NKCC1 and KCC2 Chloride Transporters Have Different Membrane Dynamics on the Surface of Hippocampal Neurons.

Na-K-2Cl cotransporter 1 (NKCC1) regulates chloride influx in neurons and thereby GABAA receptor activity in normal and pathological conditions. Here, we characterized in hippocampal neurons the membrane expression, distribution and dynamics of exogenous NKCC1a and NKCC1b isoforms and compared them to those of the chloride extruder K-Cl cotransporter 2 (KCC2). We found that NKCC1a and NKCC1b behave quite similarly. NKCC1a/1b but not KCC2 are present along the axon initial segment where they are confined. Moreover, NKCC1a/1b are detected in the somato-dendritic compartment at a lower level than KCC2, where they form fewer, smaller and less compact clusters at perisynaptic and extrasynaptic sites. Interestingly, ~60% of dendritic clusters of NKCC1a/1b are colocalized with KCC2. They are larger and brighter than those devoid of KCC2, suggesting a particular NKCC1a/1b-KCC2 relationship. In agreement with the reduced dendritic clustering of NKCC1a/1b compared with that of KCC2, NKCC1a/1b are more mobile on the dendrite than KCC2, suggesting weaker cytoskeletal interaction. NKCC1a/b are confined to endocytic zones, where they spend more time than KCC2. However, they spend less time in these compartments than at the synapses, suggesting that they can rapidly leave endocytic zones to increase the membrane pool, which can happen in pathological conditions. Thus, NKCC1a/b have different membrane dynamics and clustering from KCC2, which helps to explain their low level in the neuronal membrane, while allowing a rapid increase in the membrane pool under pathological conditions.

Dorsal raphe serotonin neurotransmission is required for the expression of nursing behavior and for pup survival.

Proper maternal care is an essential factor of reproductive success in mammals, involving a repertoire of behaviors oriented toward the feeding and care of the offspring. Among the neurotransmitters involved in the initiation of these behaviors, serotonin (5-HT) seems to play an important role. Here we compared pup-oriented maternal behaviors in mice with constitutive 5-HT depletion, the tryptophan hydroxylase 2-knock-out (Tph2-KO) and the Pet1-KO mice. We report that the only common pup-oriented defect in these 2 hyposerotoninergic models is a defective nursing in parturient mice and altered nursing-like (crouching) behavior in virgin mice, while pup retrieval defects are only present in Tph2-KO. Despite a normal mammary gland development and milk production, the defect in appropriate nursing is responsible for severe growth retardation and early lethality of pups born to hyposerotonergic dams. This nursing defect is due to acute rather constitutive 5-HT depletion, as it is reproduced by adult knockdown of Tph2 in the dorsal raphe nucleus in mothers with a prior normal maternal experience. We conclude that 5-HT innervation from the dorsal raphe is required for both the initiation and maintenance of a normal nursing behavior. Our findings may be related to observations of reduced maternal/infant interactions in human depression.

Constitutive and Acquired Serotonin Deficiency Alters Memory and Hippocampal Synaptic Plasticity.

Serotonin (5-HT) deficiency occurs in a number of brain disorders that affect cognitive function. However, a direct causal relationship between 5-HT hypo-transmission and memory and underlying mechanisms has not been established. We used mice with a constitutive depletion of 5-HT brain levels (Pet1KO mice) to analyze the contribution of 5-HT to different forms of learning and memory. Pet1KO mice exhibited a striking deficit in novel object recognition memory, a hippocampal-dependent task. No alterations were found in tasks for social recognition, procedural learning, or fear memory. Viral delivery of designer receptors exclusively activated by designer drugs was used to selectively silence the activity of 5-HT neurons in the raphe. Inhibition of 5-HT neurons in the median raphe, but not the dorsal raphe, was sufficient to impair object recognition in adult mice. In vivo electrophysiology in behaving mice showed that long-term potentiation in the hippocampus of 5-HT-deficient mice was altered, and administration of the 5-HT1A agonist 8-OHDPAT rescued the memory deficits. Our data suggest that hyposerotonergia selectively affects declarative hippocampal-dependent memory. Serotonergic projections from the median raphe are necessary to regulate object memory and hippocampal synaptic plasticity processes, through an inhibitory control mediated by 5-HT1A receptors.

Conditional anterograde tracing reveals distinct targeting of individual serotonin cell groups (B5-B9) to the forebrain and brainstem.

Serotoninergic innervation of the central nervous system is provided by hindbrain raphe nuclei (B1-B9). The extent to which each raphe subdivision has distinct topographic organization of their projections is still unclear. We provide a comprehensive description of the main targets of the rostral serotonin (5-HT) raphe subgroups (B5-B9) in the mouse brain. Adeno-associated viruses that conditionally express GFP under the control of the 5-HT transporter promoter were used to label small groups of 5-HT neurons in the dorsal (B7d), ventral (B7v), lateral (B7l), and caudal (B6) subcomponents of the dorsal raphe (DR) nucleus as well as in the rostral and caudal parts of the median raphe (MR) nucleus (B8 and B5, respectively), and in the supralemniscal (B9) cell group. We illustrate the distinctive and largely non-overlapping projection areas of these cell groups: for instance, DR (B7) projects to basal parts of the forebrain, such as the amygdala, whereas MR (B8) is the main 5-HT source to the hippocampus, septum, and mesopontine tegmental nuclei. Distinct subsets of B7 have preferential brain targets: B7v is the main source of 5-HT for the cortex and amygdala while B7d innervates the hypothalamus. We reveal for the first time the target areas of the B9 cell group, demonstrating projections to the caudate, prefrontal cortex, substantia nigra, locus coeruleus and to the raphe cell groups. The broad topographic organization of the different raphe subnuclei is likely to underlie the different functional roles in which 5-HT has been implicated in the brain. The present mapping study could serve as the basis for genetically driven specific targeting of the different subcomponents of the mouse raphe system.

Fragile X Mental Retardation Protein and Dendritic Local Translation of the Alpha Subunit of the Calcium/Calmodulin-Dependent Kinase II Messenger RNA Are Required for the Structural Plasticity Underlying Olfactory Learning.

BACKGROUND: In the adult brain, structural plasticity allowing gain or loss of synapses remodels circuits to support learning. In fragile X syndrome, the absence of fragile X mental retardation protein (FMRP) leads to defects in plasticity and learning deficits. FMRP is a master regulator of local translation but its implication in learning-induced structural plasticity is unknown. METHODS: Using an olfactory learning task requiring adult-born olfactory bulb neurons and cell-specific ablation of FMRP, we investigated whether learning shapes adult-born neuron morphology during their synaptic integration and its dependence on FMRP. We used alpha subunit of the calcium/calmodulin-dependent kinase II (αCaMKII) mutant mice with altered dendritic localization of αCaMKII messenger RNA, as well as a reporter of αCaMKII local translation to investigate the role of this FMRP messenger RNA target in learning-dependent structural plasticity. RESULTS: Learning induces profound changes in dendritic architecture and spine morphology of adult-born neurons that are prevented by ablation of FMRP in adult-born neurons and rescued by an metabotropic glutamate receptor 5 antagonist. Moreover, dendritically translated αCaMKII is necessary for learning and associated structural modifications and learning triggers an FMRP-dependent increase of αCaMKII dendritic translation in adult-born neurons. CONCLUSIONS: Our results strongly suggest that FMRP mediates structural plasticity of olfactory bulb adult-born neurons to support olfactory learning through αCaMKII local translation. This reveals a new role for FMRP-regulated dendritic local translation in learning-induced structural plasticity. This might be of clinical relevance for the understanding of critical periods disruption in autism spectrum disorder patients, among which fragile X syndrome is the primary monogenic cause.

Paradoxical increase in survival of newborn neurons in the dentate gyrus of mice with constitutive depletion of serotonin.

Increased adult neurogenesis is a major neurobiological correlate of the beneficial effects of antidepressants. Indeed, selective serotonin (5-HT) re-uptake inhibitors, which increase 5-HT transmission, enhance adult neurogenesis in the dentate gyrus (DG) of the hippocampus. However, the consequences of 5-HT depletion are still unclear as studies using neurotoxins that target serotonergic neurons reached contradictory conclusions on the role of 5-HT on DG cell proliferation. Here, we analysed two genetic models of 5-HT depletion, the Pet1(-/-) and the VMAT2(f/f) ; SERT(cre/+) mice, which have, respectively, 80 and 95% reductions in hippocampal 5-HT. In both models, we found unchanged cell proliferation of the neural precursors in the DG subgranular zone, whereas a significant increase in the survival of newborn neurons was noted 1 and 4 weeks after BrdU injections. This pro-survival trait was phenocopied pharmacologically with 5-HT synthesis inhibitor PCPA treatment in adults, indicating that this effect was not developmental. Furthermore, a 1-week administration of the 5-HT1A receptor agonist 8-OH-DPAT in Pet1(-/-) and PCPA-treated mice normalised hippocampal cell survival. Overall, our results indicate that constitutive 5-HT depletion does not alter the proliferation of neural precursors in the DG but promotes the survival of newborn cells, an effect which involves activation of postsynaptic 5-HT1A receptors. The role of 5-HT in selective neuronal elimination points to a new facet in its multiple effects in controlling neural circuit maturation.

A subpopulation of serotonergic neurons that do not express the 5-HT1A autoreceptor.

5-HT neurons are topographically organized in the hindbrain, and have been implicated in the etiology and treatment of psychiatric diseases such as depression and anxiety. Early studies suggested that the raphe 5-HT neurons were a homogeneous population showing similar electrical properties, and feedback inhibition mediated by 5-HT1A autoreceptors. We utilized histochemistry techniques in ePet1-eGFP and 5-HT1A-iCre/R26R mice to show that a subpopulation of 5-HT neurons do not express the somatodendritic 5-HT1A autoreceptor mRNA. In addition, we performed patch-clamp recordings followed by single-cell PCR in ePet1-eGFP mice. From 134 recorded 5-HT neurons located in the dorsal, lateral, and median raphe, we found lack of 5-HT1A mRNA expression in 22 cells, evenly distributed across raphe subfields. We compared the cellular characteristics of these neuronal types and found no difference in passive membrane properties and general excitability. However, when injected with large depolarizing current, 5-HT1A-negative neurons fired more action potentials, suggesting a lack of autoinhibitory action of local 5-HT release. Our results support the hypothesis that the 5-HT system is composed of subpopulations of serotonergic neurons with different capacity for adaptation.

Fragile X mental retardation protein regulates new neuron differentiation in the adult olfactory bulb.

The fragile X mental retardation protein (FMRP) is an RNA-binding protein essential for multiple aspects of neuronal mRNA metabolism. Its absence leads to the fragile X syndrome, the most prevalent genetic form of mental retardation. The anatomical landmark of the disease, also present in the Fmr1 knock-out (KO) mice, is the hyperabundance of immature-looking lengthened dendritic spines. We used the well known continuous production of adult-born granule cells (GCs) in the mouse olfactory bulb (OB) to analyze the consequences of Fmrp loss on the differentiation of GCs. Morphological analysis of GCs in the Fmr1 KO mice showed an increase in spine density without a change in spine length. We developed an RNA interference strategy to cell-autonomously mutate Fmr1 in a wild-type OB network. Mutated GCs displayed an increase in spine density and spine length. Detailed analysis of the spines through immunohistochemistry, electron microscopy, and electrophysiology surprisingly showed that, despite these abnormalities, spines receive normal glutamatergic synapses, and thus that mutated adult-born neurons are synaptically integrated into the OB circuitry. Time-course analysis of the spine defects showed that Fmrp cell-autonomously downregulates the level and rate of spine production and limits their overgrowth. Finally, we report that Fmrp does not regulate dendritogenesis in standard conditions but is necessary for activity-dependent dendritic remodeling. Overall, our study of Fmrp in the context of adult neurogenesis has enabled us to carry out a precise dissection of the role of Fmrp in neuronal differentiation and underscores its pleiotropic involvement in both spinogenesis and dendritogenesis.

HVC interneurons are not renewed in adult male zebra finches.

Adult neurogenesis is a widespread phenomenon in many species, from invertebrates to humans. In songbirds, the telencephalic region, high vocal center (HVC), continuously integrates new neurons in adulthood. This nucleus consists of a heterogenous population of inhibitory interneurons (HVC(IN)) and two populations of projection neurons that send axons towards either the robust nucleus of the arcopallium (HVC(RA)) or the striatal nucleus area X (HVC(X)). New HVC neurons were initially inferred to be interneurons, because they lacked retrograde labelling from the HVC's targets. Later studies using different tracers demonstrated that HVC(RA) are replaced but HVC(X) are not. Whether interneurons are also renewed became an open question. As the HVC's neuronal populations display different physiological properties and functions, we asked whether adult HVC indeed recruits two neuronal populations or whether only the HVC(RA) undergo renewal in adult male zebra finches. We show that one month after being born in the lateral ventricle, 42% of the newborn HVC neurons were retrogradely labelled by tracer injections into the RA. However, the remaining 58% were not immunoreactive for the neurotransmitter GABA, nor for the calcium-binding proteins, parvalbumin (PA), calbindin (CB) and calretinin (CR) that characterize different classes of HVC(IN). We further established that simultaneous application of parvalbumin, calbindin and calretinin antibodies to HVC revealed approximately the same fraction of HVC neurons, i.e. 10%, as could be detected by GABA immunoreactivity. This implies that the sum of HVC(IN) expressing the different calcium-binding proteins constitute all inhibitory HVC(IN). Together these results strongly suggest that only HVC(RA) are recruited into the adult HVC.

Understanding the regulation and function of adult neurogenesis: contribution from an insect model, the house cricket.

Since the discovery of adult neurogenesis, a major issue is the role of newborn neurons and the function-dependent regulation of adult neurogenesis. We decided to use an animal model with a relatively simple brain to address these questions. In the adult cricket brain as in mammals, new neurons are produced throughout life. This neurogenesis occurs in the main integrative centers of the insect brain, the mushroom bodies (MBs), where the neuroblasts responsible for their formation persist after the imaginal molt. The rate of production of new neurons is controlled not only by internal cues such as morphogenetic hormones but also by external environmental cues. Adult crickets reared in an enriched sensory environment experienced an increase in neuroblast proliferation as compared with crickets reared in an impoverished environment. In addition, unilateral sensory deprivation led to reduced neurogenesis in the MB ipsilateral to the lesion. In search of a functional role for the new cells, we specifically ablated MB neuroblasts in young adults using brain-focused gamma ray irradiation. We developed a learning paradigm adapted to the cricket, which we call the "escape paradigm." Using this operant associative learning test, we showed that crickets lacking neurogenesis exhibited delayed learning and reduced memory retention of the task when olfactory cues were used. Our results suggest that environmental cues are able to influence adult neurogenesis and that, in turn, newly generated neurons participate in olfactory integration, optimizing learning abilities of the animal, and thus its adaptation to its environment. Nevertheless, odor learning in adult insects cannot always be attributed to newly born neurons because neurogenesis is completed earlier in development in many insect species. In addition, many of the irradiated crickets performed significantly better than chance on the operant learning task.

Recruitment of FoxP2-expressing neurons to area X varies during song development.

In adult songbirds, neural progenitors proliferate along the lateral ventricles. After migration, many of the subsequently formed neuroblasts integrate into the song nuclei HVC and Area X that participate in auditory-guided vocal motor learning and singing. Recruitment of postembryonically generated neurons into HVC, rodent hippocampus, and olfactory bulb has been linked to learning and memory. The cellular identity and the role of postembryonically generated neurons in Area X are unknown. Here we describe that the majority of new neurons in postembryonic Area X of male zebra finches expressed DARPP32 but not choline acetyltransferase or parvalbumin. This suggests that they are spiny neurons. Retrogradely labeled neurons projecting to thalamic nucleus DLM were not renewed. The spiny neurons in Area X were recently shown to express FoxP2, a transcription factor critical for normal speech and language development in humans. Since increased FoxP2 mRNA expression was previously observed during periods of vocal plasticity we investigated whether this increase might be associated with neuronal recruitment. Consistent with their spiny phenotype, new neurons in Area X did express FoxP2 and recruitment increased transiently during the juvenile song learning period. Moreover we found that FoxP2 was expressed in the ventricular zone of adult songbirds but was absent from the germinal zones in adult mouse brains, the hippocampus, and the subventricular zone. Together these results raise the possibility that neuronal recruitment and FoxP2 expression in Area X are associated with vocal learning.

Hormonal and sensory inputs regulate distinct neuroblast cell cycle properties in adult cricket brain.

From invertebrates to humans, it has been demonstrated that new neurons are added to specific brain structures throughout adult life. In the house cricket, adult neurogenesis occurs in the mushroom bodies, the main sensory integrative center of the brain, often considered an analogue of vertebrate hippocampus. We have previously shown that this neurogenesis can be modulated by hormones through the polyamine pathway and by environmental conditions through sensory inputs and the nitric oxide pathway. Environment-induced neurogenesis is independent of juvenile hormone levels, so we addressed the roles of sensory inputs and hormones in the control of neuroblast proliferation. Here, by using double labelling of cells specifically in S phase (5-bromo-2'-deoxyuridine) together with labelling of mitotically active cells in any phase (proliferating cell nuclear antigen), we show that juvenile hormone acts on progenitor cell proliferation by inducing quiescent neuroblasts to enter the cell cycle, whereas sensory inputs act by shortening the cell cycle. Thus, in the adult house cricket, regulation of neuroblast proliferation by hormonal and environmental cues occurs through two independent modes of action.

Suppression of adult neurogenesis impairs olfactory learning and memory in an adult insect.

Although adult neurogenesis has now been demonstrated in many different species, the functional role of newborn neurons still remains unclear. In the house cricket, a cluster of neuroblasts, located in the main associative center of the insect brain, keeps producing new interneurons throughout the animal's life. Here we address the functional significance of adult neurogenesis by specific suppression of neuroblast proliferation using gamma irradiation of the insect's head and by examining the impact on the insect's learning ability. Forty gray irradiation performed on the first day of adult life massively suppressed neuroblasts and their progeny without inducing any noticeable side effect. We developed a new operant conditioning paradigm especially designed for crickets: the "escape paradigm." Using olfactory cues, visual cues, or both, crickets had to choose between two holes, one allowing them to escape and the other leading to a trap. Crickets lacking adult neurogenesis exhibited delayed learning when olfactory cues alone were used. Furthermore, retention 24 hr after conditioning was strongly impaired in irradiated crickets. By contrast, when visual cues instead of olfactory ones were provided, performance of irradiated insects was only slightly affected; when both olfactory and visual cues were present, their performance was not different from that of controls. From these results, it can be postulated that newborn neurons participate in the processing of olfactory information required for complex operant conditioning.

Sensory inputs stimulate progenitor cell proliferation in an adult insect brain.

Although most brain neurons are produced during embryonic and early postnatal development, recent studies clearly demonstrated in a wide range of species from invertebrates to humans that new neurons are added to specific brain structures throughout adult life. Hormones, neurotransmitters, and growth factors as well as environmental conditions modulate this neurogenesis. In this study, we address the role of sensory inputs in the regulation of adult neural progenitor cell proliferation in an insect model. In some insect species, adult neurogenesis occurs in the mushroom bodies, the main sensory integrative centers of the brain, receiving multimodal information and often considered as the analog of the vertebrate hippocampus. We recently showed that rearing adult crickets in enriched sensory and social conditions enhanced neuroblast proliferation in the mushroom bodies. Here, by manipulating hormonal levels and affecting olfactory and/or visual inputs, we show that environmental regulation of neurogenesis is in direct response to olfactory and visual stimuli rather than being mediated via hormonal control. Experiments of unilateral sensory deprivation reveal that neuroblast proliferation can be inhibited in one brain hemisphere only. These results, obtained in a relatively simple brain, emphasize the role of sensory inputs on stem cell division.

The common properties of neurogenesis in the adult brain: from invertebrates to vertebrates.

Until recently, it was believed that adult brains were unable to generate any new neurons. However, it is now commonly known that stem cells remain in the adult central nervous system and that adult vertebrates as well as adult invertebrates are currently adding new neurons in some specialized structures of their central nervous system. In vertebrates, the subventricular zone and the dentate gyrus of the hippocampus are the sites of neuronal precursor proliferation. In some insects, persistent neurogenesis occurs in the mushroom bodies, which are brain structures involved in learning and memory and considered as functional analogues of the hippocampus. In both vertebrates and invertebrates, secondary neurogenesis (including neuroblast proliferation and neuron differentiation) appears to be regulated by hormones, transmitters, growth factors and environmental cues. The functional implications of adult neurogenesis have not yet been clearly demonstrated and comparative study of the various model systems could contribute to better understand this phenomenon. Here, we review and discuss the common characteristics of adult neurogenesis in the various animal models studied so far.