Cholinergic influences on cortical development and adult neurogenesis

Behavioural Brain Research 221 (2011) 379–388

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Review

Cholinergic influences on cortical development and adult neurogenesis Elodie Bruel-Jungerman a,b,c , Paul J. Lucassen d , Fiona Francis a,b,c,∗ a

INSERM UMR-S 839, F75005, Paris, France Université Pierre et Marie Curie, F75005, Paris, France c Institut du Fer à Moulin, F75005, Paris, France d Center for Neuroscience, Swammerdam Institute of Life Science, University of Amsterdam, The Netherlands b

a r t i c l e

i n f o

Article history: Received 12 February 2010 Received in revised form 10 January 2011 Accepted 13 January 2011 Available online 25 January 2011 Keywords: Corticogenesis Neuronal proliferation Neurogenesis Differentiation Synaptogenesis Cholinergic innervation Hippocampus

a b s t r a c t In this review, we focus on immature neurons and their regulation by the cholinergic system, both during cortical development as well as during adult neurogenesis. We discuss various studies that indicate roles for acetylcholine in precursor development and neuronal differentiation. Cholinergic neurons projecting from the basal forebrain innervate the cerebral cortex during critical periods of neuronal development. Acetylcholine stimulation may help to promote a favourable environment for neuronal maturation. Afferents and their cortical target cells interact and are likely to influence each other during the establishment and refinement of connections. Intracortical cholinergic interneurons similarly have a local effect on cortical circuits. Reduced cholinergic innervation during development hence leads to reduced cortical thickness and dendritic abnormalities. Acetylcholine is also likely to play a critical role in neuronal plasticity, as shown in the visual and barrel cortices. Spontaneous nicotinic excitation is also important during a brief developmental window in the first postnatal weeks leading to waves of neural activity, likely to have an effect on neurite extension, target selection and synaptogenesis. In the hippocampus such activity plays a role in the maturation of GABAergic synapses during the developmental shift from depolarizing to hyperpolarizing transmission. The cholinergic system also seems likely to regulate hippocampal neurogenesis in the adult, positively promoting proliferation, differentiation, integration and potentially survival of newborn neurons. © 2011 Elsevier B.V. All rights reserved.

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Basic steps of cortical development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cholinergic innervation of the developing cortex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Basal forebrain cholinergic projections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Cholinergic interneurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Neurotrophins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Accumulating evidence for a role of the cholinergic system in cortical development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Maturation, synaptogenesis and plasticity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. A role for spontaneous cholinergic excitation in postnatal development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Ontogeny of chloride homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adult hippocampal neurogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. A multicellular complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Adult neurogenesis, cholinergic system and cognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Cholinergic regulation of adult hippocampal neurogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author at: INSERM UMR-S 839, F75005, Paris, France. Tel.: +33 1 45 87 61 45; fax: +33 1 45 87 61 32. E-mail address: fi[email protected] (F. Francis). 0166-4328/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2011.01.021

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1. Basic steps of cortical development Cortical development is characterized by a massive generation, migration and differentiation of neurons in a short time window, that eventually leads to the formation of an extensive network of afferent and efferent connections that form the complex, characteristic six-layered mammalian cortex (reviewed in [109]). Cortical axons grow towards their target cells either within the cortex or within other structures, with neurons from the thalamus and basal ganglia sending axons towards the cortex (reviewed by Molnar et al. [96]). Interestingly, the innervation of the cortex contributes to neuronal maturation. Also, interneurons originating from the ventral telencephalon arrive in the cortex during development and further contribute to these processes. The first postnatal month in the rodent is a time period of intensive axonal outgrowth and synaptogenesis during which numerous synapses are formed either on dendrites, or on perisomatic regions of target cells. The mechanisms underlying the specificity of this targeting are still unclear but likely involve compartmentalized adhesion molecules and receptors (reviewed in [49]). Eventually, the resulting cortical layers are characterized by a variety of different neuronal cell types that are highly interconnected with other cortical and subcortical regions. The hippocampal formation, an extension of the isocortex in the rodent, develops in a very similar manner. A specialized domain of this structure, the dentate gyrus, however, develops later than the pyramidal cells of the cornu ammonis (CA) fields. In contrast to the other postnatal regions, the dentate gyrus even continues to produce new neurons in the adult, a process known as adult neurogenesis.

2. Cholinergic innervation of the developing cortex 2.1. Basal forebrain cholinergic projections Large areas of the cerebral cortex are innervated by basal forebrain neurons. It has been estimated that between 14,000 and 18,000 nucleus basalis cholinergic neurons project to the adult rat cerebral cortex [93,115] while cholinergic projection neurons may make up one third of the total projections toward the rat somatosensory and prefrontal cortices [54]. Tracing studies in rodents established that cholinergic neurons in the basal forebrain innervate the entire cortex, including the hippocampus and amygdala, with a characteristic mediolateral and anteroposterior distribution pattern (reviewed in [5,10,138]). Axons from the medial septum and vertical diagonal band project to the hippocampus (Fig. 1); from the horizontal diagonal band projections run to the olfactory bulb and from the nucleus basalis of Meynert to the medial cortex, including the cingulate gyrus, and from the substantia innominata and the medial globus pallidus to the lateral neocortex. There are regional differences in this laminar pattern of innervation between cortical regions [128,138]. In some regions of the cat visual cortex, GABAergic neurons seem to be the preferential target of cholinergic input [12,39]. Cholinergic afferents innervate the cerebral cortex during extremely dynamic periods of neuronal differentiation and synaptogenesis, and play a critical role in these events (reviewed in [14,118]). Mechawar and Descarries [93] studied the timing of the cholinergic innervation of the rat cerebral cortex using a highly specific antibody and found some growth-cone tipped ChAT positive axons in the subplate of frontal, parietal and occipital cortices from birth onwards (P0, Fig. 2). By P4 there was an increased number, with some entering the cortical plate and marginal zone. A few faintly stained interneurons were also observed at this age. By P8 the number of labeled interneurons had increased and cholinergic

axons with varicose termini had formed a network in every cortical layer. A mature-like laminar pattern was already observed at P16, but it increased further in density to reach an adult appearance at P32. In terms of fiber densities between the different regions, this study showed an early rostral-caudal gradient. At P4, more fibers (with few varicosities) were observed in frontal and parietal cortices than in the occipital cortex. In all three areas and layers, the number of varicosities per micrometer of ChAT-immunostained axon increased sharply between P4 and P16 and stabilized thereafter to reach adult values. Basal forebrain neurons indeed show an early and rapid growth, axons increasing 2 cm in length and with 9000 varicosities per day [93]. The parietal cortex showed a more-or-less adult density of cholinergic axons at P16, ahead of the frontal and occipital cortices. At no time was there an indication of an overproduction or elimination of cholinergic axons or varicosities. The regional density attained in the adult frontal cortex was significantly higher than that attained in the parietal and occipital cortices. The differences in density of the innervation later observed between layers of a given area and/or between areas, may reflect the degree of branching of ACh axons. 2.2. Cholinergic interneurons Most of the cholinergic innervation in the cortex comes from fibers originating from the basal forebrain nuclei. However, early immunocytochemical studies suggested the existence of intracortical cholinergic neurons in the rat [38]. Indeed, Consonni et al. [31] have recently further characterized that ChAT positive neurons exist among a dense and intricate network of varicose cholinergic fibers. They are particularly dense in sensory cortical areas, especially in upper layers (II and III), present in the somatosensory cortex and also found in visual and auditory regions. Cells appear between P4 and P6 and there is an upsurge in the second postnatal week. Thus, the timing of apparition parallels the rapid invasion of cholinergic fibers [93]. Of interest, cells are often found close to the vessels and could therefore contribute to the local regulation of the cortical microvascular bed [38,45]. They have a variable GABAergic phenotype, with cells in the supragranular layers having a fusiform bipolar shape and those in deeper layers having a multipolar morphology. 2.3. Receptors Neuronal nicotinic acetylcholine (ACh) receptors belong to the family of excitatory ligand-gated channels and are pentameric assembled from different combinations of 12 different subunits. Nicotinic receptor subunit mRNAs appear from mid to late embryogenesis throughout the rat cerebral cortex and hence precede the cholinergic innervation from the basal forebrain [8,99,145]. Muscarinic receptors on the other hand are G-protein coupled and appear later. Muscarinic receptors gradually appear in postnatal cortex and may be more dependent on the presence of ACh [8]. 2.4. Neurotrophins Several studies have suggested that basal cholinergic neurons are influenced by, and to some extent dependent on, neurotrophic factors including NGF, neurotrophin 4/5, BDNF and NT3, which may attract or stabilize projections and promote survival of cells [3,35,56,113,134]. For example, Robertson et al. [113] tested the role of neurotrophin 3 (NT3) for axonal ingrowth into limbic cortical regions (including frontal, cingulate and insular cortex, dentate gyrus) that receive early, and relatively dense, cholinergic axons. Septal cholinergic neurons are formed during the third fetal week [117] and begin to send out axons by the late fetal stages. They

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Fig. 1. Cholinergic innervation of the cortex. Schemas originally published in [103], permission obtained for reproduction and modification. Sagittal sections of adult mouse brain showing origins of cholinergic projections. Axons from the medial septum (MS) and vertical diagonal band (VDB) project to the hippocampus (as depicted by arrows in the upper schema) and axons from the nucleus basalis (B) and substantia innominata (SI) project to the isocortex (as depicted by arrows in the lower schema).

begin to reach the deep layers of the cortex and the anterior pole of the hippocampus by P0. Primarily limbic regions express NT3 during the first postnatal week, slightly preceding acetylcholinesterase (AChE) positive fiber innervation. Robertston et al. [113] suggest that axons enter first into regions expressing NT3, then later extend into other cortical regions. NT3 may directly promote the arborization of advancing cholinergic afferents, or facilitate preferential targeting and maintenance of cholinergic terminal fields. Projections to the dentate gyrus in fact display one of the more clearly layered patterns of termination by cholinergic afferents, with septal cholinergic axons forming a distinct pattern of termination in the inner molecular layer of the dentate gyrus, although there is also some termination in the outer molecular layer and hilus [90,128]. Makuch et al. [90] showed that the supragranular blade of the dentate gyrus receives afferents considerably earlier than the infragranular blade, in fitting with the earlier develop-

ment of the supragranular blade [4]. The authors hypothesized that as granule cells mature, they produce neurotrophic factors and become an attractive target for cholinergic afferents. Thus there is a correlation between the development of septal cholinergic afferents and maturation of dentate granule cells, suggesting that afferents and target cells interact and reciprocally influence each other during the establishment of connections. 3. Accumulating evidence for a role of the cholinergic system in cortical development 3.1. Maturation, synaptogenesis and plasticity The timing of cholinergic cortical innervation is of primary importance for the normal development of cognitive functions (reviewed by Berger-Sweeney [14]). ACh and AChR are present in

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Fig. 2. Timing of cholinergic innervation of the developing rodent cortex. The schema on the left shows a section of the dorsal cortex at the perinatal stage. Neurons have thus already migrated and settled in the cortical plate (CP) in 6 layers. The subplate (SP) is a transient structure which receives the first cholinergic afferents at P0, as described in Mechawar and Descarries [93]. At P4, some also enter into the CP and marginal zone (MZ). By P8 cholinergic axons with varicose termini have formed a network in every layer and labeled interneurons are also obvious. A mature-like laminar pattern is observed at P16, which increases further in density to reach adult levels by P32. IZ, intermediate zone; VZ, ventricular zone.

the brain before synaptogenesis occurs and are therefore believed to be involved in neuronal maturation. Activation of nAChRs by nicotine or endogeneous ACh may also contribute to enhance synaptic signaling. During development, ACh might be critical for the refinement of cortical connectivity acting as a trophic factor to regulate the development and the morphology of cortical neurons. Interestingly, it has been shown in the developing retina, that ACh released by starburst amacrine cells, may cause local calcium release in developing dendrites of retinal ganglion cells, thereby preventing them from retracting [84]. Thus ACh transmission stabilizes certain dendrites in contact with amacrine cells, and has an influence on dendritic remodeling during synaptogenesis. Guizzetti et al. [55] also showed that ACh in vitro can act on muscarinic receptors expressed by astrocytes, leading them to secrete larger amounts of extracellular matrix proteins such as fibronectin and laminin, ultimately leading to accelerated neuritogenesis of hippocampal neurons in culture. Thus, ACh stimulation helps promote a favourable environment for neuronal development and maturation. In line with these results, cortical cholinergic de-afferentation during development results in pronounced neuronal alterations, including a reduced size of soma, dendritic abnormalities and altered connectivity [61,114]. Robertson et al. [114] treated rats with 192 IgG-saporin by performing injections at P0 and P2, which prevented basal forebrain afferents from arriving in the cortex. Using AChE as a marker they showed a loss of cholinergic fibers in all layers of the cortex, with the medial cortex consistently showing the greatest reduction in labeled axons. With this treatment, the cortex was reduced in size, both in thickness and in medial-lateral extent. DiI labelings in the superior colliculi labeled layer V pyramidal neurons, revealed smaller pyramidal cells exhibiting shorter apical dendrites with fewer branches and fewer spines, although the basal dendrites appeared normal. The reduction of the thickness of the cortex probably reflected the decrease in cholinergic afferents as well as pyramidal cell apical dendrites. Similar changes have also been observed in some genetic models. In heterozygous reeler mice [120], haplo-insufficient for the reelin gene, rostral sub-regions of the septum and basal forebrain, and rostro-medial cortical areas show significant decreases in the density of cholinergic neurons and innervation respectively. The authors propose that reduced cholinergic innervation in motor and cingulate areas contribute to the increased neuronal packing density, decreased cortical thickness and dendritic abnormalities observed in these mice [81,120].

Furthermore, ACh has been shown to play a role in plasticity during the early postnatal period [11,144]. Bear and Singer [11] showed this in the visual cortex of kittens, performing monocular deprivation in the critical postnatal period, where under normal conditions the non-deprived eye assumes control of the ocular dominance columns normally taken care of by the other eye. Bear and Singer [11] showed that cholinergic innervation plays a role in such plasticity. Zhu and Waite also tested the role of cholinergic innervation in somatosensory cortical plasticity. Ablating a row of whisker follicles at P1 prevents the development of barrels for that row and normally allows adjacent barrels to expand into that territory. In the study by Zhu and Waite, IgG 192 saporin was injected unilaterally at P0, whisker ablation was performed at P1, and barrels were examined at P7 [144]. In animals with an intact cholinergic innervation, as expected whisker denervation was associated with a reduction in the size of ‘deprived’ barrels and a compensatory expansion of the adjacent ‘spared’ barrels. On the other hand, cholinergic depletion led to a reduction in the expansion of the ‘spared’ barrels. This suggests that plasticity of cortical barrels also requires cholinergic connections. Cholinergic interneurons may have a role in the local control of cortical circuits [135], as well as in regulating vascular tone [31]. Local ACh release by cortical cholinergic cells could therefore also contribute to the regulation of synaptic refinement in restricted regions. Through this and other mechanisms, cholinergic cells could influence the tuning to sensory inputs of the developing cortical circuitry [31]. In resume, these studies show that ACh could act through various mechanisms to favour neuritogenesis, the stabilization of dendritic processes and neuronal plasticity. 3.2. A role for spontaneous cholinergic excitation in postnatal development Spontaneous nicotinic excitation has been shown to be important for the generation of retinal waves, bursts of activity that propagate across the ganglion cell layer in the early postnatal period [9,142]. Starburst amacrine cells form the cholinergic network in the retina and have been shown to be responsible for the generation and propagation of retinal excitation in the rabbit [142]. Interestingly, these cells synthesize and release both GABA and ACh in the early postnatal period forming a network based on recurrent connections, important for the generation of waves. Nicotinic AChR synapses become greatly diminished though over the first

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postnatal week, following a time-course similar to the transition from a nicotinic to a glutamatergic mode of propagation in the second postnatal week. This occurs at a similar time as the emergence of a strong inhibitory ionotropic GABA action on the waves (see below for a further discussion on the potential role of the cholinergic system in switching GABA from excitatory to inhibitory during development). Bansal et al. [9] showed that ␤2 nAChR knockout mice completely lack retinal waves between P1 and P8 and show a delayed refinement of individual retinal ganglion cell dendrites [9]. Retinal slices bathed in specific ␤2 nAChRs antagonists similarly showed greatly reduced waves. In the same study, ␣3 −/− mice were also shown to have altered retinal waves. Thus ␤2 and to a lesser extent ␣3 nAChRs are important for the maturation of the visual system. In the developing cortex, both heteromeric ␣4 and ␤2, as well as homomeric ␣7 nAChRs, are expressed at high levels [78]. In the prefrontal cortex, Kassam et al. [68] studied pyramidal neurons in layer VI previously shown to exhibit high affinity nicotine binding in the first postnatal weeks [126]. This layer contains corticothalamic neurons known to express nAChRs. Whole-cell recordings showed that the application of ACh or nicotine excited the pyramidal neurons, with currents generated that were higher in the first postnatal month, but then declined significantly and progressively during adolescence. An antagonist for post-synaptic ␣4 ␤2 receptors suppressed these nicotinic currents but they were not sensitive to an ␣7 antagonist. ␣5 subunits, a relatively rare accessory subunit known to be expressed in layer VI, were also shown to play a role in the generation of these currents. Thus there is a robust, developmentally regulated nicotinic excitation of layer VI pyramidal neurons in the prefrontal cortex in the first postnatal weeks. This layer contains the corticothalamic neurons which gate thalamic activity and play a critical role in attention. This is important because perinatal exposure to high levels of nicotine in rodents and humans impairs cognitive functions, supporting the idea that an excessive activation of nAChRs by nicotine during brain maturation interferes with the development of brain areas involved in attention, learning and memory ([66] reviewed by Linnet et al. [80]). Peinado [105] studied spontaneous muscarinic activity in the developing parietal cortex. Coronal slices were prepared from immature rat brains (P0–P10) and bathed in either carbachol or muscarine. Fluorescence imaging of a calcium indicator dye revealed waves of neural activity within the cortex after this treatment. M1 and M3 muscarinic ACh receptors were shown to be involved in the induction of the waves. The cholinergic response was characterized by an initial low amplitude event followed by a high amplitude wave, the latter involving depolarization. These bursts of electrical activity, involving large numbers of neurons in restricted cortical regions and occurring during a brief developmental window, are likely to affect the different aspects of neuronal wiring, including neurite extension, target selection and synaptogenesis mentioned previously. In addition, specific ACh receptors may be important in different regions of the developing cortex or brain. 3.3. Ontogeny of chloride homeostasis Spontaneous nicotinic excitation also helps to regulate giant depolarizing currents (GDPs) in hippocampal neurons during the postnatal period, which are dependent on a transient, excitatory role of GABA [78]. GDPs are thought to be instructive in enhancing synaptic efficacy and in “unsilencing” silent connections at emerging synapses [69]. They may contribute to the selective stabilization of neuronal circuits as in the visual cortex. The age-dependent reduction of GDPs can be attributed to the shift in the action of GABA from depolarizing to hyperpolarizing. Indeed, spontaneous

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Fig. 3. Development of the mature chloride gradient. In an wild type (WT) immature neuron there are very low levels of the K–Cl cotransporter, KCC2, but higher levels of the Na–K–Cl cotransporter, NKCC1. During maturation there is an increased expression of KCC2 and a decreased expression of NKCC1. In ␣7 nAChR knockout mice this maturation does not occur with neurons continuing to show abnormally high levels of NKCC1 and too low levels of KCC2. This suggests that cholinergic activity via the ␣7 nACh receptor has an effect on the expression of these transporters and neuronal maturation.

nicotinic excitation has an effect on the maturing GABAergic system during this conversion [82] as described in more detail here. GABAergic signaling is excitatory during late embryogenesis and early postnatal life due to an immature chloride gradient (reviewed in [13,104]). This excitatory phase is essential for the normal development and integration of neurons in circuits [48,110]). Increased expression of the neuron-specific K–Cl cotransporter, KCC2, together with decreased expression of NKCC1 (Fig. 3), normally induces a developmental shift to render GABAergic transmission hyperpolarizing after a period of depolarizing activity [112]. This GABAergic conversion occurs in the first weeks of postnatal life, and spontaneous nicotinic cholinergic activity has been shown to be important for this conversion [82]. ␣7 nicotinic AChRs reach peak levels in the hippocampus at the time when GABAergic signaling becomes inhibitory, furthermore ␣7 nAChRs co-localize with GABAA receptors, especially on hippocampal interneurons [78,82]. The receptor co-clusters then become innervated with cholinergic and GABAergic terminals and stimulation of nAChRs leads to a post-synaptic response in such cells [78,82,83]. Interestingly, pharmacological nicotinic blockade has been shown to inhibit the maturation of the chloride gradient with neurons retaining a much higher level of NKCC1. Calcium indicator dyes in dissociated neurons show that with nicotinic blockade the activation of GABAA receptors continues to permit a calcium influx as in immature cells [83]. Also, studies using ␣7 nAChR knockout mice show that they mainly retain an excitatory GABA response, showing abnormally high levels of NKCC1 and too low levels of KCC2 ([82], Fig. 3). Thus nicotinic activity has an effect on the maturation of the chloride gradient via the expression of these transporters. Cholinergic input is also well-positioned to exert further local post-synaptic effects at GABAergic synapses. After the conversion of GABAergic signaling to an inhibitory role, nicotinic excitation combined with GABA inhibition may be important for these later stages of development [82,83]. Liu et al. have further shown that co-treatment of KCC2-expressing cells with nicotine and GABA led them to attain a bipolar morphology with restricted innervation, whereas treatment of the same cells with GABA alone, cells did not change their morphology from multipolar. Thus nicotine in combination with an inhibitory role of GABA can have an effect on neuronal maturation and innervation. Accumulating evidence shows that cholinergic innervation of the immature cortex during neuronal differentiation and synapse formation plays an important regulatory role in these processes. Spontaneous nicotinic excitation induces the propagation of waves

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which occur during defined developmental windows in the first postnatal week in rodent. Cholinergic neuromodulation during this period is critical for synaptogenesis and plasticity. Alterations in cholinergic innervation during the postnatal period perturb neuronal maturation, leading to an abnormal cortical cytoarchitecture and permanent effects on learning and memory processes. 4. Adult hippocampal neurogenesis Contrary to traditional dogma which claimed that neurogenesis – the birth and growth of new functional neurons in the adult central nervous system – was a strictly developmental phenomenon, it is now clearly established that new neurons continue to be added to the adult brain throughout life. In the adult mammalian brain, neurogenesis is to date thought to be largely confined to two main regions: the subventricular zone (svz) of the lateral ventricle, and the subgranular zone (sgz) of the dentate gyrus of the hippocampus although there are indications that also in other cortical regions, limited neurogenesis may occur, but only under specific conditions [51,139,140]. In the sgz, neural progenitor cells give rise to transit amplifying progenitors that in turn differentiate into new immature neurons which migrate a short distance into the granular cell layer of the dentate gyrus and mature locally into dentate granule cells (DGCs) within the period of a few weeks (for reviews, [7,23,86,140]. Newly generated DGCs mature and become functionally integrated into hippocampal circuitry, receiving synaptic inputs from entorhinal cortex neurons via the perforant pathway and extend their axons to establish synapses onto CA3 pyramidal cells [125,132]. Although thousands of new neurons are produced and selected each day in the adult rat hippocampus [25] that have been implicated in specific roles in cognition (see below), their exact functional role remains poorly understood. 4.1. A multicellular complex The process of neurogenesis consists of several steps: proliferation of progenitor cells; selection and elimination by apoptosis, commitment to a neuronal phenotype; morphological and physiological maturation with the development of functional neuronal characteristics, and synaptic integration into pre-existing corticohippocampal circuitry. During this slow maturation process, many of the newborn neurons are selected and detailed analysis has shown that more than 50% of the newborn DGCs in rodents [26,70], will die within the first few weeks after birth [15]. Extensive morphological and electrophysiological analyses of nestin-GFP transgenic mice have led to the identification of a heterogeneous population of precursor cells in the dentate gyrus of the hippocampus [41,44]. The first type of precursor cells described (type-1 cells, according to the nomenclature of [71]) are multipotent, GFAP-expressing astrocytic-like cells characterized by a triangular soma localized in the sgz of the dentate gyrus with a thick apical process reaching the inner molecular layer where it branches massively. These radial processes from type-1 cells are in close proximity to immature neurons and have been suggested to play a role in mediating exchange of signaling molecules between the compartment where mature granule cells reside and the germinal zone within which hippocampal neurogenesis occurs ([44]; see also [119]). Type-1 cells express the class IV intermediate filament nestin [102] and share many features with astrocytes such as the expression of GFAP (but not the astrocytic marker S100␤), vascular end-feet and electrophysiological properties of astrocytes [41,44,74,119]. Type-1 cells have been reported to be rarely dividing. In contrast, type-2 cells lack astrocytic features and have been shown to divide at higher rates [44,74]. They do not express GFAP and have a large round or ovoid soma with short cyto-

plasm extensions which are tangentially orientated. They express nestin; some but not all express the immature neuronal markers doublecortin (DCX) and PSA-NCAM, suggesting a pre-neural state or neural lineage of these putative progenitors. They are positive for Prox1, an early-expressed neural lineage-specific homeobox transcription factor selective to dentate granule cells [74], indicating an early neuronal commitment [70]. Type-2 cells have a complex pattern of electrophysiological characteristics that are clearly different from astrocytic features; some of them show Na+ currents confirming early signs of neuronal differentiation [41] These neuronal-lineage-restricted cells appear to mature over time, first developing morphological traits of immature neurons such as a round-shaped nucleus, and extinction of nestin expression even if they are still proliferative [type-3 cells; [41,74]]. The multiple-step proliferation process of progenitor cells gives rise to immature neurons that have exited from the cell cycle and thus become post-mitotic. Immature dentate granule cells have been shown to transiently express the microtubule-associated protein DCX and the calcium-binding protein calretinin between 1 day and 1 week after birth, peaking at 3 days, which is later replaced by calbindin expression in more mature neurons [19]. Following a single pulse of a birthdating marker (tritiated thymidine or BrdU) in rats and mice, it has been shown that the number of newborn cells in the dentate gyrus starts to increase during the first two days and then decreases over time [26,70]. The highest rate of decline is between the first two weeks after birth, the population of labeled cells becoming more or less stable by 4 weeks, with no further decrease observed for at least 11 months, the longest survival time examined so far [70]. Of note, with increasing age of the animal, also the proliferation and turnover rate of the newborn cells rapidly slows down [59]. The rapid decline in the overall population during the early stages has been shown to result from active elimination by apoptosis of immature neurons [15,24,122]. This is consistent with the “use it or lose it” hypothesis [123] according to which adult neuronal survival would depend on activity-dependent regulatory mechanisms. One critical factor for the selection process during maturation might be the degree of functional integration of the new cells into cortico-hippocampal circuitry and the related presence of growth factors [21,22]. Immature neurons that receive growth factor support or those that are synaptically recruited, would have a better chance to survive, while others would be eliminated [52,72]. Clearly, life and death of the newborn cells are strongly associated to each other, and the balance between the two is delicate [37,75]. Given the massive extent of both processes during development [18,109], interfering with this turnover during early ages affects neurogenesis as well as hippocampal functions in a specific and often lasting manner [27,85,97,98]. Immature dentate granule cells migrate a short distance from the sgz to the inner part of the granular cell layer where they rapidly extend dendrites to the molecular layer and project axons through the mossy fibre pathway to reach target CA3 pyramidal cells within 4–10 days of their birth [58,91,121]. Using retroviral-based genetic tracing in mice, van Praag et al. demonstrated that 4-week old newly generated neurons can generate action potentials and are functionally integrated into hippocampal circuitry [132]. More recently, Toni et al. clearly showed that axons of adult-born granule cells establish functional synapses with hilar interneurons, mossy cells and CA3 pyramidal cells and release glutamate as their main neurotransmitter [125]. A series of well-designed studies have shown that maturation of new dentate granule cells recapitulates embryonic development [6,40,48,100,101,136,141]. During early stages of maturation, i.e. within the first week after birth, newborn cells have morphological characteristics of immature neurons, display high input resistance and low membrane capacitance and are electrophysiologically silent although

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they express both GABA and glutamatergic receptors [40,48]. In the course of maturation, during the second and third weeks, they develop a more mature appearance with the extension of an apical spineless dendritic tree expanding through the granular cell layer and reaching the middle of the molecular layer. Concomitantly with dendritic outgrowth, axonal fibres extend and reach the CA3 area [141]. In parallel with this morphological maturation, newborn cells elicit more, but not yet fully, mature electrophysiological features. New DGCs share most of the morphological and electrophysiological features with those of mature neurons, although both the complexity of dendritic arborization and spine density continue to increase, reaching comparable levels to those of fully mature granule cells by 4 months of age [125,132,141]. 4.2. Adult neurogenesis, cholinergic system and cognition Because neurogenesis was found to occur in the olfactory bulb and hippocampus, structures known to play a critical role in sensory processing and the encoding of memories, it was immediately thought neurogenesis might play a role in some forms of learning and memory processing. Clearly, the results of the past 15 years of research support the idea of a functional role of adultgenerated neurons in learning and memory processes and the relation between neurogenesis and cognition has been extensively reviewed elsewhere (for reviews, [1,2,20,79]). More recently, it was demonstrated that adult hippocampal neurogenesis plays a crucial role in long-term memory, pattern separation and anxiety [22,28,37,47,62,64,111,127], while reductions in neurogenesis have further been implicated in e.g. the vulnerability to develop depression [86,87]. Given the crucial role of the cholinergic system for normal cognitive functioning and age related dementia disorders, we focused this part of the review on the influence of ACh on the formation of new hippocampal neurons and its relation to learning and memory. Hippocampal-dependent learning and memory are profoundly influenced by the hippocampal cholinergic system (for reviews [50,57,107]). It is generally accepted that both loss and gain of cholinergic balance causes learning impairment in experimental and clinical situations, attesting to the importance of cholinergic homeostasis in hippocampal dependent learning. Indeed, ACh has been shown to induce long-term potentiation (LTP) in the dentate gyrus [16], which may account in part for this role. During aging, a loss in cholinergic neurons is likely to lead to a impairment in hippocampal functioning [33,42,43,46]. Concomitantly, adult hippocampal neurogenesis has also been shown to decline with age [59,77]. Conversely, physical exercise or hippocampal learning tasks lead to an increase in the formation and survival of newborn granule neurons [24,52,131] and have also been reported to increase ACh hippocampal levels [36,94,106,108]. All of these conditions also enhance neurogenesis in the dentate gyrus and/or svz [24,52,131], suggesting the increased ACh level to be at least partially responsible for the increased neurogenesis. Furthermore, in Alzheimer’s disease (AD) patients, degeneration of cholinergic neurons is observed [53,116]. Regarding the impact of AD on hippocampal neurogenesis, conflicting data have been obtained according to the type of animal model, the stage of the pathogenesis analysed and the methodological approaches. However, even though this is still highly debated and dependent on the stage of neurogenesis studied, A␤ pathology seems to decrease, rather than increase hippocampal neurogenesis in mouse models (for reviews, see [17,76,92,124,133]; but see [65]). 4.3. Cholinergic regulation of adult hippocampal neurogenesis The principal cholinergic innervation to the hippocampus arises from the basal forebrain, specifically from the medial septum and

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diagonal band of Broca (MSDB). ACh in the hippocampus is released at both synaptic terminals as well as at free nerve endings, where both muscarinic and nicotinic receptors are abundant and have been reported to be involved in plasticity (reviewed in [29]). The evidence that ACh does regulate adult hippocampal neurogenesis comes from studies examining the consequences of experimental cholinergic forebrain lesions on proliferation, survival and apoptosis of newborn granule neurons in rodents ([32,95,129,130]; but see, [60,63]). To selectively lesion the cholinergic basal forebrain, Cooper-Kuhn et al. infused in the lateral ventricles of adult rat the immunotoxin 192 IgG-saporin and showed that this led to a dramatic decline of hippocampal neurogenesis in association with an increase in the number of apoptotic cells in the sgz of the dentate gyrus. These data suggest that ACh may exert a pro-survival effect on the immature neurons to prevent them from dying [32]. However, by using the same immunotoxic lesion approach, Mohapel et al. reported rather a pro-proliferative effect of cholinergic fibers since the pronounced loss of cholinergic innervation in the hippocampus caused a large decrease in the number of proliferating BrdU immunoreactive cells in the dentate gyrus which in turn led to a lower number of 4 week-old newborn granule neurons [95]. This decrease in hippocampal neurogenesis was associated with a spatial memory deficit. Conversely, the acute systemic treatment with the AChE inhibitor physostigmine was associated with an increase in the number of proliferative cells [95]. In addition, a recent study showed that eserine, another type of cholinesterase inhibitor, in both adult and aged mice resulted in an increase in proliferation [63]. Furthermore, a recent study has confirmed these results showing that 192 IgG-saporin-induced cholinergic depletion was associated with a decrease in proliferation of hippocampal progenitors in the dentate gyrus [130]. Interestingly, this study showed that the selective activation of M1 muscarinic receptors mediated by oxotremorine is capable of triggering an induction of cell proliferation not only in the dentate gyrus, but also the non-neurogenic CA1 region of the hippocampus. This restoration of cell proliferation is accompanied by an increase in the number of newly-generated cells expressing neuronal markers in both dentate gyrus and CA1 and by a reversal of cognitive deficits characteristic of this model [130]. These data confirm that ACh positively regulates the proliferation of hippocampal progenitors [95,130]. Moreover, transgenic mice continuously overexpressing a recombinantly inactivated form of synaptic AChE, TgSin mice, exhibited significantly reduced ACh hydrolysis and demonstrated increased proliferation of sgz progenitor cells. This identifies ACh and AChE as important regulators of proliferative activity [30]. Two recent studies, using chronic pharmacological agents to modulate central cholinergic transmission have confirmed the positive effect of ACh on the survival of newly generated granule neurons [67,73]. Increases in extracellular ACh levels following one-month administration of donepezil, a potent, non competitive, selective and long-lasting AChE inhibitor which is widely prescribed to AD patients, enhanced the survival of newborn hippocampal neurons without influencing the proliferation rate nor the neuronal versus astroglial differentiation balance [67,73]. In contrast, inhibition of central cholinergic transmission with scopolamine, which is a potent mAChR blocker, dramatically suppressed the survival of newborn neurons [73]. Overall, these studies demonstrate that ACh positively influences hippocampal neurogenesis. In contrast with these data, two recent studies have failed to report any influence of cholinergic immunotoxic lesion in mice on either proliferation or survival of newborn hippocampal neurons [60,63]. The difference of species (rat versus mice) together with the extent of the lesion may account for these discrepancies. Nevertheless, those reports show that selective, partial lesion of cholinergic afferents, while not suffi-

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cient to affect basal hippocampal neurogenesis, did reduce the exercise-induced increased neurogenesis [60,63]. Altogether, these data suggest that ACh may regulate several steps of hippocampal neurogenesis and promote both the proliferation of progenitors and the integration and survival of the immature neurons in the neuronal circuitry following exercise. The molecular mechanisms by which ACh regulates adult hippocampal neurogenesis are still elusive. Interestingly, immunohistological labeling of progenitors and newborn neurons in the dentate gyrus showed that they are in contact with cholinergic fibers (BrdU-vAChT double positive cells; [73]) and express various ACh receptor subtypes such as the muscarinic receptor M1, M4, ␣7, ␤2 nicotinic receptors (BrdU-M1 and BrdU-M2 mAChR coexpressing newborn cells, [95], PSA-NCAM-␣7, PSA-NCAM-␤2 nAChR, PSA-NCAM-M1 and PSA-NCAM-M2 mAChR coexpressing immature neurons; [67]. These data suggest that the diffuse availability of ACh may affect directly both the progenitors and immature differentiating granule neurons or alternatively through the differentiated granule cell layer neurons in the dentate gyrus which are known to express ␣7 and ␤2 nAChRs and M1 and M4 mAChRs [29]. With regard to signaling through mAChRs, a recent publication indicates that ACh modulates the function of neural progenitor cells via various signaling pathways, such as Ras-mitogen-activated protein kinase, phosphatidylinositol 3-kinase-Akt, protein kinase C, c-Src and Ca2+ signaling [88]. The positive effect of ACh on proliferation of progenitor cells is corroborated by the in vitro evidence indicating that ACh directly stimulates the proliferation of embryonic neural stem cells and cortical precursor cells via muscarinic receptors [89,143]. Interestingly, a recent study has shown that focal application of ACh and muscarine induced a rapid calcium response in progenitor cells from aged and young adult mice [63]. ACh has therefore been shown to have an influence on progenitor cell proliferation and differentiation, as well as neurite elongation and differentiation [34,137]. Since the cholinergic system has potentially broad influences, additional experiments are needed to clarify the exact pathways involved in the pro-survival effect of ACh on newborn neurons. 5. Conclusions The studies mentioned here confirm the diverse effects of ACh, nicotine and muscarine on progenitor cell proliferation, and immature neuron development and survival in the developing and adult brains. Projecting cholinergic innervation and local cortical cholinergic interneurons provide a source of ACh giving rise to waves of excitation most probably having an effect on neuritogenesis, targeting and stabilization. Future work will reveal the molecular mechanisms and pathways contributing to these processes. Acknowledgements FF is supported by the Inserm Avenir program, the ANR (ANR-08MNPS-013), which has funded EBJ’s salary, the Fondation Jérôme Lejeune and the Fondation Bettencourt Schueller. FF’s team is also affiliated with the Paris School of Neuroscience (ENP). PJL is supported by the Royal Dutch Academy of Sciences, the Nederlandse HersenStichting, the Volkswagen Stiftung Germany, Corcept Inc., the European Union (NEURAD Program) and the Internationale Stichting Alzheimer Onderzoek (ISAO). References [1] Abrous DN, Koehl M, Le Moal M. Adult neurogenesis: from precursors to network and physiology. Physiol Rev 2005;85:523–69. [2] Aimone JB, Wiles J, Gage FH. Computational influence of adult neurogenesis on memory encoding. Neuron 2009;61(2):187–202.

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