The effects of cholinergic and dopaminergic neurons on hippocampal learning and memory processes

Dysfunction of cholinergic and dopaminergic systems has been implicated in memory function de cits that are core pathology and associated features of several neurological disorders. However, in order to develop more effective treatments, it is crucial to better understand how different aspects of learning and memory are modulated by these neuromodulatory systems. Using optogenetic stimulation or silencing, this thesis aims to investigate the effects of cholinergic and dopaminergic modulation on various hippocamal learning and memory processes. To understand how these neuromodulatory systems modulate hippocampal network activity, I first examined their effects on hippocampal local field potentials in urethane-anaesthetised mice. I demonstrated that optogenetic cholinergic activation suppressed slow oscillations, shifting brain activity to a state dominated by theta and gamma oscillations. In contrast, dopaminergic activation suppressed gamma oscillations. Second, to directly probe the effects of neuromodulation on different stages of spatial learning, I acutely activated or inactivated cholinergic or dopaminergic neurons during various behavioural tasks. My findings suggested that cholinergic activation, solely during the reward phase of a long-term spatial memory task, slowed place learning, highlighting the importance of temporally-precise neuromodulation. Moreover, dopaminergic stimulation may enhance place learning of a food rewarded task, supporting a role for dopamine in spatial learning. In addition, I tested the effects of cholinergic and dopaminergic modulation on reversal learning and found that cholinergic inactivation and dopaminergic activation appear to impair this process. Together, these findings emphasise the importance of cholinergic and dopaminergic modulation in learning and memory. They suggest that precise timing of neuromodulator action is critical for optimal learning and memory performance, and that acetylcholine and dopamine support complementary processes that allow for effective learning and adaptation to changing environments.Gulbenkian Yuval Cambridge Studentshi

Persistent barrage firing in cortical interneurons can be induced in vivo and may be important for the suppression of epileptiform activity

Neural circuits are typically maintained in a state of dynamic equilibrium by balanced synaptic excitation and inhibition. However, brain regions that are particularly susceptible to epilepsy may have evolved additional specialized mechanisms for inhibiting over-excitation. Here we identify one such possible mechanism in the cerebral cortex and hippocampus of mice. Recently it was reported that some types of GABAergic interneurons can slowly integrate excitatory inputs until eventually they fire persistently in the absence of the original stimulus. This property, called persistent firing or retroaxonal barrage firing (BF), is of unknown physiological importance. We show that two common types of interneurons in cortical regions, neurogliaform (NG) cells and fast-spiking (FS) cells, are unique in exhibiting BF in acute slices (~85 and ~23% success rate for induction, respectively). BF can also be induced in vivo, although the success rate for induction is lower (~60% in NG cells). In slices, BF could reliably be triggered by trains of excitatory synaptic input, as well as by exposure to proconvulsant bath solutions (elevated extracellular K+, blockade of GABAA receptors). Using pair recordings in slices, we confirmed that barrage-firing NG cells can produce synaptic inhibition of nearby pyramidal neurons, and that this inhibition outlasts the original excitation. The ubiquity of NG and FS cells, together with their ability to fire persistently following excessive excitation, suggests that these interneurons may function as cortical sentinels, imposing an activity-dependent brake on undesirable neuronal hyperexcitability.This work was supported by the National Health and Medical Research Council of Australia (Project Grants 1009382 and 1050832 to John M. Bekkers)

Depletion of the IKBKAP ortholog in zebrafish leads to hirschsprung disease-like phenotype

© The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved. AIM: To investigate the role of IKBKAP (inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase complex-associated protein) in the development of enteric nervous system (ENS) and Hirschsprung disease (HSCR). METHODS: In this study, we injected a morpholino that blocked the translation of ikbkap protein to 1-cell stage zebrafish embryos. The phenotype in the ENS was analysed by antibody staining of the pan-neuronal marker HuC/D followed by enteric neuron counting. The mean numbers of enteric neurons were compared between the morphant and the control. We also studied the expressions of ret and phox2bb, which are involved in ENS development, in the ikbkap morpholino injected embryos by quantitative reverse transcriptase polymerase chain reaction and compared them with the control. RESULTS: We observed aganglionosis (χ2, P < 0.01) and a reduced number of enteric neurons (38.8 ± 9.9 vs 50.2 ± 17.3, P < 0.05) in the zebrafish embryos injected with ikbkap translation-blocking morpholino (morphant) when compared with the control embryos. Specificity of the morpholino was confirmed by similar results obtained using a second non-overlapping morpholino that blocked the translation of ikbkap. We further studied the morphant by analysing the expression levels of genes involved in ENS development such as ret, phox2bb and sox10, and found that phox2bb, the ortholog of human PHOX2B, was significantly down-regulated (0.51 ± 0.15 vs 1.00 ± 0, P < 0.05). Although we also observed a reduction in the expression of ret, the difference was not significant. CONCLUSION: Loss of IKBKAP contributed to HSCR as demonstrated by functional analysis in zebrafish embryos.Link_to_subscribed_fulltex

Targeted next-generation sequencing on hirschsprung disease: A pilot study exploits DNA pooling

To adopt an efficient approach of identifying rare variants possibly related to Hirschsprung disease (HSCR), a pilot study was set up to evaluate the performance of a newly designed protocol for next generation targeted resquencing. In total, 20 Chinese HSCR patients and 20 Chinese sex-matched individuals with no HSCR were included, for which coding sequences (CDS) of 62 genes known to be in signaling pathways relevant to enteric nervous system development were selected for capture and sequencing. Blood DNAs from eight pools of five cases or controls were enriched by PCR-based RainDance technology (RDT) and then sequenced on a 454 FLX platform. As technical validation, five patients from case Pool-3 were also independently enriched by RDT, indexed with barcode and sequenced with sufficient coverage. Assessment for CDS single nucleotide variants showed DNA pooling performed well (specificity/sensitivity at 98.4%/83.7%) at the common variant level; but relatively worse (specificity/sensitivity at 65.5%/61.3%) at the rare variant level. Further Sanger sequencing only validated five out of 12 rare damaging variants likely involved in HSCR. Hence more improvement at variant detection and sequencing technology is needed to realize the potential of DNA pooling for large-scale resequencing projects. © 2014 John Wiley & Sons Ltd/University College London.postprin

Sequencing of a Chinese tetralogy of Fallot cohort reveals clustering mutations in myogenic heart progenitors

Tetralogy of Fallot (TOF) is the most common cyanotic heart defect, yet the underlying genetic mechanisms remain poorly understood. Here, we performed whole-genome sequencing analysis on 146 nonsyndromic TOF parent-offspring trios of Chinese ethnicity. Comparison of de novo variants and recessive genotypes of this data set with data from a European cohort identified both overlapping and potentially novel gene loci and revealed differential functional enrichment between cohorts. To assess the impact of these mutations on early cardiac development, we integrated single-cell and spatial transcriptomics of early human heart development with our genetic findings. We discovered that the candidate gene expression was enriched in the myogenic progenitors of the cardiac outflow tract. Moreover, subsets of the candidate genes were found in specific gene coexpression modules along the cardiomyocyte differentiation trajectory. These integrative functional analyses help dissect the pathogenesis of TOF, revealing cellular hotspots in early heart development resulting in cardiac malformations

Fine Mapping of the NRG1 Hirschsprung's Disease Locus

The primary pathology of Hirschsprung's disease (HSCR, colon aganglionosis) is the absence of ganglia in variable lengths of the hindgut, resulting in functional obstruction. HSCR is attributed to a failure of migration of the enteric ganglion precursors along the developing gut. RET is a key regulator of the development of the enteric nervous system (ENS) and the major HSCR-causing gene. Yet the reduced penetrance of RET DNA HSCR-associated variants together with the phenotypic variability suggest the involvement of additional genes in the disease. Through a genome-wide association study, we uncovered a ∼350 kb HSCR-associated region encompassing part of the neuregulin-1 gene (NRG1). To identify the causal NRG1 variants contributing to HSCR, we genotyped 243 SNPs variants on 343 ethnic Chinese HSCR patients and 359 controls. Genotype analysis coupled with imputation narrowed down the HSCR-associated region to 21 kb, with four of the most associated SNPs (rs10088313, rs10094655, rs4624987, and rs3884552) mapping to the NRG1 promoter. We investigated whether there was correlation between the genotype at the rs10088313 locus and the amount of NRG1 expressed in human gut tissues (40 patients and 21 controls) and found differences in expression as a function of genotype. We also found significant differences in NRG1 expression levels between diseased and control individuals bearing the same rs10088313 risk genotype. This indicates that the effects of NRG1 common variants are likely to depend on other alleles or epigenetic factors present in the patients and would account for the variability in the genetic predisposition to HSCR

RET Mutational Spectrum in Hirschsprung Disease: Evaluation of 601 Chinese Patients

Rare (RVs) and common variants of the RET gene contribute to Hirschsprung disease (HSCR; congenital aganglionosis). While RET common variants are strongly associated with the commonest manifestation of the disease (males; short-segment aganglionosis; sporadic), rare coding sequence (CDS) variants are more frequently found in the lesser common and more severe forms of the disease (females; long/total colonic aganglionosis; familial)

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Roles of Enteric Neural Stem Cell Niche and Enteric Nervous System Development in Hirschsprung Disease

The development of the enteric nervous system (ENS) is highly modulated by the synchronized interaction between the enteric neural crest cells (ENCCs) and the neural stem cell niche comprising the gut microenvironment. Genetic defects dysregulating the cellular behaviour(s) of the ENCCs result in incomplete innervation and hence ENS dysfunction. Hirschsprung disease (HSCR) is a rare complex neurocristopathy in which the enteric neural crest-derived cells fail to colonize the distal colon. In addition to ENS defects, increasing evidence suggests that HSCR patients may have intrinsic defects in the niche impairing the extracellular matrix (ECM)-cell interaction and/or dysregulating the cellular niche factors necessary for controlling stem cell behaviour. The niche defects in patients may compromise the regenerative capacity of the stem cell-based therapy and advocate for drug- and niche-based therapies as complementary therapeutic strategies to alleviate/enhance niche-cell interaction. Here, we provide a summary of the current understandings of the role of the enteric neural stem cell niche in modulating the development of the ENS and in the pathogenesis of HSCR. Deciphering the contribution of the niche to HSCR may provide important implications to the development of regenerative medicine for HSCR