hiPSC-derived neuronal model for studying chemical-induced neurotoxicity resulting from pathway perturbations.

Genomic and phenotypic alterations of the neuronal-like cells derived from human embryonal carcinoma stem cells (NT2) caused by exposure to organophosphorus compounds paraoxon and mipafox.

Historically, only few chemicals have been identified as neurodevelopmental toxicants, however, concern remains, and has recently increased, based upon the association between chemical exposures and increased developmental disorders. Diminution in motor speed and latency has been reported in preschool children from agricultural communities. Organophosphorus compounds (OPs) are pesticides due to their acute insecticidal effects mediated by the inhibition of acetylcholinesterase, although other esterases as neuropathy target esterase (NTE) can also be inhibited. Other neurological and neurodevelopmental toxic effects with unknown targets have been reported after chronic exposure to OPs in vivo. We studied the initial stages of retinoic acid acid-triggered differentiation of pluripotent cells towards neural progenitors derived from human embryonal carcinoma stem cells to determine if neuropathic OP, mipafox, and non-neuropathic OP, paraoxon, are able to alter differentiation of neural precursor cells in vitro. Exposure to 1 µM paraoxon (non-cytotoxic concentrations) altered the expression of different genes involved in signaling pathways related to chromatin assembly and nucleosome integrity. Conversely, exposure to 5 µM mipafox, a known inhibitor of NTE activity, showed no significant changes on gene expression. We conclude that 1 µM paraoxon could affect the initial stage of in vitro neurodifferentiation possibly due to a teratogenic effect, while the absence of transcriptional alterations by mipafox exposure did not allow us to conclude a possible effect on neurodifferentiation pathways at the tested concentration.

Learning-related long-term potentiation of inhibitory synapses in the cerebellar cortex.

Despite the widespread distribution of inhibitory synapses throughout the central nervous system, plasticity of inhibitory synapses related to associative learning has never been reported. In the cerebellum, the neural correlate of fear memory is provided by a long-term potentiation (LTP) of the excitatory synapse between the parallel fibers (PFs) and the Purkinje cell (PC). In this article, we provide evidence that inhibitory synapses in the cerebellar cortex also are affected by fear conditioning. Whole-cell patch-clamp recordings of spontaneous and miniature GABAergic events onto the PC show that the frequency but not the amplitude of these events is significantly greater up to 24 h after the conditioning. Adequate levels of excitation and inhibition are required to maintain the temporal fidelity of a neuronal network. Such fidelity can be evaluated by determining the time window for multiple input coincidence detection. We found that, after fear learning, PCs are able to integrate excitatory inputs with greater probability within short delays, but the width of the whole window is unchanged. Therefore, excitatory LTP provides a more effective detection, and inhibitory potentiation serves to maintain the time resolution of the system.

Synapse formation and clustering of neuroligin-2 in the absence of GABAA receptors.

GABAergic synapses are crucial for brain function, but the mechanisms underlying inhibitory synaptogenesis are unclear. Here, we show that postnatal Purkinje cells (PCs) of GABA(A)alpha1 knockout (KO) mice express transiently the alpha3 subunit, leading to the assembly of functional GABA(A) receptors and initial normal formation of inhibitory synapses, that are retained until adulthood. Subsequently, down-regulation of the alpha3 subunit causes a complete loss of GABAergic postsynaptic currents, resulting in a decreased rate of inhibitory synaptogenesis and formation of mismatched synapses between GABAergic axons and PC spines. Notably, the postsynaptic adhesion molecule neuroligin-2 (NL2) is correctly targeted to inhibitory synapses lacking GABA(A) receptors and the scaffold molecule gephyrin, but is absent from mismatched synapses, despite innervation by GABAergic axons. Our data indicate that GABA(A) receptors are dispensable for synapse formation and maintenance and for targeting NL2 to inhibitory synapses. However, GABAergic signaling appears to be crucial for activity-dependent regulation of synapse density during neuronal maturation.

Frailty Syndromes in Persons With Cerebrovascular Disease: A Systematic Review and Meta-Analysis.

Background: Frailty can change the prognosis and treatment approach of chronic diseases. Among others, frailty has been associated with cerebrovascular diseases such as stroke. However, the extent to which the two conditions are related is unclear, and no systematic review of the literature has been conducted. Objectives: To conduct a systematic review and meta-analysis assessing the association of cerebrovascular diseases and frailty, as well as prefrailty, in observational studies. The project was carried out on behalf of the Joint Action ADVANTAGE WP4 group. Methods: The review was performed according to PRISMA guidelines. We searched PubMed, Web of Science, and Embase from 01/01/2002-26/05/2019. Pooled estimates were obtained through random effect models and Mantel-Haenszel weighting. Homogeneity was assessed with the I2 statistic. Publication bias was assessed with Egger's and Begg's tests. Results: Of 1027 studies searched, 18 studies were included (n = 48,009 participants). Stroke was the only cerebrovascular disease studied in relation to frailty syndromes. All studies except one reported an association between stroke and prefrailty or frailty. However, most studies were not of high quality and there was heterogeneity between results. The pooled prevalence of prefrailty and frailty in stroke patients was 49% (95% CI = 42-57) and 22% (95% CI = 16-27), respectively. The prevalence of frailty was 2-fold in persons with stroke compared to those without stroke (pooled odds ratio = 2.32, 95% CI = 2.11-2.55). Only two studies longitudinally examined the association between stroke and frailty, producing conflicting results. Conclusions: Frailty and prefrailty are common in persons with stroke. These results may have clinical implications, as they identify the need to assess frailty in post-stroke survivors and assess how it may affect prognosis. Better quality, longitudinal research that examines the temporal relationship between stroke and frailty are needed, as well as studies on other types of cerebrovascular disease.

Long-term synaptic changes induced in the cerebellar cortex by fear conditioning.

To better understand learning mechanisms, one needs to study synaptic plasticity induced by behavioral training. Recently, it has been demonstrated that the cerebellum is involved in the consolidation of fear memory. Nevertheless, how the cerebellum contributes to emotional behavior is far from known. In cerebellar slices at 10 min and 24 hr following fear conditioning, we found a long-lasting potentiation of the synapse between parallel fibers and Purkinje cells in vermal lobules V-VI, but not in the climbing fiber synapses. The mechanism is postsynaptic, due to an increased AMPA response. In addition, in hotfoot mice with a primary deficiency of the parallel fiber to Purkinje cell synapse, cued (but not contextual) fear conditioning is affected. We propose that this synapse plays an important role in the learned fear and that its long-term potentiation may represent a contribution to the neural substrate of fear memory.

Activity-dependent presynaptic and postsynaptic structural plasticity in the mature cerebellum.

Two models of spine formation have been proposed. Spines can derive from emerging dendritic filopodia that have encountered presynaptic partners, or presynaptic molecules may induce the spine maturation event directly from the dendritic shaft. The first model applies better to the Purkinje cell (PC), because numerous free spines have been described in several conditions, particularly when granule cells degenerate before parallel fiber (PF) synapses are formed. A large number of new spines, many of them being free, appear in the proximal dendritic domain after blockage of electrical activity by tetrodotoxin (TTX). A complete blockage of the AMPA receptors by NBQX (2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzoquinoxaline-7-sulfonamide), leading to a complete absence of PF- and climbing fiber (CF)-evoked EPSCs and of spontaneous glutamatergic quantal events, mimics the TTX effect. In contrast, metabotropic glutamate receptor blockage by MCPG [(S)-alpha-methyl-4-carboxyphenylglycine] is ineffective. In normal conditions, in the proximal dendritic domain of the PC, clusters of a few spines are present only under each CF varicosity. It has been proposed that the active CF is responsible for spine pruning in the territory surrounding the CF synapses. Here, we show that such a pruning is mediated by AMPA but not by metabotropic receptors. Finally, after AMPA receptor blockage, there is a reduced number of spines in each spine cluster underlying CF varicosity. In conclusion, PCs tend to express spines over the entire dendritic territory. CF activity reinforces the CF synaptic contacts and actively suppresses spines in the surrounding territory, which is an effect mediated by AMPA receptors.

A multi-laboratory evaluation of microelectrode array-based measurements of neural network activity for acute neurotoxicity testing.

There is a need for methods to screen and prioritize chemicals for potential hazard, including neurotoxicity. Microelectrode array (MEA) systems enable simultaneous extracellular recordings from multiple sites in neural networks in real time and thereby provide a robust measure of network activity. In this study, spontaneous activity measurements from primary neuronal cultures treated with three neurotoxic or three non-neurotoxic compounds was evaluated across four different laboratories. All four individual laboratories correctly identifed the neurotoxic compounds chlorpyrifos oxon (an organophosphate insecticide), deltamethrin (a pyrethroid insecticide) and domoic acid (an excitotoxicant). By contrast, the other three compounds (glyphosate, dimethyl phthalate and acetaminophen) considered to be non-neurotoxic ("negative controls"), produced only sporadic changes of the measured parameters. The results were consistent across the different laboratories, as all three neurotoxic compounds caused concentration-dependent inhibition of mean firing rate (MFR). Further, MFR appeared to be the most sensitive parameter for effects of neurotoxic compounds, as changes in electrical activity measured by mean frequency intra burst (MFIB), and mean burst duration (MBD) did not result in concentration-response relationships for some of the positive compounds, or required higher concentrations for an effect to be observed. However, greater numbers of compounds need to be tested to confirm this. The results obtained indicate that measurement of spontaneous electrical activity using MEAs provides a robust assessment of compound effects on neural network function.

The cerebellum: synaptic changes and fear conditioning.

In addition to coordinating movement, the cerebellum participates in motor learning, emotional behavior, and fear memory. Fear learning is reflected in a change of autonomic and somatic responses, such as heart rate and freezing, elicited by a neutral stimulus that has been previously paired with a painful one. Manipulation of the vermis affects these responses, and its reversible inactivation during the consolidation period impairs fear memory. The neural correlate of cerebellar involvement in fear consolidation is provided by a behaviorally induced long-term increase of synaptic efficacy between parallel fibers and a Purkinje cell. Similar synaptic changes after fear conditioning are well documented in the amygdala and hippocampus, providing a link between emotional experiences and changes in neural activity. In addition, in hotfoot mice, with a primary deficiency of parallel fiber to Purkinje cell synapse, short- and long-term fear memories are affected. All these data support the idea that the cerebellum participates in fear learning. The functional interconnection of the vermis with hypothalamus, amygdala, and hippocampus suggests a more complex role of the cerebellum as part of an integrated network regulating emotional behavior.

A human pluripotent carcinoma stem cell-based model for in vitro developmental neurotoxicity testing: effects of methylmercury, lead and aluminum evaluated by gene expression studies.

The major advantage of the neuronal cell culture models derived from human stem cells is their ability to replicate the crucial stages of neurodevelopment such as the commitment of human stem cells to the neuronal lineage and their subsequent stages of differentiation into neuronal and glial-like cell. In these studies we used mixed neuronal/glial culture derived from the NTERA-2 (NT-2) cell line, which has been established from human pluripotent testicular embryonal carcinoma cells. After characterization of the different stages of cell differentiation into neuronal- and glial-like phenotype toxicity studies were performed to evaluate whether this model would be suitable for developmental neurotoxicity studies. The cells were exposed during the differentiation process to non-cytotoxic concentrations of methylmercury chloride, lead chloride and aluminum nitrate for two weeks. The toxicity was then evaluated by measuring the mRNA levels of cell specific markers (neuronal and glial). The results obtained suggest that lead chloride and aluminum nitrate at low concentrations were toxic primarily to astrocytes and at the higher concentrations it also induced neurotoxicity. In contrast, MetHgCl was toxic for both cell types, neuronal and glial, as mRNA specific for astrocytes and neuronal markers were affected. The results obtained suggest that a neuronal mixed culture derived from human NT2 precursor cells is a suitable model for developmental neurotoxicity studies and gene expression could be used as a sensitive endpoint for initial screening of potential neurotoxic compounds.

Application of multielectrode array (MEA) chips for the evaluation of mixtures neurotoxicity.

Cortical neurons grown on multielectrode array (MEA) chips have been shown to be a valuable alternative method to study electrophysiological properties of the central nervous system neurons and to perform functional toxicological screening. Here we studied the effects of binary mixtures on neuronal networks cultured on MEAs. We have considered compounds with similar and different mode-of-action (MoA) to characterize and assess their combined effects. Individual and binary mixture dose-response curves based on spontaneous neuronal activity have been generated and the IC(50) has been considered as the end-point for neurotoxicity assessment. The two classical approaches of mixtures toxicity studies: concentration addition (CA) and independent action (IA) have been applied to compare calculated and experimental results. Nuclear magnetic resonance (NMR) spectroscopy has been employed to confirm no chemical reaction or complexation between mixtures components. The results suggest that both CA and IA are able to predict the toxicity of the mixture and that the combination of in vitro test methods with theoretical dose-response models has a strong potential as an alternative tool for the prediction of mixtures neurotoxicity.

Development of micro-electrode array based tests for neurotoxicity: assessment of interlaboratory reproducibility with neuroactive chemicals.

Neuronal assemblies within the nervous system produce electrical activity that can be recorded in terms of action potential patterns. Such patterns provide a sensitive endpoint to detect effects of a variety of chemical and physical perturbations. They are a function of synaptic changes and do not necessarily involve structural alterations. In vitro neuronal networks (NNs) grown on micro-electrode arrays (MEAs) respond to neuroactive substances as well as the in vivo brain. As such, they constitute a valuable tool for investigating changes in the electrophysiological activity of the neurons in response to chemical exposures. However, the reproducibility of NN responses to chemical exposure has not been systematically documented. To this purpose six independent laboratories (in Europe and in USA) evaluated the response to the same pharmacological compounds (Fluoxetine, Muscimol, and Verapamil) in primary neuronal cultures. Common standardization principles and acceptance criteria for the quality of the cultures have been established to compare the obtained results. These studies involved more than 100 experiments before the final conclusions have been drawn that MEA technology has a potential for standard in vitro neurotoxicity/neuropharmacology evaluation. The obtained results show good intra- and inter-laboratory reproducibility of the responses. The consistent inhibitory effects of the compounds were observed in all the laboratories with the 50% Inhibiting Concentrations (IC(50)s) ranging from: (mean ± SEM, in µM) 1.53 ± 0.17 to 5.4 ± 0.7 (n = 35) for Fluoxetine, 0.16 ± 0.03 to 0.38 ± 0.16 µM (n = 35) for Muscimol, and 2.68 ± 0.32 to 5.23 ± 1.7 (n = 32) for Verapamil. The outcome of this study indicates that the MEA approach is a robust tool leading to reproducible results. The future direction will be to extend the set of testing compounds and to propose the MEA approach as a standard screen for identification and prioritization of chemicals with neurotoxicity potential.

The effects of fear conditioning on cerebellar LTP and LTD.

Long-term potentiation (LTP) and depression (LTD) at parallel fibre-Purkinje cell synapses have been described in vitro in the cerebellar cortex, but the physiological roles of these two forms of plasticity have not been well defined. Here we show that, in cerebellar slices taken from rats that had undergone fear conditioning, there was a significant occlusion of electrically induced LTP at parallel fibre-Purkinje cell synapses. This effect was long-lasting and related to associative processes, as LTP was not occluded in unpaired animals. Notably, in conditioned animals the LTP-inducing protocol produced LTD in some cells instead of LTP. Conversely, synaptic depression induced by conjunctive stimulation of parallel fibers and climbing fibres was impaired in tissue taken immediately following aversive stimulation in both paired and unpaired subjects. This effect was not, however, long-lasting as the incidence and extent of LTD returned to normal levels 24 h after behavioural testing. These findings suggest that LTP takes part in the mechanisms underlying aversive associative memories in the cerebellum.

Correlation between multiple climbing fibre regression and parallel fibre response development in the postnatal mouse cerebellum.

At the neuromuscular junction elimination of supernumerary synaptic connections during development is owing to a competitive process between single neuronal populations, whereas in the central nervous system the interaction of different types of input could affect the process. In the cerebellum, the regression from multiple- to mono-innervation of the Purkinje cells by climbing fibres is virtually completed during the first two weeks of postnatal development. While it is clear that parallel fibres are important in the control of the regression, there are conflicting results in relation to whether an early phase of regression is independent of parallel fibre effects. We studied the precise timing of climbing fibre synapse development and decline and the relationship with the functional maturation of parallel fibres. Until postnatal day (P) 6 or 7, the synaptic currents generated by different climbing fibres become progressively more uniform in amplitude. However, between P7 and 14, the amplitudes of the currents increasingly diverge until only one fibre remains connected. These data are taken as evidence that, in multiply innervated Purkinje cells, competition between different climbing fibres appears at P7 and continues during the second postnatal week. Morphological and electrophysiological data demonstrate that parallel fibres synapses appear at P7 and their development is significantly correlated with the time course of the climbing fibre regression. These results provide no evidence for climbing fibre regression independent of parallel fibres before P7 and also suggest a dominant role of the parallel fibre input in the later phase.

Sodium imaging of climbing fiber innervation fields in developing mouse Purkinje cells.

Maturation of specific neuronal connections in the mature nervous system includes elimination of redundant synapses formed earlier during development. In the cerebellum of adult animals, each Purkinje cell (PC) is innervated by a single climbing fiber (CF). In early postnatal development each PC is innervated by multiple CFs and elimination of synapses formed by supernumerary CFs occurs until monoinnervation is established at around postnatal day 20 (P20) in mice. It is not clear whether multiple CFs, or only a single CF, translocate from the cell body of immature PCs to the developing dendrite and, in case several CFs translocate, whether they share or segregate their innervation fields. To localize CF innervation fields, we imaged changes in postsynaptic sodium concentration resulting from CF-mediated postsynaptic currents. We found that more than one CF translocates from an innervation field on the cell body of the PC to the developing dendrite and that these CFs share rather than segregate their innervation fields. We concluded that both the soma and the proximal dendrite of the PC are territories of competition for the developing CFs and that the overlapping of their termination fields may be the prerequisite for a local process of elimination of all but one CF, as previously demonstrated in the developing neuromuscular junction.