Heavy ethanol use causes neurodegeneration manifested by neuronal loss and dysfunction. It is becoming imperative to delineate the underlying mechanism to promote the treatment of ethanol-induced neurodegeneration. Endoplasmic reticulum (ER) stress is a hallmark and an underlying mechanism of many neurodegenerative diseases. This study aims to investigate the role of ER stress in ethanol-induced neurodegeneration. In experimental design, adult mice were exposed to binge ethanol drinking by daily gavage for 1, 5, or 10 days and the response of ER stress was examined. We found the induction of ER stress appeared at 5 days and remained at 10 days. Moreover, the induction of ER stress was accompanied by an increase in neurodegeneration. With cell culture, we demonstrated that ethanol exposure resulted in neuronal apoptosis and that blocking ER stress by sodium phenylbutyrate (4-PBA) abolished ethanol-induced neuronal apoptosis, suggesting that ER stress contributes to ethanol-induced neurodegeneration. Mesencephalic astrocyte-derived neurotrophic factor (MANF) responds to ER stress and has been identified as a protein upregulated in ethanol-exposed developmental mouse brains. To investigate its implication in ethanol-induced neurodegeneration, we established a central nervous system (CNS)-specific Manf knockout mouse model and examined the effects of MANF deficiency on ethanol-induced neuronal apoptosis and ER stress using a third-trimester equivalent mouse model. We found MANF deficiency worsened ethanol-induced neuronal apoptosis and ER stress and that blocking ER stress abrogated the harmful effects of MANF deficiency on ethanol-induced neuronal apoptosis. Moreover, a whole transcriptome RNA sequencing supported the involvement of MANF in ER stress modulation and revealed candidates that may mediate the ER stress-buffering capacity of MANF. Collectively, these data suggest that MANF is neuroprotective against ethanol-induced neurodegeneration via ameliorating ER stress. Because MANF is a neurotrophic factor, we also examined the effects of MANF deficiency on neurogenesis. We observed that MANF deficiency increased neurogenesis in the subgranular zone of the hippocampal dentate gyrus and subventricular zone of the lateral ventricles in the mouse brain. Mechanistically, this finding was supported by a decrease of cell cycle inhibitors (p15 and p27), an increase of G2/M marker (phospho-histone H3), and an increase of neural progenitor markers (Sox2 and NeuroD1) in the brain of conditional Manf knockout mice. Our in vitro studies demonstrated that the gain-of-function of MANF inhibited cell cycle progression, whereas the loss-of-function of MANF promoted cell cycle progression. Taken together, these data suggest that MANF may affect the process of neurogenesis through altering cell cycle progression.

Ischemic injury leeds to sequence of pathophysiological events, which are accompanied by a release of growth factors and morphogens that significantly affect cell proliferation, migration and also their differentiation. Following ischemia, besides enhanced neurogenesis and gliogenesis in subventricular zone of the lateral ventricles and gyrus dentatus of the hippocampus, neurogenesis/gliogenesis also occurs in non-neurogenic regions, such as cortex or striatum. Recently, the attention was turned to a new glial cell type, termed polydendrocytes or NG2 glia. Under physiological conditions, these cells are able to divide and differentiate into mature oligodendrocytes due to they have often been equated with oligodendrocyte precursor cells. Based on recent reports, polydendrocytes are also able to generate protoplasmic astrocytes (Zhu et al., 2008) and neurons in vitro (Belachew et al., 2003), however their ability to differentiate into astrocytes or neurons under physiological or pathological conditions is still highly debated. Therefore, we have investigated the effect of different growth factors and morphogens, specifically brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and a morphogen sonic hedgehog (Shh), on...

Morfin is a clinically used analgesic drug but also an abusive drug. It has an impact on a wide range of CNS regions (nucleus accumbens, ventral tegmentum, hippocampus, etc.) and affects their functions, e.g. cognitive functions or anxiety. Although the results of so far published studies are often contradictory, the effects on cell death and proliferation in the CNS have been demonstrated. In this work, we focused on how chronic administration of morphine and subsequent withdrawal of this drug affects neurogenesis and neurodegeneration in the rat brain and how it affects some markers involved in the addiction and post-drug-induced condition. We have succeeded in introducing immunohistochemical markers for monitoring neurogenesis (bromodeoxyuridine and doublecortin) and neurodegeneration (Fluoro-Jade C) and for detection of selected neuromodulatory peptides (cholecystokinin and neuropeptide Y). We have found that morphine may influence the process of neurogenesis and neurodegeneration, but its effects differ in different CNS structures (nucleus accumbens, hippocampus, and amygdala). Key words: Morphine, brain, rat, withdrawal syndrom, neurogenesis, neurodegeneration

Die Bildung neuen Nervenzellen im erwachsenen Gehirn—adulte Neurogenese—ist bei Säugetieren auf spezifische Regionen beschränkt. Eine der beiden bekannten ist der Hippokampus, eine Gehirnstruktur, die eine wichtige Rolle beim Lernen sowie der Gedächtnisbildung spielt. Ein Reservoir von neuralen Stammzellen befindet sich in der subgranulären Zone des hippokampalen Gyrus dentatus. Diese Zellen teilen sich fortwährend und bilden neue Nervenzellen. Die Regulation adulter hippokampaler Neurogenese wird sowohl von der Umgebung beeinflusst als auch von mehreren Genen gesteuert. In der vorliegenden Arbeit wurden mittels Hochdurchsatz- Genexpressionsverfahren die an der Neurogenese beteiligten Gene identifiziert und ihr Zusammenspiel untersucht. Anhand von genetischen, umgebungsbedingten und zeitlichen Angaben und Variationen wurde ein vielseitiger Datensatz erstellt, der einen multidimensionalen Blick auf den proliferativen Phänotyp verschafft. Netzwerke aus Gen-Gen und Gen-Phänotyp Interaktionen wurden beschrieben und in einer mehrschichtigen Ressource zusammengefasst. Ein Kern-Netzwerk bestehend aus immerwiederkehrenden Modulen aus verschiedenen Ebenen wurde anhand von Proliferation als Keim-Phänotyp identifiziert. Aus diesem Kern-Netzwerk sind neue Gene und ihre Interaktionen hervorgegangen, die potentiell bei der Regulierung adulter Neurogenesis beteiligt sind.:Zusammenfassung i Abstract iii Acknowledgements vii Contents ix Preface xiii General Introduction 1 Adult Neurogenesis 1 Historical setting 1 Neurogenesis exists in two regions of the adult mammalian brain 1 Implications of neurogenesis in the hippocampus 1 The Hippocampal Formation 2 Function of the hippocampus in learning and memory 2 The functional role of adult neurogenesis 2 Anatomy of the hippocampal formation 2 Neural Precursor Biology 3 The subgranular zone as a neurogenic niche 3 Neuronal maturation is a multi-step pathway 3 Regulation of Adult Neurogenesis 3 Neurogenesis is modulated by age 3 Neurogenesis is modulated by environmental factors 4 Neurogenesis is modulated by genetic background 4 Genetics of the BXD RI Cross 5 C57BL/6 and DBA/2 5 Recombinant Inbred Lines 5 The BXD panel 6 Quantitative genetics 6 Microarray Analysis 7 The concept of ‘whole genome’ expression analysis 7 Technical considerations 8 Theoretical considerations 9 Current Analytical Methods 9 Network Analysis 10 Network Description and Terminology 10 Graph Theory 10 Multiple-Network Comparison 11 Biological networks 11 Types of Biological Network 11 Sources of Network Data 12 Biological Significance of Networks 12 Aim of the current work 13 Methods and Materials 15 Animals 15 BXD panel 15 Progenitor strains 15 Animal behaviour 15 Running wheel activity 15 Enriched environment 16 Morris water maze 16 Open field test 16 Corticosterone assay 16 Histology 17 Tissue collection 17 BrdU staining 17 Statistics 17 Cell culture 18 Maintenance and differentiation 18 Immunostaining 18 RNA isolation 18 Microarray processing 18 Affymetrix arrays 18 M430v2 probe reannotation 19 Illumina arrays 19 Illumina probe reannotation 19 Bioinformatics 19 Translating the STRING network 19 QTL mapping 20 Network graph layout 20 Triplot 20 Enrichment analysis 20 Mammalian Adult Neurogenesis Gene Ontology 21 Introduction 21 Results 25 The cell stage ontology 25 The process ontology 25 Genes known to regulate hippocampal adult neurogenesis 26 Enrichment analysis 27 The MANGO gene network 27 Discussion 28 Hippocampal Coexpression Networks from the BXD Panel 31 Introduction 31 Results 32 Variation and covariation of gene expression across a panel of inbred lines 32 A hippocampal expression correlation network 32 Diverse neurogenesis phenotypes associate with discrete transcript networks 34 Discussion 34 Interactions Between Gene Expression Phenotypes and Genotype 37 Introduction 37 Results 39 QTL analysis and interval definitions 39 Pleiotropic loci and ‘trans-bands’ 39 Transcript expression proxy-QTLs can help in dissection of complex phenotypes 41 Interaction network 43 Discussion 43 Strain-Dependent Effects of Environment 47 Introduction 47 Results 48 Effects of strain and environment on precursor cell proliferation 48 Effects of strain and environment on learning behaviour 52 Transcript expression associated with different housing environments 53 Strain differences in transcript regulation 55 Distance-weighted coexpression networks 57 Discussion 58 Expression Time Course from Differentiating Cell Culture 61 Introduction 61 Results 63 Differentiation of proliferating precursors into neurons in vitro 63 Transcripts associated with stages of differentiation 63 Early events in NPC differentiation 64 A network of transcript coexpression during in vitro differentiation 66 Discussion 67 Integrated Gene Interaction Networks 71 Introduction 71 Results 72 Description of network layers 72 Merging of network layers to a multigraph 74 A network of genes controls neural precursor proliferation in the adult hippocampus 75 Novel candidate regulators of adult hippocampal neurogenesis 77 Novel pathways regulating adult hippocampal neurogenesis 77 Discussion 79 General Discussion 81 References 89 Selbständigkeitserklärung 107 / Neurogenesis, the production of new neurons, is restricted in the adult brain of mammals to only a few regions. One of these sites of adult neurogenesis is the hippocampus, a structure essential for many types of learning. A pool of stem cells is maintained in the subgranular zone of the hippocampal dentate gyrus which proliferate and can differentiate into new neurons, astrocytes and oligodendroctytes. Regulation of adult hippocampal neurogenesis occurs in response to en- vironmental stimuli and is under the control of many genes. This work employs high-throughput gene expression technologies to identify these genes and their interactions with each other and the neurogenesis phenotype. Harnessing variation from genetic, environmental and temporal sources, a multi-faceted dataset has been generated which offers a multidimensional view of the neural precursor proliferation phenotype. Networks of gene-gene and gene-phenotype interac- tions have been described and merged into a multilayer resource. A core subnetwork derived from modules recurring in the different layers has been identified using the proliferation phenotype as a seed. This subnetwork has suggested novel genes and interactions potentially involved in the regulation of adult hippocampal neurogenesis.:Zusammenfassung i Abstract iii Acknowledgements vii Contents ix Preface xiii General Introduction 1 Adult Neurogenesis 1 Historical setting 1 Neurogenesis exists in two regions of the adult mammalian brain 1 Implications of neurogenesis in the hippocampus 1 The Hippocampal Formation 2 Function of the hippocampus in learning and memory 2 The functional role of adult neurogenesis 2 Anatomy of the hippocampal formation 2 Neural Precursor Biology 3 The subgranular zone as a neurogenic niche 3 Neuronal maturation is a multi-step pathway 3 Regulation of Adult Neurogenesis 3 Neurogenesis is modulated by age 3 Neurogenesis is modulated by environmental factors 4 Neurogenesis is modulated by genetic background 4 Genetics of the BXD RI Cross 5 C57BL/6 and DBA/2 5 Recombinant Inbred Lines 5 The BXD panel 6 Quantitative genetics 6 Microarray Analysis 7 The concept of ‘whole genome’ expression analysis 7 Technical considerations 8 Theoretical considerations 9 Current Analytical Methods 9 Network Analysis 10 Network Description and Terminology 10 Graph Theory 10 Multiple-Network Comparison 11 Biological networks 11 Types of Biological Network 11 Sources of Network Data 12 Biological Significance of Networks 12 Aim of the current work 13 Methods and Materials 15 Animals 15 BXD panel 15 Progenitor strains 15 Animal behaviour 15 Running wheel activity 15 Enriched environment 16 Morris water maze 16 Open field test 16 Corticosterone assay 16 Histology 17 Tissue collection 17 BrdU staining 17 Statistics 17 Cell culture 18 Maintenance and differentiation 18 Immunostaining 18 RNA isolation 18 Microarray processing 18 Affymetrix arrays 18 M430v2 probe reannotation 19 Illumina arrays 19 Illumina probe reannotation 19 Bioinformatics 19 Translating the STRING network 19 QTL mapping 20 Network graph layout 20 Triplot 20 Enrichment analysis 20 Mammalian Adult Neurogenesis Gene Ontology 21 Introduction 21 Results 25 The cell stage ontology 25 The process ontology 25 Genes known to regulate hippocampal adult neurogenesis 26 Enrichment analysis 27 The MANGO gene network 27 Discussion 28 Hippocampal Coexpression Networks from the BXD Panel 31 Introduction 31 Results 32 Variation and covariation of gene expression across a panel of inbred lines 32 A hippocampal expression correlation network 32 Diverse neurogenesis phenotypes associate with discrete transcript networks 34 Discussion 34 Interactions Between Gene Expression Phenotypes and Genotype 37 Introduction 37 Results 39 QTL analysis and interval definitions 39 Pleiotropic loci and ‘trans-bands’ 39 Transcript expression proxy-QTLs can help in dissection of complex phenotypes 41 Interaction network 43 Discussion 43 Strain-Dependent Effects of Environment 47 Introduction 47 Results 48 Effects of strain and environment on precursor cell proliferation 48 Effects of strain and environment on learning behaviour 52 Transcript expression associated with different housing environments 53 Strain differences in transcript regulation 55 Distance-weighted coexpression networks 57 Discussion 58 Expression Time Course from Differentiating Cell Culture 61 Introduction 61 Results 63 Differentiation of proliferating precursors into neurons in vitro 63 Transcripts associated with stages of differentiation 63 Early events in NPC differentiation 64 A network of transcript coexpression during in vitro differentiation 66 Discussion 67 Integrated Gene Interaction Networks 71 Introduction 71 Results 72 Description of network layers 72 Merging of network layers to a multigraph 74 A network of genes controls neural precursor proliferation in the adult hippocampus 75 Novel candidate regulators of adult hippocampal neurogenesis 77 Novel pathways regulating adult hippocampal neurogenesis 77 Discussion 79 General Discussion 81 References 89 Selbständigkeitserklärung 107

The neocortex is the most recently evolved part of the mammalian brain which is involved in a repertoire of higher order brain functions, including those that separate humans from other animals. Humans have evolved an expanded neocortex over the course of evolution through a massive increase in neuron number (compared to our close relatives-­‐‑ the chimpanzees) in spite of sharing similar gestation time frames. So what do humans do differently compared to chimpanzees within the same time frame during their development? This dissertation addresses this question by comparing the developmental progression of neurogenesis between humans and chimpanzees using cerebral organoids as the model system. The usage of cerebral organoids, has enabled us to compare the development of both the human neocortex, and the chimpanzee neocortex from the very initiation of the neural phase of embryogenesis until very long periods of time. The results obtained so far suggest that the genetic programs underlying the development of the chimpanzee neocortex and the human neocortex are not very different, but rather the difference lies in the timing of the developmental progression. These results show that the chimpanzee neocortex spends lesser time in its proliferation phase, and allots lesser time to the generation of its neurons than the human neocortex. In more scientific terms, the neurogenic phase of the neocortex is shorter in chimpanzees than it is in humans. This conclusion is supported by (1) an earlier onset of gliogenesis in chimpanzees compared to humans which is indicative of a declining neurogenic phase, (2) an earlier increase in the chimpanzee neurogenic progenitors during development, compared to humans, (3) a higher number of stem cell– like progenitors in human cortices compared to chimpanzees, (4) a decline in neurogenic areas within the chimpanzee cerebral organoids over time compared to human cerebral organoids.

Der Hippocampus ist ein Hirnareal welches fundamental für die Generierung von bewussten Erinnerungen und für die Etablierung von flexiblen Beziehungen zwischen kontextuellen Repräsentationen ist. Hippocampale Fehlfunktion während des Alterns wurde als ein Schlüsselfaktor für den kognitiven Abbau vorgeschlagen und im Kontext einer schnell alternden Bevölkerung wird es zwingend notwenig, mögliche Mechanismen zu verstehen, die diese Einschränkungen verhindern oder rückgängig machen können. Der Hippocampus ist ein von nur zwei Arealen im Gehirn, in dem neue Neuronen ständig im Erwachsenenleben erzeugt werden. Deren Rolle beim Lernen und bei Gedächtnisfunktionen ist jedoch nicht gut verstanden. Interessanterweise wurde adulte hippocampale Neurogenese als eine zelluläre Komponente eines Gehirnreservenmechanismus vorgeschlagen, mit dem Potenzial kognitive Fähigkeiten ein Leben lang zu erhalten sowie ein mögliches Ziel für therapeutische Ansätze darzustellen. In dieser Arbeit habe ich eine spezifische, genetisch-bedingte Expandierung von hippocampalen Nervenstammzellen genutzt, um deren intrinsisches Potenzial, neugeborene Neuronen zu erzeugen, auszuschöpfen, was zu einer lebenslangen erhöhten Neurogenese geführt hat. Dies hat die hippocampale Funktion auf mehreren Ebenen gefördert, vom verbesserten flexiblen Lernen in Navigationsaufgaben in der Jugend, über Kompensation des altersbedingten kognitiven Abbaus bis hin zur Verjüngung von kontextuellem Gedächtnis beim Altern. Zusammengefasst stellt meine Arbeit ein besseres Verständnis des funktionellen Beitrags der Neurogenese zu Lernen und Gedächtnis zur Verfügung and zeigt, dass kritische Aspekte hippocampaler kognitiver Beeinträchtigung im Alter rückgängig gemacht oder ein Leben lang durch extrinsische Ausnutzung der endogenen Hirnreserven kompensiert werden können.

Among mammals, primates are exceptional for their large brain size relative to body size. Relative brain size, or encephalization, is particularly striking among humans and their direct ancestors. Since the human-chimp split 5 to 7 million years ago, brain size has tripled in the human lineage (Wood & Collard 1999). The focus of this doctoral work is to investigate some of the cell biological mechanisms responsible for this increase in relative brain size. In particular, the processes that regulate symmetric cell division (ultimately generating more progenitors), the constraints on progenitor proliferation, and how neural progenitors have overcome these constraints in the process of primate encephalization are the primary questions of interest. Both functionally analyses in the mouse model system and comparative neurobiology of rodents and primates are used here to address these questions. Using the mouse model system, the cell biological role of the Aspm (abnormal spindle-like microcephaly associated) protein in regulating brain size was investigated. Specifically, Aspm function in symmetric, proliferative divisions of neuroepithelial (NE) cells was analyzed. It was found that Aspm expression in the mouse neuroepithelium correlates in time and space with symmetric, proliferating divisions. The Aspm protein localizes to NE cell spindle poles during all phases of mitosis, and is down-regulated in cells that undergo asymmetric (neurogenic) cell divisions. Aspm RNAi alters the division plane in NE cells, increasing the likelihood of premature asymmetric division resulting in an increase in non-NE progeny. At least some of the non-NE progeny generated by Aspm RNAi migrate to the neuronal layer and express neuronal markers. Importantly, whatever the fate of the non-NE progeny, their generation comes at the expense of the expansion of the proliferative pool of NE progenitor cells. These data have contributed to the generation of an hypothesis regarding evolutionary changes in the regulation of spindle orientation in vertebrate and mammalian neural progenitors and their impact on brain size. Specifically, in contrast to invertebrates that regulate the switch from symmetric to asymmetric division through a rotation of the spindle (horizontal versus vertical cleavage), asymmetric NE cell division in vertebrates is accomplished by only a slight deviation in the cleavage plane away from the vertical, apical-basal axis. The requirement for the precise alignment of the spindle along the apical-basal axis in symmetric cell divisions may have contributed to selection on spindle “precision” proteins, thus increasing the number of symmetric NE cell division, and contributing to brain size increases during mammalian evolution. Previous comparative neurobiological analyses have revealed an increase in basally dividing NE cells in the brain regions of highest proliferation and in species with the largest brains (Smart 1972a,b; Martinez-Cerdeno et al. 2006). The cell biological characteristics of these basally dividing cells are still largely unknown. We found that primate basal progenitors, similar to rodent apical progenitors, are Pax6+. This suggests that primate basal progenitors may share other properties with rodent apical progenitors, such as maintenance of apical contact. Our previous finding that artificial alteration of cleavage plane in NE cells affects their ability to continue proliferating supports the hypothesis that the apical membrane and junctional complexes are cell fate determinants (Huttner & Kosodo 2005). As such, the need to maintain apical membrane contact appears to be a constraint on proliferation (Smart 1972a,b; Smart et al. 2002). Together, these data favor the hypothesis that primate basally dividing cells maintain apical contact and are epithelial in nature.

Degeneration of the noradrenergic locus coeruleus (LC) in aging and neurodegenerative diseases is well documented. Slowing or reversing this effect may have therapeutic implications. Phox2a and Phox2b are homeodomain transcriptional factors that function as determinants of the noradrenergic phenotype during embryogenesis. In the present study, recombinant lentiviral eGFP-Phox2a and -Phox2b (vPhox2a and vPhox2b) were constructed to study the effects of Phox2a/2b over-expression on dopamine β-hydroxylase (DBH) and norepinephrine transporter (NET) levels in central noradrenergic neurons. Microinjection of vPhox2 into the LC of adult rats significantly increased Phox2 mRNA levels in the LC region. Over-expression of either Phox2a or Phox2b in the LC was paralleled by significant increases in mRNA and protein levels of DBH and NET in the LC. Similar increases in DBH and NET protein levels were observed in the hippocampus following vPhox2 microinjection. In the frontal cortex, only NET protein levels were significantly increased by vPhox2 microinjection. Over-expression of Phox2 genes resulted in a significant increase in BrdU-positive cells in the hippocampal dentate gyrus. The present study demonstrates an upregulatory effect of Phox2a and Phox2b on the expression of DBH and NET in noradrenergic neurons of rat brains, an effect not previously shown in adult animals. Phox2 genes may play an important role in maintaining the function of the noradrenergic neurons after birth, and regulation of Phox2 gene expression may have therapeutic utility in aging or disorders involving degeneration of noradrenergic neurons.

Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. Stroke is the leading cause of disability worldwide. HSPA12B, a heat-shock protein recently identified expression specifically in endothelial cells, is able to promote angiogenesis. Here, we have investigated its effects on functional recovery at chronic phase of ischaemic stroke. Ischaemic stroke was induced by 60 min. of middle cerebral artery occlusion in transgenic mice with overexpression of HSPA12B (HSPA12B Tg) and wild-type littermates (WT). HSPA12B Tg mice demonstrated a significant higher survival rate than WT mice within 28 days post-stroke. Significant improved neurological functions, increased spontaneous locomotor activity and decreased anxiety were detected inHSPA12B Tg mice compared with WT controls within 21 days post-stroke. Stroke-induced hippocampal degeneration was attenuated in HSPA12B Tg mice examined at day 28 post-stroke. Interestingly, HSPA12B Tg mice showed enhanced peri-infarct angiogenesis (examined 28 days post-stroke) and hippocampal neurogenesis (examined 7 days post-stroke), respectively, compared to WT mice. The stroke-induced eNOS phosphorylation and TGF-β1 expression were augmented in HSPA12B Tg mice. However, administration with eNOS inhibitor L-NAME diminished the HSPA12B-induced protection in neurological functional recovery and mice survival post-stroke. The data suggest that HSPA12B promoted functional recovery and survival after stroke in an eNOS-dependent mechanism. Targeting HSPA12B expression may have a therapeutic potential for the stroke-evoked functional disability and mortality.

In the adult nervous system, neurotransmitters mediate cellular communication within neuronal circuits. In developing tissues and primitive organisms, neurotransmitters subserve growth regulatory and morphogenetic functions. Accumulated evidence suggests that acetylcholine, (ACh), released from growing axons, regulates growth, differentiation, and plasticity of developing central nervous system neurons. In addition to intrinsic cholinergic neurons, the cerebral cortex and hippocampus receive extensive innervation from cholinergic neurons in the basal forebrain, beginning prenatally and continuing throughout the period of active growth and synaptogenesis. Acute exposure to ethanol in early gestation (which prevents formation of basal forebrain cholinergic neurons) or neonatal lesioning of basal forebrain cholinergic neurons, significantly compromises cortical development and produces persistent impairment of cognitive functions. Neonatal visual deprivation alters developmental expression of muscarinic acetylcholine receptors (mAChR) in visual cortex, whereas local infusion of mAChR antagonists impairs plasticity of visual cortical neurons. These findings raise the possibility that exposure to environmental neurotoxins that affect cholinergic systems may seriously compromise brain development and have long-lasting morphologic, neurochemical, and functional consequences.