Cerebellar control of a unitary head direction sense.

The head-direction (HD) system, a key neural circuit for navigation, consists of several anatomical structures containing neurons selective to the animal's head direction. HD cells exhibit ubiquitous temporal coordination across brain regions, independently of the animal's behavioral state or sensory inputs. Such temporal coordination mediates a single, stable, and persistent HD signal, which is essential for intact orientation. However, the mechanistic processes behind the temporal organization of HD cells are unknown. By manipulating the cerebellum, we identify pairs of HD cells recorded from two brain structures (anterodorsal thalamus and retrosplenial cortex) that lose their temporal coordination, specifically during the removal of the external sensory inputs. Further, we identify distinct cerebellar mechanisms that participate in the spatial stability of the HD signal depending on sensory signals. We show that while cerebellar protein phosphatase 2B-dependent mechanisms facilitate the anchoring of the HD signal on the external cues, the cerebellar protein kinase C-dependent mechanisms are required for the stability of the HD signal by self-motion cues. These results indicate that the cerebellum contributes to the preservation of a single and stable sense of direction.

Zif268/egr1 gene controls the selection, maturation and functional integration of adult hippocampal newborn neurons by learning.

New neurons are continuously added to the dentate gyrus of the adult mammalian brain. During the critical period of a few weeks after birth when newborn neurons progressively mature, a restricted fraction is competitively selected to survive in an experience-dependent manner, a condition for their contribution to memory processes. The mechanisms that control critical stages of experience-dependent functional incorporation of adult newborn neurons remain largely unknown. Here, we identify a unique transcriptional regulator of the functional integration of newborn neurons, the inducible immediate early gene zif268/egr1. We show that newborn neurons in zif268-KO mice undergo accelerated death during the critical period of 2-3 wk around their birth and exhibit deficient neurochemical and morphological maturation, including reduced GluR1 expression, increased NKCC1/KCC2b chloride cotransporter ratio, altered dendritic development, and marked spine growth defect. Investigating responsiveness of newborn neurons to activity-dependent expression of zif268 in learning, we demonstrate that in the absence of zif268, training in a spatial learning task during this critical period fails to recruit newborn neurons and promote their survival, leading to impaired long-term memory. This study reveals a previously unknown mechanism for the control of the selection, functional maturation, and experience-dependent recruitment of dentate gyrus newborn neurons that depends on the inducible immediate early gene zif268, processes that are critical for their contribution to hippocampal-dependent long-term memory.

Cerebellar contribution to spatial navigation: new insights into potential mechanisms.

The contribution of the cerebellum to the non-motor aspects of spatial navigation is now established, but the mechanisms of its participation remain unclear. The L7-PKCI mouse model, in which inhibited PKC activity suppresses parallel fiber-Purkinje cell long-term depression (LTD), provides the opportunity to study their spatial abilities in the absence of any motor impairment. L7-PKCI mice are deficient in the spatial but not the cued version of the watermaze task. Their performances are preserved when alleys guide their trajectories in the starmaze task, suggesting that cerebellar PKC-dependent mechanisms are required for the production of an optimal trajectory toward a goal. Furthermore, electrophysiological recordings in freely moving L7-PKCI mice revealed that their hippocampal place cell properties are affected when they have to rely on self motion information: in the absence of external information as well as in a conflicting situation between self-motion and external information. This suggests that the cerebellum is involved in the processing of self-motion information and is required for the construction of the spatial representation in the hippocampus.

Mature Purkinje cells require the retinoic acid-related orphan receptor-α (RORα) to maintain climbing fiber mono-innervation and other adult characteristics.

Neuronal maturation during development is a multistep process regulated by transcription factors. The transcription factor RORα (retinoic acid-related orphan receptor α) is necessary for early Purkinje cell (PC) maturation but is also expressed throughout adulthood. To identify the role of RORα in mature PCs, we used Cre-lox mouse genetic tools in vivo that delete it specifically from PCs between postnatal days 10-21. Up to 14 d of age, differences between mutant and control PCs were not detectable: both were mono-innervated by climbing fibers (CFs) extending along their well-developed dendrites with spiny branchlets. By week 4, mutant mice were ataxic, some PCs had died, and remaining PC soma and dendrites were atrophic, with almost complete disappearance of spiny branchlets. The innervation pattern of surviving RORα-deleted PCs was abnormal with several immature characteristics. Notably, multiple functional CF innervation was reestablished on these mature PCs, simultaneously with the relocation of CF contacts to the PC soma and their stem dendrite. This morphological modification of CF contacts could be induced even later, using lentivirus-mediated depletion of rora from adult PCs. These data show that the late postnatal expression of RORα cell-autonomously regulates the maintenance of PC dendritic complexity, and the CF innervation status of the PC (dendritic vs somatic contacts, and mono-innervation vs multi-innervation). Thus, the differentiation state of adult neurons is under the control of transcription factors; and in their absence, adult neurons lose their mature characteristics and acquire some characteristics of an earlier developmental stage.

The cerebellum promotes sequential foraging strategies and contributes to the directional modulation of hippocampal place cells.

The cerebellum contributes to goal-directed navigation abilities and place coding in the hippocampus. Here we investigated its contribution to foraging strategies. We recorded hippocampal neurons in mice with impaired PKC-dependent cerebellar functions (L7-PKCI) and in their littermate controls while they performed a task where they were rewarded for visiting a subset of hidden locations. We found that L7-PKCI and control mice developed different foraging strategies: while control mice repeated spatial sequences to maximize their rewards, L7-PKCI mice persisted to use a random foraging strategy. Sequential foraging was associated with more place cells exhibiting theta-phase precession and theta rate modulation. Recording in the dark showed that PKC-dependent cerebellar functions controlled how self-motion cues contribute to the selectivity of place cells to both position and direction. Thus, the cerebellum contributes to the development of optimal sequential paths during foraging, possibly by controlling how self-motion and theta signals contribute to place cell coding.

Excessive self-grooming, gene dysregulation and imbalance between the striosome and matrix compartments in the striatum of Shank3 mutant mice.

Autism is characterized by atypical social communication and stereotyped behaviors. Mutations in the gene encoding the synaptic scaffolding protein SHANK3 are detected in 1-2% of patients with autism and intellectual disability, but the mechanisms underpinning the symptoms remain largely unknown. Here, we characterized the behavior of Shank3 Δ11/Δ11 mice from 3 to 12 months of age. We observed decreased locomotor activity, increased stereotyped self-grooming and modification of socio-sexual interaction compared to wild-type littermates. We then used RNAseq on four brain regions of the same animals to identify differentially expressed genes (DEGs). DEGs were identified mainly in the striatum and were associated with synaptic transmission (e.g., Grm2, Dlgap1), G-protein-signaling pathways (e.g., Gnal, Prkcg1, and Camk2g), as well as excitation/inhibition balance (e.g., Gad2). Downregulated and upregulated genes were enriched in the gene clusters of medium-sized spiny neurons expressing the dopamine 1 (D1-MSN) and the dopamine 2 receptor (D2-MSN), respectively. Several DEGs (Cnr1, Gnal, Gad2, and Drd4) were reported as striosome markers. By studying the distribution of the glutamate decarboxylase GAD65, encoded by Gad2, we showed that the striosome compartment of Shank3 Δ11/Δ11 mice was enlarged and displayed much higher expression of GAD65 compared to wild-type mice. Altogether, these results indicate altered gene expression in the striatum of Shank3-deficient mice and strongly suggest, for the first time, that the excessive self-grooming of these mice is related to an imbalance in the striatal striosome and matrix compartments.

Incomplete and inaccurate vocal imitation after knockdown of FoxP2 in songbird basal ganglia nucleus Area X.

The gene encoding the forkhead box transcription factor, FOXP2, is essential for developing the full articulatory power of human language. Mutations of FOXP2 cause developmental verbal dyspraxia (DVD), a speech and language disorder that compromises the fluent production of words and the correct use and comprehension of grammar. FOXP2 patients have structural and functional abnormalities in the striatum of the basal ganglia, which also express high levels of FOXP2. Since human speech and learned vocalizations in songbirds bear behavioral and neural parallels, songbirds provide a genuine model for investigating the basic principles of speech and its pathologies. In zebra finch Area X, a basal ganglia structure necessary for song learning, FoxP2 expression increases during the time when song learning occurs. Here, we used lentivirus-mediated RNA interference (RNAi) to reduce FoxP2 levels in Area X during song development. Knockdown of FoxP2 resulted in an incomplete and inaccurate imitation of tutor song. Inaccurate vocal imitation was already evident early during song ontogeny and persisted into adulthood. The acoustic structure and the duration of adult song syllables were abnormally variable, similar to word production in children with DVD. Our findings provide the first example of a functional gene analysis in songbirds and suggest that normal auditory-guided vocal motor learning requires FoxP2.

Activity-dependent adjustments of the inhibitory network in the olfactory bulb following early postnatal deprivation.

The first-order sensory relay for olfactory processing, the main olfactory bulb (MOB), retains the ability to acquire new interneurons throughout life. It is therefore a particularly appropriate region for studying the role of experience in sculpting neuronal networks. We found that nostril closure decreased the number of newborn granule cells in the MOB, the complexity of their dendritic arborization, and their spine density, without affecting the preexisting population of granule cells. Accordingly, the frequency of miniature synaptic inhibitory events received by mitral cells was reduced. However, due to a compensatory increase in newborn granule cell excitability, action potential-dependent GABA release was dramatically enhanced, thus counteracting the reduction in spine density and leading to an unaltered synchronization of mitral cell firing activity. Together, this study reveals a unique form of adaptive response brought about exclusively by the cohort of newborn cells and used to maintain normal functioning of the MOB.

Anatomical and physiological foundations of cerebello-hippocampal interaction.

Multiple lines of evidence suggest that functionally intact cerebello-hippocampal interactions are required for appropriate spatial processing. However, how the cerebellum anatomically and physiologically engages with the hippocampus to sustain such communication remains unknown. Using rabies virus as a retrograde transneuronal tracer in mice, we reveal that the dorsal hippocampus receives input from topographically restricted and disparate regions of the cerebellum. By simultaneously recording local field potential from both the dorsal hippocampus and anatomically connected cerebellar regions, we additionally suggest that the two structures interact, in a behaviorally dynamic manner, through subregion-specific synchronization of neuronal oscillations in the 6-12 Hz frequency range. Together, these results reveal a novel neural network macro-architecture through which we can understand how a brain region classically associated with motor control, the cerebellum, may influence hippocampal neuronal activity and related functions, such as spatial navigation.

Impaired cerebellar Purkinje cell potentiation generates unstable spatial map orientation and inaccurate navigation.

Cerebellar activity supported by PKC-dependent long-term depression in Purkinje cells (PCs) is involved in the stabilization of self-motion based hippocampal representation, but the existence of cerebellar processes underlying integration of allocentric cues remains unclear. Using mutant-mice lacking PP2B in PCs (L7-PP2B mice) we here assess the role of PP2B-dependent PC potentiation in hippocampal representation and spatial navigation. L7-PP2B mice display higher susceptibility to spatial map instability relative to the allocentric cue and impaired allocentric as well as self-motion goal-directed navigation. These results indicate that PP2B-dependent potentiation in PCs contributes to maintain a stable hippocampal representation of a familiar environment in an allocentric reference frame as well as to support optimal trajectory toward a goal during navigation.

Enriched odor exposure increases the number of newborn neurons in the adult olfactory bulb and improves odor memory.

In the mammalian forebrain, most neurons originate from proliferating cells in the ventricular zone lining the lateral ventricles, including a discrete area of the subventricular zone (SVZ). In this region, neurogenesis continues into adulthood. Most of the cells generated in the SVZ are neuronal precursors with progeny that migrate rostrally along a pathway known as the rostral migratory stream before they reach the main olfactory bulb (MOB) where they differentiate into local interneurons. The olfactory system thus provides an attractive model to investigate neuronal production and survival, processes involving interplay between genetic and epigenetic influences. The present study was conducted to investigate whether exposure to an odor-enriched environment affects neurogenesis and learning in adult mice. Animals housed in either a standard or an odor-enriched environment for 40 d were injected intraperitoneally with bromodeoxyuridine (BrdU) to detect proliferation among progenitor cells and to follow their survival in the MOB. The number of BrdU-labeled neurons was not altered 4 hr after a single BrdU injection. In contrast, the number of surviving progenitors 3 weeks after BrdU injection was markedly increased in animals housed in an enriched environment. This effect was specific because enriched odor exposure did not influence hippocampal neurogenesis. Finally, we showed that adult mice housed in odor-enriched cages display improved olfactory memory without a change in spatial learning performance. By maintaining a constitutive turnover of granule cells subjected to modulation by environmental cues, ongoing bulbar neurogenesis could be associated with improved olfactory memory.

Making scents of olfactory neurogenesis.

Olfaction was long considered to belong more to the realm of art than to that of science. As a result, how the brain perceives, discriminates, and recognizes odorant molecules is still a mystery. Recent progress has nonetheless been made at early stages of the olfactory pathway when olfactory studies entered into the molecular era to elucidate the first contact of an odor molecule with a receptor. Our group focuses on the analysis of odor information in the olfactory bulb, the first processing relay in the mammalian brain. Using this model, we are attempting to decipher the code for odorant information. Furthermore, the olfactory bulb also provides an attractive model to investigate neuronal proliferation, differentiation, migration, and neuronal death, processes involving an interplay between genetic and epigenetic influences. Finally, our goal is to explore the possible consequences of the olfactory bulb plasticity, in olfactory performance. For these purposes, we aim to combine morphological, electrophysiological and behavioral approaches to investigate: (1) how the olfactory bulb processes odor molecule information, (2) how neural precursors differentiate into olfactory bulb interneurons, (3) how these newly-generated neurons integrate into an operational neural network, (4) what role they play in the adult olfactory bulb, and (5) how are basic olfactory functions maintained in such a sensory system subjected to continuous renewal of a large percentage of its neuronal population. These questions should provide new fuel for the molecular and cellular bases of sensory perception and shed light onto cellular bases of learning and memory.

The cerebellum: a new key structure in the navigation system.

Early investigations of cerebellar function focused on motor learning, in particular on eyeblink conditioning and adaptation of the vestibulo-ocular reflex, and led to the general view that cerebellar long-term depression (LTD) at parallel fiber (PF)-Purkinje cell (PC) synapses is the neural correlate of cerebellar motor learning. Thereafter, while the full complexity of cerebellar plasticities was being unraveled, cerebellar involvement in more cognitive tasks-including spatial navigation-was further investigated. However, cerebellar implication in spatial navigation remains a matter of debate because motor deficits frequently associated with cerebellar damage often prevent the dissociation between its role in spatial cognition from its implication in motor function. Here, we review recent findings from behavioral and electrophysiological analyses of cerebellar mutant mouse models, which show that the cerebellum might participate in the construction of hippocampal spatial representation map (i.e., place cells) and thereby in goal-directed navigation. These recent advances in cerebellar research point toward a model in which computation from the cerebellum could be required for spatial representation and would involve the integration of multi-source self-motion information to: (1) transform the reference frame of vestibular signals and (2) distinguish between self- and externally-generated vestibular signals. We eventually present herein anatomical and functional connectivity data supporting a cerebello-hippocampal interaction. Whilst a direct cerebello-hippocampal projection has been suggested, recent investigations rather favor a multi-synaptic pathway involving posterior parietal and retrosplenial cortices, two regions critically involved in spatial navigation.

Young and intense: FoxP2 immunoreactivity in Area X varies with age, song stereotypy, and singing in male zebra finches.

FOXP2 is a transcription factor functionally relevant for learned vocalizations in humans and songbirds. In songbirds, FoxP2 mRNA expression in the medium spiny neurons of the basal ganglia song nucleus Area X is developmentally regulated and varies with singing conditions in different social contexts. How individual neurons in Area X change FoxP2 expression across development and in social contexts is not known, however. Here we address this critical gap in our understanding of FoxP2 as a link between neuronal networks and behavior. We used a statistically unbiased analysis of FoxP2-immunoreactivity (FoxP2-IR) on a neuron-by-neuron basis and found a bimodal distribution of FoxP2-IR neurons in Area X: weakly-stained and intensely-stained. The density of intensely-stained FoxP2-IR neurons was 10 times higher in juveniles than in adults, exponentially decreased with age, and was negatively correlated with adult song stability. Three-week old neurons labeled with BrdU were more than five times as likely to be intensely-stained than weakly-stained. The density of FoxP2-IR putative migratory neurons with fusiform-shaped nuclei substantially decreased as birds aged. The density of intensely-stained FoxP2-IR neurons was not affected by singing whereas the density of weakly-stained FoxP2-IR neurons was. Together, these data indicate that young Area X medium spiny neurons express FoxP2 at high levels and decrease expression as they become integrated into existing neural circuits. Once integrated, levels of FoxP2 expression correlate with singing behavior. Together, these findings raise the possibility that FoxP2 levels may orchestrate song learning and song stereotypy in adults by a common mechanism.

Cerebellum shapes hippocampal spatial code.

Spatial representation is an active process that requires complex multimodal integration from a large interacting network of cortical and subcortical structures. We sought to determine the role of cerebellar protein kinase C (PKC)-dependent plasticity in spatial navigation by recording the activity of hippocampal place cells in transgenic L7PKCI mice with selective disruption of PKC-dependent plasticity at parallel fiber-Purkinje cell synapses. Place cell properties were exclusively impaired when L7PKCI mice had to rely on self-motion cues. The behavioral consequence of such a deficit is evidenced here by selectively impaired navigation capabilities during a path integration task. Together, these results suggest that cerebellar PKC-dependent mechanisms are involved in processing self-motion signals essential to the shaping of hippocampal spatial representation.

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.

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.

Short-term survival of newborn neurons in the adult olfactory bulb after exposure to a complex odor environment.

In the olfactory bulb of adult mice, new neurons are continually integrated into existing neuronal networks. Previous studies have demonstrated that exposure to a complex odor environment increases the incorporation of newborn bulbar neurons without modifying the proliferation rate. Whether this incorporation is transient or leads to the long-lasting presence of new neurons has not yet been answered. Because a transient increase of new neurons impacts olfactory information processing differently than a long-lasting increase, we conducted experiments to investigate the time course of survival and cell death of newly generated bulbar neurons following exposure to an enriched olfactory environment. Dividing cells were labeled with bromodeoxyuridine (BrdU) and were counted at several survival time points thereafter. Interestingly, whereas the number of surviving BrdU-labeled cells was elevated at the time when animals were withdrawn from their enriched housing, this number returned to control level 1 month later. Similarly, when olfactory memory was investigated, we found that the improvement of short-term memory, induced by enriched odor exposure, lasted less than 1 month. These findings indicate not only that the recruitment of newborn neurons closely followed the degree of environment complexity, but also that olfactory memory is tightly associated with the level of ongoing neurogenesis in the adult olfactory bulb.

[Neurogenesis in the adult brain. Functional consequences]. / Neurogenèse dans le cerveau adulte. Conséquences fonctionnelles.

In the adult mammalian brain, neuroblasts are continuously produced within the subgranular zone of the hippocampus and the subventricular zone (SVZ) of the forebrain. In this review we describe how some physiological and environmental factors play important roles in regulating neurogenesis in the hippocampus. Neuroblasts in the SVZ network migrate rostrally into the olfactory bulb where they differentiate into local interneurons. We focus on the production, survival and functional consequences of these newly generated interneurons. We show that enriched odor-exposure enhances the number of newborn neurons in the adult olfactory bulb but not in the hippocampus. This effect did not result from changes in cell proliferation but rather was due to greater neuronal survival. Furthermore, the enriched condition was found to dramatically extend the olfactory memory. By maintaining a constitutive turnover of interneurons subjected to regulation by bulbar activity, ongoing neurogenesis plays a key role in olfactory memory.