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Behavioural Brain Research 221 (2011) 367–378

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Behavioural Brain Research

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Review

Developmental aspects of the cholinergic system

Yael Abreu-Villaca ∗, Cláudio C. Filgueiras, Alex C. ManhãesLaboratório de Neurofisiologia, Departamento de Ciências Fisiológicas, Instituto de Biologia Roberto Alcantara Gomes, Centro Biomédico, Universidade do Estado do Rio de Janeiro,Av. Prof. Manoel de Abreu 444, 5 andar, Vila Isabel, Rio de Janeiro, RJ 20550-170, Brazil

a r t i c l e i n f o

Article history:Received 22 December 2009Accepted 26 December 2009Available online 6 January 2010

Keywords:AcetylcholineCholine acetyltransferaseVesicular acetylcholine transporterHigh-affinity choline transporterAcetylcholinesteraseNicotinic receptorsMuscarinic receptorsDevelopment

a b s t r a c t

Beyond its importance in sustaining or modulating different aspects of the activity of the central nervoussystem (CNS), the cholinergic system plays important roles during development. In the current review,we focus on the developmental aspects associated with major components of the cholinergic system:Acetylcholine, choline acetyltransferase, vesicular acetylcholine transporter, high-affinity choline trans-porter, acetylcholinesterase, nicotinic and muscarinic receptors. We describe when and where each oneof these components is first identified in the CNS and the changes in their levels that occur during thecourse of prenatal and postnatal development. We also describe how these components are relevantto many events that occur during the development of the CNS, including progenitor cells proliferationand differentiation, neurogenesis, gliogenesis, neuronal maturation and plasticity, axonal pathfinding,regulation of gene expression and cell survival. It will be noticed that evidence regarding the develop-mental aspects of the cholinergic system comes mostly from studies that used agonists, such as nicotine,and antagonists, such as hemicholinium-3. Studies using immunohistochemistry and genetically alteredmice also provided valuable information.

© 2010 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3672. Choline acetyltransferase, vesicular acetylcholine transporter and acetylcholine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3683. Cholinergic receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369

3.1. Nicotinic AChRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3693.2. Muscarinic AChRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371

4. Acetylcholinesterase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3725. High-affinity choline transporter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3736. Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374

1. Introduction

Acetylcholine (ACh) is a neurotransmitter widely distributed inthe central (and also peripheral, autonomic and enteric) nervoussystem. It is synthesized from choline and acetyl CoA by the cholineacetyltransferase (ChAT) enzyme and stored in presynaptic vesi-cles. The vesicular acetylcholine transporter (VAChT) is responsiblefor loading ACh into these vesicles. When released in the synapticcleft, ACh binds to two distinct types of receptors: Nicotinic acetyl-choline receptors (nAChR) and muscarinic acetylcholine receptors(mAChRs). Neuronal nAChR subtypes are located in pre- and post-

∗ Corresponding author. Tel.: +55 21 2587 6295; fax: +55 21 2587 6129.E-mail address: yael a v@yahoo.com.br (Y. Abreu-Villaca).

synaptic membranes of neurons and also in extrasynaptic locationsthroughout the CNS. In the cleft, ACh may also be hydrolyzed byacetylcholinesterase (AChE) into choline and acetate and almost50% of the choline derived from ACh hydrolysis is recovered bya presynaptic high-affinity choline transporter (CHT) supportingcontinued ACh production and release (Fig. 1) [8].

ACh, ChAT, VAChT, nAChRs, mAChRs, AChE and CHT are com-ponents of the cholinergic system and, as described below, eachone of them follows a characteristic developmental plan, from themoment they first appear in the different regions of the CNS to thetime when adult levels are achieved. Besides, these componentsplay important roles in many events that occur during the devel-opment of the central nervous system (CNS) (Fig. 2). In the ensuingparagraphs, we also review how events such as progenitor cells pro-liferation and differentiation, neurogenesis, gliogenesis, neuronal

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Fig. 1. Schematic representation of a cholinergic synapse. The neurotransmitteracetylcholine (ACh) is synthesized from choline and acetyl CoA by the enzymecholine acetyltransferase (ChAT) and is loaded by the vesicular acetylcholine trans-porter (VAChT) into presynaptic vesicles. ACh remains stored in these vesicles untilthe presynaptic terminal is activated. When released in the synaptic cleft, ACh maybind to two distinct types of receptors: Ionotropic nicotinic acetylcholine recep-tors (nAChR) and metabotropic muscarinic acetylcholine receptors (mAChRs), thelast ones coupled to G-protein. Neuronal nAChR subtypes are located mainly inpre- and postsynaptic membranes of neurons throughout the CNS. In the cleft, AChmay also be hydrolyzed by acetylcholinesterase (AChE) into choline and acetate.The choline derived from ACh hydrolysis is recovered by a presynaptic high-affinitycholine transporter (CHT).

Fig. 2. Components of the cholinergic system and events that occur during thedevelopment of the central nervous system that are affected by each one ofthem. Acetylcholine (ACh) has direct (solid lines) and indirect (dashed lines)routes of action. The latter one is associated with developmental events thatare affected by the action of acetylcholine on nicotinic and muscarinic receptors(respectively nAChR and mAChR). Acetylcholinesterase (AChE) also has direct (non-classical—solid lines) and indirect (classical—dashed line) effects on development.However, in the latter case, the effects are related to the enzymatic activity of AChEon ACh. As for the presynaptic high-affinity choline transporter (CHT), the cholineacetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT), thescarcity of data regarding direct effects of these components makes it difficult toclearly associate them with any of the listed developmental events.

maturation and plasticity, axonal pathfinding, regulation of geneexpression and cell survival are influenced by the aforementionedcholinergic system components.

2. Choline acetyltransferase, vesicular acetylcholinetransporter and acetylcholine

Choline acetyltransferase is the enzyme that catalyzes thebiosynthesis of acetylcholine [146]. In spite of the fact thatChAT has been used as a specific marker for cholinergic neu-rons [99,138,153,205], this enzyme is constitutively expressed inpractically all living cells [211–213]. In non-neuronal cells, theACh, synthesized by ChAT, is assumed to be continuously releasedin small amounts into the immediate extracellular space of thecell to maintain cellular homeostasis, regulating several basic cellfunctions, such as mitosis, cell differentiation, organization of thecytoskeleton, cell–cell interactions and regulation of immune func-tions [212,213].

Regarding the nervous system, ChAT has an extensive intra-neuronal distribution in many cholinergic neurons, being presentin cell bodies, dendrites, axons and axon terminals [85–86].Immunohistochemical studies in humans and other mammals havedemonstrated that ChAT-immunoreactive neurons are distributedin many areas of the adult brain [94,99,153,205]. In the 80s, Mesu-lam and collaborators proposed the nomenclature of Ch1 to Ch8 todesignate the cholinergic neurons of the basal forebrain and upperbrainstem [137–139,144]. Briefly, the Ch1 and Ch2 groups, whichmainly project to hippocampus, are located within the medial sep-tal nucleus and the vertical limb nucleus of the diagonal band ofBroca, respectively. The Ch3 group is located within the horizon-tal limb nucleus of the diagonal band of Broca and projects to theolfactory bulb, pyriform and entorhinal cortices. Neurons of theCh4 group, mainly located in substantia innominata and magno-cellular basal nucleus, provide the major cholinergic innervation ofthe entire neocortex and the basolateral nucleus of the amygdala.Groups Ch5 and Ch6 provide the major cholinergic innervation ofthe thalamus and are contained mostly within the pedunculopon-tine nucleus and the laterodorsal tegmental nucleus, respectively.The Ch7 group is located within the medial habenular nucleusand provides the cholinergic innervation of the interpeduncularnucleus. The Ch8 group, located within the parabigeminal nucleus,provides a major source of extrinsic cholinergic projections to thesuperior colliculus. Other groups of cholinergic neurons in the cen-tral nervous system include the intrinsically organized cholinergicneurons of striatum [21,203], hippocampus [63,214], and cerebralcortex [42,207]. Populations of intrinsic cholinergic neurons havealso been described in some cranial nerve nuclei [85] and in theanterior horn of the spinal cord [89].

The expression of ChAT during brain development has beenextensively used as an indicator for the development of the cholin-ergic system. In rats, ChAT immunoreactive cells appear 1–2 daysfollowing generation of neurons from dividing progenitor cells[109]. In mice, however, the presence of some ChAT immunore-active mitotic cells has been observed in the germinative zonesof the forebrain [109,181]. These cells had been used to track thedevelopment of cholinergic neurons, providing a comprehensiveview of the places of origin, routes of migration and final desti-nations in the forebrain [181]. ChAT immunoreactive neurons inthe basal forebrain originate mainly from the medial ganglioniceminence and appear in a caudal-to-rostral gradient between E12and E18 (E1: first embryonic day) in rats [28,183] and betweenE14 and E17 in mice [181]. It has been demonstrated that theexpression of L3/Lhx8, a member of the LIM homeobox gene fam-ily, is required for the formation of cholinergic neurons in thebasal forebrain [129,130,225]. Accordingly, loss of Lhx8 function,

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via a targeted mutation in the mouse, blocks the formation of mostcholinergic neurons in the forebrain [225]. In addition, suppressionof L3/Lhx8 expression results in a dramatic decrease in the num-ber of ChAT-positive cells obtained from cultured mouse stem cells,while overexpression of L3/Lhx8 reverts this effect [129]. The Olig2,a basic helix–loop–helix transcription factor expressed in germi-native zones associated with generation of ChAT-positive cells hasalso been implicated in the development of cholinergic neurons inthe basal forebrain [65].

As compared to adults, ChAT levels remain relatively low untilbirth [14,46,72]. In the majority of ACh groups of the basal fore-brain, the expression of ChAT in cholinergic neurons and terminals,as well as of ChAT activity, rises progressively until adult levelsare attained approximately between the third and fourth weeksafter birth [43,51,72,84,141,151]. In the rat laterodorsal tegmen-tal nucleus for instance, ChAT activity increases strikingly duringthe second postnatal week and moderately thereafter, reaching theadult level in the fourth postnatal week [151]. This time courseof change in ChAT activity has been related to the time course ofthe development of some locomotor patterns and to decreases inthe amount of paradoxal sleep and frequency of muscle twitchesafter birth [150,151]. Of note, different patterns of developmentalchanges have been described in other cholinergic populations. Inthe striatum, ChAT activity increases slightly during the first twoweeks, rises strikingly at P15 (P1: first postnatal day), and reachesadult levels after P25 [141,198]. In the cortex and hippocampus, asharp increase in ChAT activity occurs ten days after birth, reachingclose to adult levels at three weeks of age [107].

In general, the levels of ChAT activity reflects developmentalchanges in ChAT mRNA levels, with the increase in mRNA precedingthat of enzyme activity by about 2 days [84]. Of note, the ChAT andthe vesicular acetylcholine transporter (VAChT) genes are arrangedin a common locus, referred to as “the cholinergic locus”, wherethe gene encoding the VAChT is located within the first intron ofthe gene encoding ChAT and is in the same transcriptional orienta-tion as ChAT [56,209]. The VAChT and ChAT co-expression, whichis required for the characterization of the cholinergic phenotype[34,209], has been studied during development. It was reportedthat VAChT protein levels parallels that of ChAT during develop-ment, whereas VAChT gene transcription begins much earlier indevelopment than does ChAT, with greater than half of adult VAChTmRNA levels present in the rat at birth, compared with only 20% ofadult levels of ChAT mRNA at this time [84].

Taken together, the aforementioned studies indicate that theearliest development of ACh machinery take place in regions con-taining cholinergic cell bodies, followed later by their target areas.For instance, ChAT activity in the medial septum, diagonal bandand magnocellular preoptic region preceded detectable enzymeactivity in their targets at hippocampus and cortex by some days[198]. Several studies have demonstrated that cholinergic neuronscan synthesize ACh during their migration and that these neuronscan release ACh from their growth cones prior to target contact orsynapse formation [91,196,220]. Interestingly, ACh secretion dur-ing development is not restricted to the growth cone, but has alsobeen reported along the neurite and at the soma [7,11,222]. Indeveloping Xenopus neurons, ACh secretion is mediated by vesiclesthat are recycled locally within the axon and that can be detectedthroughout the entire axonal surface [222]. In cultured Drosophilaneurons, the machinery for spontaneous quantal ACh secretionis present throughout the entire neuron, whereas ACh packets atthe soma are considerably greater than those at the growth cones[219]. It has been suggested that ACh released in the extracellu-lar medium from growing axons contributes to the developmentof neuronal networks [11,177]. Accordingly, it has been demon-strated that ACh participates in axonal pathfinding and navigationby modulating the rate and direction of axonal growth [226] and

that it affects axonal outgrowth as well as growth cone morphology[158,177].

There is a large body of literature demonstrating that the activityof both ChAT and VAChT are developmentally regulated. Partic-ularly, several extracellular factors are capable of regulating thecholinergic phenotype and of exerting trophic effects on devel-oping cholinergic neurons [6,20,123,152,199,231,232]. One of thebest characterized factors is the nerve growth factor (NGF). NGFis a member of the neurotrophin family of structurally relatedpolypeptides crucial for differentiation, survival and function ofcertain neuronal populations [17,116]. Large et al. [107] demon-strated that the developmental increase in NGF content of thebasal forebrain parallels that of the neocortex and hippocampus,and that it precedes an increase of similar magnitude in ChATactivity. NGF released from targets upregulates the expressionof the ChAT and VAChT genes in developing cholinergic neu-rons [22,120,127,161,199] interacting with two receptors: Thelow-affinity neurotrophin receptor p75NTR and the high-affinityreceptor TrkA [22,120]. In addition to NGF, other related neu-rotrophins, such as the brain-derived neurotrophic factor (BDNF),also promote the basal forebrain cholinergic phenotype [6,152].

Studies with genetically modified mice have shown that bothVAChT and ChAT play an essential role in the normal developmentof the cholinergic system. ChAT homozygous knockout mice arestillborn and present several neuromuscular alterations such asincreased number of motoneurons, muscle hyperinnervation andalterations in ACh receptor clusters in muscle [29]. Mice lackingVAChT (VAChT knockout) present neuromuscular abnormalitiessimilar to those found in ChAT knockout and also die shortly afterbirth [48]. However, mice with reduced levels of VAChT (VAChTknockdown) are viable and have been used to evaluate the role ofthis transporter [49,163]. For instance, VAChT knockdown homozy-gous mice with 65% reduction in the expression levels of VAChT aremyasthenic and have cognitive deficits in tasks involving objectand social recognition during adulthood [163]. Similarly, humanspresenting mutations in cholinergic locus present congenital myas-thenic syndrome associated with frequently fatal episodes of apnea[154]. Despite of the fact that the abnormalities observed in mutantmice and humans are likely attributable to the loss of ACh func-tion, it is not possible to discard the possibility that ChAT and/orVAChT have other nonenzymatic activities during development.The abnormal phenotype observed in knockdown and knockoutmodels may also be caused by some compensatory mechanismtriggered by the early absence of ACh. In this sense, the post-transcriptional gene knockdown through RNA interference (RNAi),which was recently developed to promptly disrupt CHAT expres-sion [178], could provide important contributions.

3. Cholinergic receptors

ACh receptors are differentially expressed throughout the CNS,where they play a role in the modulation of cell proliferation andsurvival, neuronal differentiation, regulation of gene expression,synapse formation and maturation, axonal pathfinding and neu-rotransmitter release. As described below, studies on the effectsof both nAChRs and mAChRs agonists and antagonists have beenextremely helpful to the understanding of the importance of thesereceptors to brain development (Table 1).

3.1. Nicotinic AChRs

Nicotinic receptors are pentameric ligand-gated ion channelswith different � (�2–10) and � (�2–4) subunits [71]. Multiple sub-types of neuronal nAChR can be formed from various combinationsof subunits and these subtypes can be classified into two major

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Table 1Frequently used central acetylcholine receptors (AChRs) agonists and antagonists in experimental studies.

Nicotinic AChR Muscarinic AChR

Agonists Antagonists Agonists Antagonists

Carbachol �-bungarotoxin (�7) Carbachol AtropineCytisine (�4�2) Mecamylamine Muscarine PirenzepineDihydro-�-erythroidine D-tubocurarine Oxotremorine sesquifumarate Quinuclidinyl benzilateEpibatidine Methyllycaconitine (�7) ScopolamineNicotine5-iodo-3-(2(S)-azetidinylmethoxy)pyridine dihydrochloride (�2)

Into brackets subtype or subunit specificity.

classes containing either a heteropentameric or a homopentamericstructure. Among the heteromeric receptors, the �4�2 nAChRs arethe predominant subtype in mammalian brain [230], while amongthe homomeric class, the �7 is the one most highly expressed [39].Receptors containing �9 and/or �10 subunits are usually classifiedwith �7 [166].

Nicotinic receptors subunits are among the first membrane pro-teins to appear during CNS development. Functional nAChRs wererevealed by patch-clamp measurements in fetal mouse cerebralcortex as early as on E10 [16]. In the rat cortex, the �2 subunithas been detected on E12–E13 while the �4 subunit is initiallyexpressed on E17–19 [229]. Additionally, the �7 binding sites havebeen observed in the hippocampus and posterior hypothalamusas early as on E12 [200]. The temporal and regional expression ofindividual subunits may also vary during postnatal development.For example, in the rat cortex and hippocampus, �2 mRNA is lowon P7 and increases by P14, whereas �7 mRNA peaks on P7. Cor-tical �4 mRNA peaks on P14 and remains high on P28, whereashippocampal/septal �4 mRNA is higher on P7 and P14 when com-pared to P1 and P28 [184]. The expression of nAChRs is still modifiedlate during postnatal development so that adolescents expressgreater numbers of �4�2* and �7* nAChRs compared to the samebrain regions of adults. [53], even thought there is also evidence ofregion-dependent effects [169]. In the human fetal brain, nAChRsare detected in the first trimester, gradually increasing up to mid-gestation. Even though there is evidence that nAChR levels remainconstant or decrease throughout postnatal life depending on thebrain region and receptor composition [58,59,70,83], there is scantinformation regarding infancy and adolescence.

These changes in nAChR expression occur during critical pre-natal and postnatal periods of neuronal development, involvingmechanisms associated with neurogenesis, cell migration and dif-ferentiation, and synaptogenesis. Accordingly, the critical rolesthese receptors are believed to play in many aspects of brain devel-opment seem to depend on their regional and temporal expression.In addition, as detailed below, distinct nAChRs present particularpermeability to ions and activate distinct signaling pathways, sothat the ability of these receptors to modulate brain development isalso dependent on the pharmacology of their subunit composition.

Although the cation permeability of the subtypes is influ-enced by their subunit composition, the nAChRs are permeableto the monovalent Na+ and K+ ions, as well as to Ca2+. Ca2+

signals are pivotal in shaping nAChR-mediated effects. Two dif-ferent classes of neuronal nAChRs may be identified accordingto their Ca2+ permeability, which correlates with other pharma-cological and structural properties: (1) homopentameric nAChRscontaining subunits (�7–�9) that exhibit the highest measuredCa2+ permeability values; (2) heteropentameric nAChRs, alwayscomprising at least one � (out of �2–�6) and one � (out of �2–�4)subunits, with lower measured Ca2+ permeability [47,64]. Fol-lowing nAChR stimulation, Ca2+ can enter cells either directly,through the intrinsic ion channel, or indirectly following voltage-gated Ca2+ channel activation. Additionally, Ca2+ levels can be

amplified via Ca2+-induced Ca2+ release from intracellular stores[47].

Indeed, Ca2+ influx through nAChR channels has been shownto modulate several Ca2+-sensitive events [47,64]. Interestingly,release of Ca2+ from intracellular stores contributes to nicotine-elicited increases in intracellular Ca2+ [40]. Nicotine was shownto stimulate proliferation of neural progenitor cells derived fromthe P19 murine embryonal carcinoma, and this effect was depen-dent on Ca2+ mobilization from intracellular stores [167]. NicotinicAChRs-mediated mobilization of Ca2+ is also relevant to otherdevelopmental events. At immature synapses from neonatal rathippocampal slices, activation of presynaptic intracellular Ca2+

stores contributes to nicotine-elicited increase of glutamate release[114]. The �7 nAChR is believed to be involved in the regulationof neuronal growth, differentiation and synapse formation duringdevelopment. By modulating intracellular Ca2+ levels, this recep-tor may regulate events such as activation of second messengersystems and induction of immediate-early gene expression [5,30].The �7 nAChRs modulate neurite outgrowth. In this regard, bothstimulatory and inhibitory effects have been described. In the chickciliary ganglion from E8 embryos, where �7 nAChRs have a pre-dominantly nonsynaptic location on neurons, pulses of ACh andnicotine were shown to restrict neurite growth in cell culture andinduce retraction in a Ca2+-dependent way. This effect was blockedby the �7 nAChR antagonist �-bungarotoxin, a poisonous com-ponent present in snake venom [164]. Similar inhibitory effectson neuritic extension mediated by �7 nAChRs were described inPC12 cells [36]. Interestingly, this effect seems to be dependenton the stage of development. For instance, an opposite effect wasdescribed in cultures of dorsal root ganglia neurons obtained fromE12 chick embryos, in which treatment with the agonists muscarineand carbachol elicited longer fibers, a higher number of fibers perneuron and increased neurofilament protein expression. In addi-tion, concomitant use of agonists and antagonists, such as atropineor mecamylamine, counteracted the increase in fiber outgrowth,indicating that neurite outgrowth is mediated by both muscarinicand nicotinic receptors. In contrast, modulated expression of neu-rofilament proteins by cholinergic agonists was not evident on E18,when sensory neurons have completed their differentiation [195].Finally, in a primary culture model of neonatal rat olfactory bulbs,the agonist carbachol significantly increased neuritic length, aneffect that was abolished by co-treatment with �-bungarotoxin,which argues for a positive role for �7 nAChRs in neuritic outgrowthalso in the rat olfactory bulb [44].

Activation of nAChRs by ACh can also affect the behavior ofneuronal growth cones, regulating their morphology, motility andpathfinding. In explant cultures of thalamic neurons, ACh exposurechanged the morphology of growth cones; they became larger andextended more filopodia. A quantitative analysis of growth arearevealed that growth cones increased by almost up to 50% in sizein the presence of ACh in the culture medium [177]. In develop-ing oocytes, d-tubocurarine, a nicotinic AChR antagonist, inhibitedelectric field-induced orientation of cultured neurites toward the

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cathode, whereas atropine, a muscarinic AChR blocker, markedlyenhanced the guidance properties of the applied field [57].

Variations in the expression of particular nAChR subtypes pro-foundly influence the developmental outcome across differentbrain regions. For instance, cell survival is modulated by nAChRs,and both neurotoxic and neuroprotective effects of nAChRs activa-tion have been described. In this regard, �4�2 nAChRs have beenshown to promote survival, while �7 nAChRs seem to mediateneurotoxic effects of nicotine. Nicotine was shown to be neu-roprotective in vivo, in a rodent model of neonatal excitotoxicbrain injury, and in vitro, in primary cultures of cortical neurons.Inhibition of �4�2 nAChRs by using the antagonist dihydro-�-erythroidine (DH�E) or by using �2−/− knockout mice was shownto abolish the protective effect of nicotine both in vivo and in vitro.In contrast, activation of �7 nAChR was deleterious in neonates,whereas its blockade, either by using the �7 antagonist methyl-lycaconitine (MLA) or by using �7−/− knockout mice providedneuroprotection [108]. A genetic mutation that prolongs the dura-tion of �7 channel opening results in dramatic increases in celldeath, possibly due to increased Ca2+ influx [157]. Berger et al.[19] have shown that �7 nAChR activation by low doses of nico-tine results in apoptotic cell death of primary and immortalizedhippocampal progenitor cells.

Interestingly, neurodevelopmental actions of AChR activationmay also depend on the differentiation state of the cell. Distinctstages of differentiation are associated with differential expressionof nAChRs subunits, which, in turn, were shown to affect cell sur-vival. In HC2S2-immortalized hippocampal progenitors, nicotineis cytotoxic to undifferentiated cells, whereas it spares the samecells once differentiation has been induced. Nicotine, acting vianAChRs, inhibits proliferation in embryonal carcinoma P19 cells,but promotes it following induction of neuronal differentiation andtransition to neural progenitor cells [167]. Evidence from studiesthat used mutant mouse embryos with hypersensitive �4 subunitsshows that the continuous activation of hypersensitive nAChRson neurons in the substantia nigra and ventral tegmental area isassociated with severe reduction of nigral dopaminergic cells asmaturation progresses during the course of prenatal development[105]. In accordance with these data, nicotine was shown to exertpositive trophic effects primarily on undifferentiated pheochro-mocytoma PC12 cells, whereas with differentiation, the effectsundergo a transition to neurotoxicity [2]. Nicotine exposure wasshown to upregulate mRNA expression of growth factors in the hip-pocampus during the first postnatal week of rats, a period of intenseneurogenesis in this region [192], and this upregulation is hypoth-esized to reduce programmed cell death [90]. Finally, increasedapoptotic cell death due to nicotine exposure was identified evenduring the adolescence of rodents, resulting in reduced density ofboth neuronal and glial cell populations. These findings suggest thatnAChR-mediated modulation of cell survival persists at late stagesof brain development. [95,155].

Regulation of gene expression is an important aspect of braindevelopment. Nicotinic AChRs may directly or indirectly lead to thephosphorylation of cAMP response element binding protein (CREB),which results in gene transcription. CREB controls the transcriptionof many gene products, and it is implicated in many impor-tant developmental events [89]. Nicotinic AChR-mediated CREBphosphorylation requires both Ca2+/calmodulin-dependent pro-tein kinase (CAMK) and mitogen-activated protein (MAP) kinaseactivation as well as Ca2+ release from internal stores [37,88]. Inthe chick ciliary ganglion, this activation is opposed by L-typevoltage-gated Ca2+ channels, which induce phosphatase activitythat dephosphorylate CREB, thereby extinguishing the nAChR sig-nal [37]. In contrast, in the developing hippocampus, voltage-gatedCa2+ channels do not cause dephosphorylation of CREB and ter-mination of signal. In fact, nAChR-mediated gene transcription in

the hippocampus has both glutamate-independent and glutamate-dependent components, the latter one possibly resulting frompresynaptic facilitation of glutamate release [88]. The low levels ofsustained nicotinic stimulation required for transcriptional effectsare consistent with those achievable either by the normal septalcholinergic innervation of the hippocampus or by repeated tobaccoexposure [88].

Interestingly, both nicotine administration and exposure to anenvironment previously associated with rewarding properties ofnicotine results in an increase of CREB phosphorylation in sev-eral brain regions [31,208]. Nicotine activates a broad array ofnAChRs subtypes. Therefore, it excites different types of neurons,thus perturbing baseline neuronal communication, interferingwith synaptic properties and modulating synaptic plasticity. [131].These nicotine-elicited alterations seem to reorganize patterns ofsynaptic connectivity which, in turn, alters the operation of brainregions involved in incentive motivation and reward (such as thenucleus accumbens), judgment and inhibitory control of behavior(such as the prefrontal cortex), thus contributing to some of thepersistent sequela associated with drug use, including addiction[171].

Among the different types of neuronal plasticity, CREB has beenimplicated in those associated with learning and memory [189]. Inthis regard, long-term changes seen after in utero nicotine exposurein rodents reproduce behavioral deviations that are seen in chil-dren of smokers, including cognitive impairment [160,202]. Indeed,the period of susceptibility to nicotine extends into adolescence,since learning/memory detrimental effects of adolescent nicotineexposure were also described in mice [1].

Studies in animal models have also shown that ubiquitouslydistributed presynaptic nAChRs serve as ligand-gated ion chan-nels that promote neurotransmitter release. Thus, nAChR appearto exert a predominantly modulatory influence on brain mecha-nisms. Both Na+ and Ca2+ fluxes associated with nAChR activationcan influence neurotransmitter release [216]. In fact, the release ofclassical neurotransmitters such as dopamine, noradrenaline, ACh,glutamate and GABA are facilitated by nicotine exposure [215]. Theabove-mentioned facilitation of neurotransmitter release mediatedby nAChRs is believed to be relevant to the proper maturation ofseveral neurotransmitter systems. In this regard, during the earlypostnatal period of rats, most glutamatergic synapses in the hip-pocampus are immature and functionally silent. However, bothACh and nicotine strongly enhance neurotransmitter release andconvert presynaptically silent synapses into conductive ones viapresynaptic �7 nAChR-mediated increases in Ca2+ [128]. It is likelythat a similar facilitation of neurotransmitter release occurs inother brain regions and during distinct phases of developmentand that presynaptic nAChRs determine the maturation of distinctneurotransmitter systems. Besides, the inappropriate activationof presynaptic nAChRs may be an important mechanism throughwhich cholinergic agonists such as nicotine disturb several aspectsof brain development.

3.2. Muscarinic AChRs

Five subtypes of mAChRs, M1, M2, M3, M4 and M5 have beendescribed. The receptor protein is composed of seven transmem-brane domains. The third inner cytoplasmic mAChR loop interactswith the G-protein and this portion of the protein is believed toinfer different functional characteristics to the receptor [26,35,103].Distinct signaling pathways are activated depending on the sub-type of mAChR. The M2 and M4 subtypes were shown to inhibitadenylyl cyclase activity through activation of a pertussis toxin(PTX)-sensitive inhibitory G-protein (Gi), a mechanism that leadsto a reduction in the levels of cyclic AMP. In turn, M1, M3 andM5 subtypes are generally coupled to a PTX-insensitive G-protein

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(Gq) which activates phospholipase C. Activation of phospholipaseC leads to the formation of inositol 1,4,5-trisphosphate (IP3) anddiacylglycerol (DAG). IP3 causes mobilization of Ca2+ from intracel-lular stores, while DAG activates protein kinase C (PKC) isozymes[45,206]. Muscarinic AChRs activation modulates several types ofion channels: For example, M1 and M2 receptors in the mouseand M1 and M4 receptors in the rat depress Ca2+ currents whilemuscarinic suppression of the M-type K+ current in the rat is alsomediated by M1 receptors [185]. MAP kinases and small GTPases,such as rho and rac, are also activated by mAChRs [45,206].

M1, M2 and M4 mAChRs are the predominate mAChR sub-types expressed in the adult brain. As examples, approximately40% of total mAChRs in the cortex of the rodent brain representM1, 37% M2 and approximately 15% M4. In the hippocampus, 36%represent M1, 33% M2 and 27% M4 [115]. The mAChR is presentin both the forebrain and the hindbrain during early develop-mental stages. Binding of [3H]-quinuclidinyl benzilate (QNB), anon-selective mAChR antagonist, has been demonstrated in humanfetal brain from early gestation [75]. In the rat, mAChR binding sitesappear in the spinal cord on E13. On E15, binding sites are evi-dent in the rhombencephalon and mesencephalic structures andon E16, in forebrain regions [182]. There is evidence of changesin receptor expression during postnatal life. As an example, in thevisual cortex of rats, the laminar distribution of the mAChRs mRNAvaries depending on the receptor subtype and on the age. In general,there are increases in mRNA expression during postnatal develop-ment and the final adult distribution is achieved during adolescence[174,175]. In the cat, both M1 and M2 subtypes of AChRs bindingsites are detected in the striatum as early as on E40 and in thesubstantia nigra on E45 (birth on E65). During fetal and perinataldevelopment, overall muscarinic binding increases in the striatumbut is stable in the substantia nigra [148,149].

When compared to nAChRs, much less is known about what theactivation of M1–M5 receptors does in the brain [162]. However,studies on the effects of mAChRs agonists and antagonists havealready demonstrated that these receptors mediate several effectsof ACh in the developing CNS. In this regard, mAChRs have beenshown to mediate effects of ACh on proliferation, differentiationand survival. In embryonic P19 carcinoma cells, a widely acceptedin vitro model for early neurogenesis, muscarine induces prolifer-ation in progenitor cells by activation of M1, M3 and M5 receptorsand mobilization of intracellular Ca2+ stores, whereas M2 receptoractivity mediates neuronal differentiation [167,168]. Neuroepithe-lial cells in the ventricular and subventricular zones of embryonicrat cortex express M2, M3 and M4 subtypes of mAChRs and acti-vation of mAChRs stimulates proliferation of not only multipotentprecursor cells, but also those cells progressing along neuronal lin-eages [125,126]. The exact intracellular mechanisms underlyingmAChR-modulated DNA synthesis and neurogenesis are not fullyunderstood. However, several pathways through PTX-sensitive G-proteins, cytosolic Ca2+ signaling, protein kinase C, MAP kinasephosphorylation, phosphatidylinositol-3 kinase (PI-3K) and c-Srchave been shown to play roles [125,126]. Li et al. [118] demon-strated that stimulation of mAChRs with the agonist carbacholactivates the extracellular signal-regulated kinase (ERK)1/2 and PI-3K. This, in turn, stimulates DNA synthesis in neural progenitorcells. These findings demonstrate that the PI-3K and MAP kinasesignaling pathways via mAChRs are involved in neural progenitorcell proliferation during early neurogenesis. [118]. The nonreceptorprotein tyrosine kinase c-src activity was also shown to be requiredfor the mAChR-mediated proliferation and neurogenesis in neuralprecursor cells via ERK1/2 and CREB [224]. Moreover, the mAChRantagonist atropine blocks the enhancement and inhibits the sub-sequent differentiation of progenitor cells [227]. The hippocampusis one of the few brain regions that maintains neurogenesis duringpostnatal life and newborn cells in the hippocampus of adult rats

express both M1 and M4 subtypes of mAChRs [142]. Lesion of septalcholinergic nuclei projecting to hippocampus suppresses prolifera-tion and short-term survival of newborn cells in the dentate gyrus,with a concurrent impairment in spatial memory [142]. In mousehippocampal slices, the activation of ERK, which is crucial for manyneural functions, including learning, memory, and synaptic plas-ticity, is mediated by the M1 mAChR subtype [20]. Donepezil, aselective AChE inhibitor, enhances survival of newborn neurons inthe hippocampal dentate gyrus and scopolamine, a mAChR blocker,suppresses it. Furthermore, donepezil enhances and scopolaminesuppresses phosphorylation of CREB, which is involved in cell sur-vival in this brain region. These results suggest that the activationof the central cholinergic transmission enhances the survival ofnewborn neurons in the dentate gyrus via CREB signaling. [102].Activation of mAChRs also plays important roles in the proliferationof rat cortical astrocytes [15,76,217] and oligodendrocyte progen-itor cells [41].

Neurite outgrowth is also modulated by mAChRs. In thalamusexplant cultures from E14 mice, application of ACh reduces thelength of axons in a dose dependent manner, an effect mediatedby both nicotinic and muscarinic AChRs, but with strongest effectsmediated by mAChR, as indicated by adding either nicotinic or mus-carinic agonists to the medium [177]. In the prenatal hippocampus,cholinergic stimulation of pyramidal neurons through the M1 sub-type of mAChR accelerates axonal growth through the inductionof Ca2+ mobilization and the activation of PKC and of ERK1/2[204]. Additionally, M3 mAChRs expressed in rat cortical astro-cytes stimulate neurite outgrowth in rat hippocampal neurons.These neurons, co-cultured with astrocytes previously exposed tothe cholinergic agonist carbachol, display longer neurites, whichdemonstrates that the release of permissive factors from astrocytesaccelerates neuronal development [77].

Furthermore, mAChR activity is implicated in neuronal plas-ticity, including long-term potentiation (LTP). Both M1 and M2/4subtypes of mAChR are involved in LTP induction in the dentategyrus of rats [119,124], while M2/4 subtypes regulate LTP in themouse visual cortex [156]. Evidence is also available indicating thatphosphorylation of CREB, activation of MAP kinase and ERK1/2 playroles in mAChR-mediated neuronal plasticity [68,74,80,173].

4. Acetylcholinesterase

AChE catalyses the hydrolysis of ACh, thereby interruptingcholinergic activity in the synapse. Mammalian AChE appears tobe encoded by a single gene [197]. In humans, the AChE gene islocated in chromosome 7 and in mice it is located in chromo-some 5 [67,165]. In most species, the AChE gene encodes a catalyticsubunit of about 75 kDa [176]. After translation, AChE moleculesfold forming functional monomers and assemble into disulfide-bonded dimers. The dimers can be induced to tetramerize by thenon-catalytic subunit “p” peptide in the case of neurons, or the col-lagenic ColQ protein in the case of skeletal muscle. The oligomericforms are then transported through the cells and released intothe synaptic cleft where they may attach to available sites on thespecialized synaptic basal lamina [176]. Two alternatively splicedisoforms of AChE that are particularly relevant for the nervoussystem have been well documented: the tetrameric AChE-S iso-form (‘S’, synaptic) and the ‘read-through’ variant of AChE (AChE-R)[134]. Although the enzymatic activity of the two isoforms is equiv-alent, they do differ in aspects such as ontogenetic expression,multimeric assembly, cellular localization and tissue specificity.Interestingly, it seems that AChE has an additional developmentallyregulated enzymatic activity as an aryl acylamidase [27].

AChE appears quite early during embryonal development of theCNS. In fact, its mRNA is already expressed by progenitor cells in

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the ventricular zone (VZ) and undifferentiated cells in the corti-cal plate (CP) [52]. At the beginning of neurogenesis in mice (E11),which is characterized by remarkable progenitor cell proliferation,AChE mRNA is co-localized in most VZ cells and its expression ismost intense in the apical portion, next to the ventricular lumen.At E13, clusters of adjoining cells in the basal portion of the VZpresent more pronounced cytoplasmic expression of AChE. By E15,AChE-expressing cell clusters are becoming progressively smallerso that by E17 they are limited to very small clusters or singlecells. In the intermediate zone, AChE mRNA expression, albeit lessintense than in the VZ, is observed in individual cells or in clustersof cells. These cells are radially oriented, indicating that they aremigrating from the VZ to the CP. With further development, AChEexpression in the CP appears in clusters of intensely labeled cells.These cells are more prominent in the superficial portion comparedwith the deep CP, suggesting that younger, newly arriving cells inthe CP express more AChE transcripts than cells that had alreadyundergone some differentiation. Subsequently, at E17, the intensesignals in the superficial cell layer of the CP become significantlyhigher than those of the deep CP portion [52]. In general, total AChEactivity increases about 15-fold between E9 and E19 [92]. Duringpostnatal development, up to P30, AChE activity increases in a waythat varies dramatically from one brain region to another [143,180].In humans, AChE activity also increases postnatally until it becomesfully established during adulthood [135].

In addition to this classic enzymatic role, AChE also hassome non-classical properties concerning SNC development. Forinstance, it is now accepted that AChE has functions associ-ated with adhesion, neurite growth, circuitry formation and evenapoptosis [96,113,186,188,193,223]. For instance, it was demon-strated that AChE expression correlates directly to the level ofcell–substratum adhesion of neuroblastoma cells [187]. Further-more, AChE forms a complex with amyloid precursor protein andperlecan that seems to be involved in substratum adhesion andpolarized migration of adherent cells [9]. That AChE has adhesiveproperties was also demonstrated in non-neuronal cells such asosteoblasts: blockade of some sites in the AChE molecule results ina concentration-dependent decrease in osteoblastic cell adhesion[66,93].

The adhesive properties of AChE may also help to explainanother of its non-classical roles: stimulation of neurite outgrowth.In fact, this was the first non-classical activity of AChE to beunequivocally identified [112]. As indicated above, AChE expres-sion occurs early in postmitotic neurons [111] and precedes theoutgrowth of neuritic processes [110,210]. Blocking the enzymaticactivity of AChE does not affect its role in promoting neurite growth[112]. The effects of AChE in stimulating neurite growth weredemonstrated in studies that used overexpression of this enzyme indorsal root ganglion neurons and neuroblastoma cells [24,101]. Theimportance of AChE to axon tract formation was also demonstratedin vivo: the thickness of the major longitudinal axon tract of theXenopus brain, the tract of the post-optic commissure, is reducedafter treatment with BW284C51, which blocks non-catalytic sitesin the AChE molecule [10].

In trying to explain how AChE exerts this non-classical activ-ity during development, the association between AChE and othermolecules has been a focus of study. For example, laminin, which isknown to bind to AChE [97,159], is an extracellular matrix pro-tein that participates in neuronal differentiation and adhesion.Although the exact mechanism that explains how AChE affectslaminin activity during development is yet to be described, it iswell established that laminin facilitates the migration of neuronalprecursors to their proper regions in the brain. It also elicits anddirects neurite outgrowth [73,121,170,172,190,228], possibly byinteracting with integrins, one of the most abundant family ofreceptors for molecules located in the extracellular matrix [106].

Curiously, classical and non-classical roles of AChE may associateduring development. ACh is known to arrest neurite growth afterbeing released from the growing tips. By metabolizing ACh, a clas-sical role, AChE could prevent this arresting effect, while, at thesame time, directing neurite growth toward its target through itsadhesive, non-classical, properties.

Studies using AChE knockout mice (AChE−/−) are also beginningto provide information concerning the relevance of this enzyme tothe normal development of the nervous system. In these strains,a series of adaptations allow animals to have brain and mus-cle functions that are compatible with long-term survival despitethe complete lack of AChE and abnormally high levels of ACh intheir synapses [38,54,82]. Interestingly, these animals are capableof forming central cholinergic pathways [136]. Some of the fac-tors that let animals survive postnatally are: The presence of theenzyme butyrylcholinesterase (BChE), which also metabolizes AChin the synapses; downregulation of acetylcholine (nicotinic andmuscarinic) and dopamine receptors; and morphological remod-eling of the endplate region [4,50,69,87,117]. It must be pointedout, however, that AChE−/− mice have significant behavioral andmotor deficits [55]. The analysis of the retina of AChE−/− miceindicated that AChE is fundamental to the normal development ofthis structure in a way that is unrelated to its enzymatic function,since BChE content was normal [33]. In these animals, the develop-ment of the laminar structure of the retina is disturbed during thefirst two postnatal weeks. The resulting anomalous circuits thatare assembled are not capable of sustaining photoreceptor activ-ity and long-term viability, in spite of a seemingly normal initialdevelopment of these cells. By three months, all photoreceptors,as well as cells from the inner retina, are lost due to apoptosis.In an additional study, neurons derived from dorsal root gangliaof embryonic AChE−/− and AChE+/+ mice indicated that axonalgrowth inhibition by organophosphorus pesticidesis is not relatedto the enzymatic activity of AChE [218].

5. High-affinity choline transporter

As previously mentioned, most of the ACh that is releasedinto the synaptic cleft is rapidly hydrolyzed by AChE intocholine and acetate. ACh hydrolysis interrupts cholinergic signal-ing and the resulting choline is readily transported back into thepresynaptic terminal, through the membrane, by a high-affinity,Na+-dependent, hemicholinium-3 sensitive (HC-3, a bicyclic, con-strained choline analog) mechanism. The CHT activity, supplyingthe choline that will be used by ChAT, is the rate-limiting step in thebiosynthesis of ACh. The ACh is then loaded into synaptic vesiclesby the VAChT [8,61,179]. The majority of the choline transportedby the CHT is efficiently converted to ACh [78,79,145], suggestingit is both metabolically linked and physically proximal to sites ofACh synthesis.

The CHT genes identified in mice, rats, and humans are respec-tively located on chromosomes 17, 9, and 2 [12,13]. The CHTproteins synthesized from these genes have thirteen transmem-brane domains with an extracellular N terminus and a cytoplasmicC terminus [13,201]. The development of CHT-specific antibod-ies has allowed the direct, activity-independent detection of CHTs[62,100,104,122,140]. Different pools of CHTs can be identifiedwithin cholinergic neurons [62,104,122]. At the cell body level,abundant CHT immunoreactivity is evident in association with therough endoplasmic reticulum membranes. This biosynthetic poollikely accounts for the CHT signal associated with cholinergic soma,because CHT immunoreactivity is not abundant at the cell somaplasmalemma. In contrast, CHT immunoreactivity is evident at theplasma membrane of presynaptic terminals, in line with its role insupport of ACh synthesis [32,194]. Interestingly, by far the greatest

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density of CHT labeling is surprisingly found to be associated withvesicles located inside the terminal.

In spite of its role in cholinergic systems, only a handful ofstudies have addressed developmental aspects of CHT, particularlyin the CNS. In rats [81], on E18 and right after birth, CHT levels,as demonstrated by a quantitative autoradiography method thatused [3H]HC-3, were high only in the medial habenula. In mostbrain regions, including the striatum, nucleus accumbens, olfac-tory tubercle, cortex, and hippocampus, significant increases in CHTlevels occurred primarily during the postnatal period. Although sig-nificant variations existed between brain regions, adult levels ofCHT were reached between P 15 and P21, with 2–12-fold increasesbeing observed. Differences between brain regions regarding CHTlevels is further emphasized in another study [221]. In this study, aperiod of comparatively high levels of [3H]HC-3 binding in the cere-bral cortex and midbrain + brain stem observed before birth wasfollowed by marked reductions during the first postnatal week andsubsequent increases, faster in the midbrain + brain stem, towardadult levels by the sixth postnatal week. Regarding the hippocam-pus and the striatum, the peak levels were observed during the 5thand 6th postnatal weeks and marked reductions toward adult lev-els were observed from then on. In mice, [23], the earliest, E14, andstrongest CHT mRNA expression was found in the spinal cord. Inter-estingly, mRNA levels remained more or less constant up to P30in this structure. Conversely, in the septum and striatum, a steadyincrease, faster in the former one, in mRNA expression, from hardlydetectable on E14 to a high level on P30, was observed. In the cere-bellum, the levels of CHT mRNA expression reached the highestlevel still during gestation, on E19, a peak that was followed by arapid decline from then on. Regarding the cortex and the hippocam-pus, mRNA levels remained low, with slight variations, from E14 toP30. By P30 CHT mRNA levels were highest in the spinal cord, stria-tum and septum. Lower levels were observed for the cortex andhippocampus and even lower levels were observed for the cere-bellum. A more recent study [133], using rats and restricted to thepostnatal development of the hippocampus, indicated that duringthe first postnatal week CHT mRNA expression rapidly increasedand then stabilized from P8 to P24. This period of stability was fol-lowed by a period of marked decrease in CHT mRNA levels in whichthe lowest level was reached by P34. From then on, a slight increasefollowed by a plateau (at 50% of peak levels) was observed up to P90.As for the CHT protein [23], it was detected in the spinal cord, sep-tum, striatum, cerebellum cortex and hippocampus as early as E14.However, levels were generally small. Significant accumulation ofthe protein was detected in the spinal cord by P8, in the striatum byP15 and in the septum by P30. Taken together, the aforementionedresults in mice indicate that, in most regions, protein accumulationis delayed in relation to mRNA expression.

As demonstrated in CHT−/− mice, the absence of CHT is notlethal during gestation but results in newborn animals that arehypoxic, immobile and that die within an hour after birth, pos-sibly due to the lack of ACh in cholinergic synapses that supportrespiration [60], an observation that corroborates previous find-ings that indicate that CHT function is necessary for sustained AChproduction and release [25,98]. Interestingly, these mice are mor-phologically normal an equivalent in weight when compared toCHT+/+ mice and CHT+/−. Furthermore, no differences in mor-phology and weight were observed concerning various organs,including the brain. Nissl staining also failed to indicate defectsin brain development in the CHT−/− mice. Conversely, CHT+/−mice, which display a 50% reduction in CHT protein levels, live intoadulthood and are able to reproduce. In general, these mice do notpresent significant differences in behavior (motor co-ordination,general activity, anxiety and spatial learning and memory) whencompared to CHT+/+ ones. On the other hand, a physical challenge,such as that provided by a treadmill paradigm, is able to uncover

impairments in performance in CHT+/− mice [18]. Therefore, thefew studies that are available concerning developmental aspectsof CHT in the SNC indicate that this protein is not directly criti-cal for the normal SNC development during the gestational period.The low cholinergic activity observed during this period can bemaintained by the low-affinity, Na+ independent, HC-3-insensitivecholine transport mechanism. However, CHT is indirectly respon-sible for normal SNC development after birth, since the increaseddemand on the cholinergic systems, associated with the control ofbreathing for example, depends on a constant supply of cholineto the presynaptic terminals that can only be maintained by thistransporter system.

6. Future directions

Despite the fact that there is plenty of information on severalaspects of the development of the cholinergic system, there arescant studies that aimed to investigate whether or how the devel-opment of each component of this system affects the others, i.e.to integrate information regarding all components of the system.Perhaps most importantly, as evidenced in this review (Fig. 2), thecomponents of the cholinergic system are responsible for executinginter-related events and developmental events are frequently influ-enced by more than one of these components. This suggests that theequilibrium between them is essential for their functions, there-fore, future studies are necessary to investigate how the complexnet formed by the components of the cholinergic system (whichmight go beyond the effect of each component), determines thenormal development of the CNS and how imbalances elicited forexample by environmental agents affect it.

Indeed, exposure to environmental agents that affect the cholin-ergic system may seriously compromise brain development andhave long-lasting morphologic, neurochemical, and functionalconsequences. Interestingly, the investigation of the effects ofneurotoxins is also a useful tool to identify the roles of com-ponents of this system on brain development. Until recently,most studies on developmental aspects of the cholinergic sys-tem focused on the prenatal and neonatal periods, however,it is now clear that, in several aspects, the adolescent brainis particularly vulnerable to neurotoxins. In this regard, recentstudies show that adolescents present a peculiar sensitivity tonicotine [3,191] and the fact that the maturation of central cholin-ergic system is consolidated during the periadolescent period[53,132,135,147,221] may explain, in part, this sensitivity. Accord-ingly, there has been an increasing number of studies on theeffects of neurotoxins during adolescence, which in the next fewyears may fill the gap of information regarding this period ofdevelopment.

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