Dynamic modulation of activity in cerebellar nuclei neurons during pavlovian eyeblink conditioning in mice

While research on the cerebellar cortex is crystallizing our understanding of its function in learning behavior, many questions surrounding its downstream targets remain. Here, we evaluate the dynamics of cerebellar interpositus nucleus (IpN) neurons over the course of Pavlovian eyeblink conditioning. A diverse range of learning-induced neuronal responses was observed, including increases and decreases in activity during the generation of conditioned blinks. Trial-bytrial correlational analysis and optogenetic manipulation demonstrate that facilitation in the IpN drives the eyelid movements. Adaptive facilitatory responses are often preceded by acquired transient inhibition of IpN activity that, based on latency and effect, appear to be driven by complex spikes in cerebellar cortical Purkinje cells. Likewise, during reflexive blinks to periocular stimulation, IpN cells show excitation-suppression patterns that suggest a contribution of climbing fibers and their collaterals. These findings highlight the integrative properties of subcortical neurons at the cerebellar output stage mediating conditioned behavior

CAMK2-Dependent Signaling in Neurons Is Essential for Survival

Ca2+/calmodulin-dependent protein kinase II (CAMK2) is a key player in synaptic plasticity and memory formation. Mutations in Camk2a or Camk2b cause intellectual disability in humans, and severe plasticity and learning deficits in mice, indicating unique functions for each isoform. However, considering the high homology between CAMK2A and CAMK2B, it is conceivable that for critical functions, one isoform compensates for the absence of the other, and that the full functional spectrum of neuronal CAMK2 remains to be revealed.Here we show that germline as well as adult deletion of both CAMK2 isoforms in male or female mice is lethal. Moreover, Ca2+-dependent activity as well as autonomous activity of CAMK2 is essential for survival. Loss of both CAMK2 isoforms abolished LTP, whereas synaptic transmission remained intact. The double-mutants showed no gross morphological changes of the brain, and in contrast to the long-considered role for CAMK2 in the structural organization of the postsynaptic density (PSD), deletion of both CAMK2 isoforms did not affect the biochemical composition of the PSD. Together, these results reveal an essential role for CAMK2 signaling in early postnatal development as well as the mature brain, and indicate that the full spectrum of CAMK2 requirements cannot be revealed in the single mutants because of partial overlappin

Relations between the milnor and quillen K-theory of fields

De novo mutations in specific mTOR pathway genes cause brain overgrowth in the context of intellectual disability (ID). By analyzing 101 mMTOR-related genes in a large ID patient cohort and two independent population cohorts, we show that these genes modulate brain growth in health and disease. We report the mTOR activator gene RHEB as an ID gene that is associated with megalencephaly when mutated. Functional testing of mutant RHEB in vertebrate animal models indicates pathway hyperactivation with a concomitant increase in cell and head size, aberrant neuronal migration, and induction of seizures, concordant with the human phenotype. This study reveals that tight control of brain volume is exerted through a large community of mTOR-related genes. Human brain volume can be altered, by either rare disruptive events causing hyperactivation of the pathway, or through the collective effects of common alleles

The intellectual disability-associated CAMK2G p.Arg292Pro mutation acts as a pathogenic gain-of-function

The abundantly expressed calcium/calmodulin-dependent protein kinase II (CAMK2), alpha (CAMK2A), and beta (CAMK2B) isoforms are essential for learning and memory formation. Recently, a de novo candidate mutation (p.Arg292Pro) in the gamma isoform of CAMK2 (CAMK2G) was identified in a patient with severe intellectual disability (ID), but the mechanism(s) by which this mutation causes ID is unknown. Here, we identified a second, unrelated individual, with a de novo CAMK2G p.Arg292Pro mutation, and used in vivo and in vitro assays to assess the impact of this mutation on CAMK2G and neuronal function. We found that knockdown of CAMK2G results in inappropriate precocious neuronal maturation. We further found that the CAMK2G p.Arg292Pro mutation acts as a highly pathogenic gain-of-function mutation, leading to increased phosphotransferase activity and impaired neuronal maturation as well as impaired targeting of the nuclear CAMK2G isoform. Silencing the catalytic site of the CAMK2G p.Arg292Pro protein reversed the pathogenic effect of the p.Arg292Pro mutation on neuronal maturation, without rescuing its nuclear targeting. Taken together, our results reveal an indispensable function of CAMK2G in neurodevelopment and indicate that the CAMK2G p.Arg292Pro protein acts as a pathogenic gain-of-function mutation, through constitutive activity toward cytosolic targets, rather than impaired targeting to the nucleus

Role of calcium/calmodulin-dependent kinase 2 in neurodevelopmental disorders

Neurodevelopmental disorders are a complex and heterogeneous group of neurological disorders characterized by their early-onset and estimated to affect more than 3% of children worldwide. The rapid advancement of sequencing technologies in the past years allowed the identification of hundreds of variants in several different genes causing neurodevelopmental disorders. Between those, new variants in the Calcium/calmodulin dependent protein kinase II (CAMK2) genes were recently linked to intellectual disability. Despite many years of research on CAMK2, this proves for the first time that this well-known and highly conserved molecule plays an important role in the human brain. In this review, we give an overview of the identified CAMK2 variants, and we speculate on potential mechanisms through which dysfunctions in CAMK2 result in neurodevelopmental disorders. Additionally, we discuss how the identification of CAMK2 variants might result in new exciting discoveries regarding the function of CAMK2 in the human brain

Focused ultrasound neuromodulation on a multiwell MEA

BackgroundMicroelectrode arrays (MEA) enable the measurement and stimulation of the electrical activity of cultured cells. The integration of other neuromodulation methods will significantly enhance the application range of MEAs to study their effects on neurons. A neuromodulation method that is recently gaining more attention is focused ultrasound neuromodulation (FUS), which has the potential to treat neurological disorders reversibly and precisely.MethodsIn this work, we present the integration of a focused ultrasound delivery system with a multiwell MEA plate.ResultsThe ultrasound delivery system was characterised by ultrasound pressure measurements, and the integration with the MEA plate was modelled with finite-element simulations of acoustic field parameters. The results of the simulations were validated with experimental visualisation of the ultrasound field with Schlieren imaging. In addition, the system was tested on a murine primary hippocampal neuron culture, showing that ultrasound can influence the activity of the neurons.ConclusionsOur system was demonstrated to be suitable for studying the effect of focused ultrasound on neuronal cultures. The system allows reproducible experiments across the wells due to its robustness and simplicity of operation.Electronic Components, Technology and MaterialsBio-Electronic

RHEB/mTOR hyperactivity causes cortical malformations and epileptic seizures through increased axonal connectivity.

Hyperactivation of the mammalian target of rapamycin (mTOR) pathway can cause malformation of cortical development (MCD) with associated epilepsy and intellectual disability (ID) through a yet unknown mechanism. Here, we made use of the recently identified dominant-active mutation in Ras Homolog Enriched in Brain 1 (RHEB), RHEBp.P37L, to gain insight in the mechanism underlying the epilepsy caused by hyperactivation of the mTOR pathway. Focal expression of RHEBp.P37L in mouse somatosensory cortex (SScx) results in an MCD-like phenotype, with increased mTOR signaling, ectopic localization of neurons, and reliable generalized seizures. We show that in this model, the mTOR-dependent seizures are caused by enhanced axonal connectivity, causing hyperexcitability of distally connected neurons. Indeed, blocking axonal vesicle release from the RHEBp.P37L neurons alone completely stopped the seizures and normalized the hyperexcitability of the distally connected neurons. These results provide new evidence of the extent of anatomical and physiological abnormalities caused by mTOR hyperactivity, beyond local malformations, which can lead to generalized epilepsy

RHEB/mTOR hyperactivity causes cortical malformations and epileptic seizures through increased axonal connectivity

Hyperactivation of the mammalian target of rapamycin (mTOR) pathway can cause malformation of cortical development (MCD) with associated epilepsy and intellectual disability (ID) through a yet unknown mechanism. Here, we made use of the recently identified dominant-active mutation in Ras Homolog Enriched in Brain 1 (RHEB), RHEBp.P37L, to gain insight in the mechanism underlying the epilepsy caused by hyperactivation of the mTOR pathway. Focal expression of RHEBp.P37L in mouse somatosensory cortex (SScx) results in an MCD-like phenotype, with increased mTOR signaling, ectopic localization of neurons, and reliable generalized seizures. We show that in this model, the mTOR-dependent seizures are caused by enhanced axonal connectivity, causing hyperexcitability of distally connected neurons. Indeed, blocking axonal vesicle release from the RHEBp.P37L neurons alone completely stopped the seizures and normalized the hyperexcitability of the distally connected neurons. These results provide new evidence of the extent of anatomical and physiological abnormalities caused by mTOR hyperactivity, beyond local malformations, which can lead to generalized epilepsy

COMPARAÇÃO DE METODOLOGIAS DE DETERMINAÇÃO DO COEFICIENTE DE DISPERSÃO PARA O CLORETO DE CÁLCIO EM UM LATOSSOLO VERMELHO-AMARELO

RESUMO Com o objetivo de se comparar os métodos de ajuste gráfico e a regressão não-linear na determinação do coeficiente de dispersão, montaram-se três colunas de solo, através das quais se fez passar uma solução deslocadora de cloreto de cálcio (CaCl2), o que permitiu a obtenção dos pontos da curva de eluição. Para efeito de comparação das curvas de eluição ajustadas à experimental, empregaram-se as soluções analíticas das equações diferenciais para a dispersão longitudinal, dispersão para um pulso, difusivo-convectivo e geral do transporte de solutos. Os valores dos coeficientes de dispersão obtidos pelos métodos empregados mostraram que o ajuste gráfico, quando bem empregado, é uma alternativa quando não se dispõe de recursos computacionais para usar o ajuste através de regressão não-linear. As soluções analíticas descrevem com perfeição a curva de eluição, desde que se conheça o coeficiente de dispersão

Excitatory Cerebellar Nucleocortical Circuit Provides Internal Amplification during Associative Conditioning

Closed-loop circuitries between cortical and subcortical regions can facilitate precision of output patterns, but the role of such networks in the cerebellum remains to be elucidated. Here, we characterize the role of internal feedback from the cerebellar nuclei to the cerebellar cortex in classical eyeblink conditioning. We find that excitatory output neurons in the interposed nucleus provide efference-copy signals via mossy fibers to the cerebellar cortical zones that belong to the same module, triggering monosynaptic responses in granule and Golgi cells and indirectly inhibiting Purkinje cells. Upon conditioning, the local density of nucleocortical mossy fiber terminals significantly increases. Optogenetic activation and inhibition of nucleocortical fibers in conditioned animals increases and decreases the amplitude of learned eyeblink responses, respectively. Our data show that the excitatory nucleocortical closed-loop circuitry of the cerebellum relays a corollary discharge of premotor signals and suggests an amplifying role of this circuitry in controlling associative motor learning