Intrinsic Neuronal Activity during Migration Controls the Recruitment of Specific Interneuron Subtypes in the Postnatal Mouse Olfactory Bulb.

Neuronal activity has been identified as a key regulator of neuronal network development, but the impact of activity on migration and terminal positioning of interneuron subtypes is poorly understood. The absence of early subpopulation markers and the presence of intermingled migratory and postmigratory neurons make the developing cerebral cortex a difficult model to answer these questions. Postnatal neurogenesis in the subventricular zone (SVZ) offers a more accessible and compartmentalized model. Neural stem cells regionalized along the border of the lateral ventricle produce two main subtypes of neural progenitors, granule cells and periglomerular neurons that migrate tangentially in the rostral migratory stream (RMS) before migrating radially in the olfactory bulb (OB) layers. Here, we used targeted postnatal electroporation to compare the migration of these two populations in male and female mice. We do not observe any obvious differences regarding the mode of tangential or radial migration between these two subtypes. However, we find a striking increase of intrinsic calcium activity in granule cell precursors (GC-Ps) when they switch from tangential to radial migration. By decreasing neuronal excitability in GC-Ps, we find that neuronal activity has little effect on migration but is required for normal positioning and survival of GC-Ps in the OB layers. Strikingly, decreasing activity of periglomerular neuron precursors (PGN-Ps) did not impact their positioning or survival. Altogether these findings suggest that neuronal excitability plays a subtype specific role during the late stage of migration of postnatally born OB interneurons.SIGNIFICANCE STATEMENT While neuronal activity is a critical factor regulating different aspects of neurogenesis, it has been challenging to study its role during the migration of different neuronal subpopulations. Here, we use postnatal targeted electroporation to label and manipulate the two main olfactory bulb (OB) interneuron subpopulations during their migration: granule cell and periglomerular neuron precursors (PGN-Ps). We find a very striking increase of calcium activity only in granule cell precursors (GC-Ps) when they switch from tangential to radial migration. Interestingly, blocking activity in GC-Ps affected mainly their positioning and survival while PGN-Ps were not affected. These results suggest that neuronal activity is required specifically for the recruitment of GC-Ps in the OB layers.

Role of NMDA receptors in adult neurogenesis: an ontogenetic (re)view on activity-dependent development.

It is now widely accepted that neurogenesis continues throughout life. Accumulating evidence suggests that neurotransmitters are essential signaling molecules that control the different steps of neurogenesis. Nevertheless, we are only beginning to understand the precise role of neurotransmitter receptors and in particular excitatory glutamatergic transmission in the differentiation of adult-born neurons. Recent technical advances allow single-cell gene deletion to study cell-autonomous effects during the maturation of adult-born neurons. Single-cell gene deletion overcomes some of the difficulties in interpreting global gene deletion effects on entire brain areas or systemic pharmacological approaches that might result in compensatory circuit effects. The aim of this review is to summarize recent advances in the understanding of the role of NMDA receptors (NMDARs) during the differentiation of adult-born neurons and put them in perspective with previous findings on cortical development.

Neural progenitor cells regulate capillary blood flow in the postnatal subventricular zone.

In the postnatal subventricular zone (SVZ), S phase entry of neural progenitor cells (NPCs) correlates with a local increase in blood flow. However, the cellular mechanism controlling this hemodynamic response remains unknown. We show that a subpopulation of SVZ cells, astrocyte-like cells or B-cells, sends projections ensheathing pericytes on SVZ capillaries in young mice. We examined whether calcium increases in pericytes or B-cells led to a vascular response in acute slices using the P2Y(2/4) receptor (P2Y(2/4)R) agonist UTP, electrical stimulation, or transgenic mice expressing exogenous Gq-coupled receptors (MrgA1) in B-cells. UTP increased calcium in pericytes leading to capillary constrictions. Electrical stimulation induced calcium propagation in SVZ cells followed by capillary constrictions involving purinergic receptors. In transgenic mice, selective calcium increases in B-cells induced P2Y(2/4)R-dependent capillary constrictions, suggesting that B-cells release ATP activating purinergic receptors on pericytes. Interestingly, in the presence of a P2Y(2/4)R blocker, dilation was observed. Intraventricular UTP injection transiently decreased blood flow monitored in vivo using laser Doppler flowmetry. Using neonatal electroporation, we expressed MrgA1 in slow cycling radial glia-derived B1 cells, i.e., NPCs. Intraventricular injection of an MrgA1 ligand increased blood flow in the SVZ. Thus, upon intracellular calcium increases B-cells/NPCs release ATP and vasodilating factors that activate purinergic receptors on pericytes triggering a vascular response and blood flow increase in vivo. Considering that NPCs receive signals from other SVZ cells, these findings further suggest that NPCs act as transducers of neurometabolic coupling in the SVZ.

Effects of sensory deprivation on glomerular interneurons in the mouse olfactory bulb: differences in mortality and phenotypic adjustment of dopaminergic neurons.

Neurogenesis persists in the mammalian subventricular zone after birth, producing various populations of olfactory bulb (OB) interneurons, including GABAergic and mixed dopaminergic/GABAergic (DA) neurons for the glomerular layer. While olfactory sensory activity is a major factor controlling the integration of new neurons, its impact on specific subtypes is not well understood. In this study we used genetic labeling of defined neuron subsets, in combination with reversible unilateral sensory deprivation and longitudinal in vivo imaging, to examine the behavior of postnatally born glomerular neurons. We find that a small fraction of GABAergic and of DA neurons die after 4 weeks of sensory deprivation while surviving DA-neurons exhibit a substantial decrease in tyrosine hydroxylase (TH) expression levels. Importantly, after reopening of the naris, cell death is arrested and TH levels go back to normal levels, indicating a specific adaptation to the level of sensory activity. We conclude that sensory deprivation induces adjustments in the population of glomerular neurons, involving both, cell death and adaptation of neurotransmitter use in specific neuron types. Our study highlights the dynamic nature of glomerular neurons in response to sensory deprivation and provide valuable insights into the plasticity and adaptability of the olfactory system.

Prominent in vivo influence of single interneurons in the developing barrel cortex.

Spontaneous synchronous activity is a hallmark of developing brain circuits and promotes their formation. Ex vivo, synchronous activity was shown to be orchestrated by a sparse population of highly connected GABAergic 'hub' neurons. The recent development of all-optical methods to record and manipulate neuronal activity in vivo now offers the unprecedented opportunity to probe the existence and function of hub cells in vivo. Using calcium imaging, connectivity analysis and holographic optical stimulation, we show that single GABAergic, but not glutamatergic, neurons influence population dynamics in the barrel cortex of non-anaesthetized mouse pups. Single GABAergic cells mainly exert an inhibitory influence on both spontaneous and sensory-evoked population bursts. Their network influence scales with their functional connectivity, with highly connected hub neurons displaying the strongest impact. We propose that hub neurons function in tailoring intrinsic cortical dynamics to external sensory inputs.

Gap junction-mediated calcium waves define communication networks among murine postnatal neural progenitor cells.

In the postnatal neurogenic niche, two populations of astrocyte-like cells (B cells) persist, one acting as neural progenitor cells (NPCs, B1 cells) and one forming a structural boundary between the neurogenic niche and the striatum (B2 cells, niche astrocytes). Despite being viewed as two distinct entities, we found that B1 and B2 cells express the gap junction protein connexin 43 and display functional coupling involving 50-60 cells. Using neonatal electroporation to label slowly cycling radial glia-derived B1 cells, which send a basal process onto blood vessels, we further confirmed dye coupling between NPCs. To assess the functionality of the coupling, we used calcium imaging in a preparation preserving the three-dimensional architecture of the subventricular zone. Intercellular calcium waves were observed among B cells. These waves travelled bidirectionally between B1 and B2 cells and propagated on blood vessels. Inter-B-cell calcium waves were absent in the presence of a gap junction blocker but persisted with purinergic receptor blockers. These findings show that privileged microdomains of communication networks exist among NPCs and niche astrocytes. Such functional coupling between these two cell types suggests that niche astrocytes do not merely have a structural role, but may play an active role in shaping the behavior of NPCs.

GFAP-GFP neural progenitors are antigenically homogeneous and anchored in their enclosed mosaic niche.

Study of the different stages of postnatal neurogenesis relies on using antigenic markers and transgenic mice. In particular, neural stem cells that express GFAP are studied using mice expressing GFP under the human GFAP promoter (GFAP-GFP). However, it remains unclear whether GFP and the commonly used progenitor markers label different cell populations in the neurogenic subventricular zone (SVZ) and its rostral extension into the olfactory bulb (i.e. rostral migratory stream, RMS). Here, we found that all GFP-fluorescent cells express GFAP, the radial glia marker brain lipid-binding protein (BLBP), Lewis X (LeX), and the astrocytic marker GLAST. Faint GFP fluorescence could be detected in a few cells expressing EGF receptors (EGFRs), Olig2, or S100, suggesting that GFAP-GFP cells generate these diverse cell types. GFP-fluorescent cells were slowly cycling, as shown by their long-term retention of BrdU, and less than 10% expressed the proliferative markers Ki67 and Mcm2. The majority of EGFR-expressing cells and Olig2-expressing cells were cycling. NG2 and EGFR identified distinct progenitor populations while Olig2 labeled a subset of EGFR-expressing cells. The entire neurogenic zone contained a mosaic of different cell types and was ensheathed by processes of GFAP-expressing cells and NG2 cells. Finally, using time-lapse imaging in acute slices, we show that GFP-fluorescent cells are stationary within the SVZ. Our findings collectively highlight the cellular mosaic of the neurogenic niche, show that the slowly-cycling GFAP-expressing cells are stationary and generate distinct intermediate progenitors.

Characterization of perinatally born glutamatergic neurons of the mouse olfactory bulb based on NeuroD6 expression reveals their resistance to sensory deprivation.

During postnatal olfactory bulb (OB) neurogenesis, predetermined stem cells residing in the ventricular-subventricular zone continuously generate progenitors that migrate in the rostral migratory stream and integrate into the OB. Although the vast majority of these postnatally generated interneurons are inhibitory, a sub-fraction represents glutamatergic neurons that integrate into the superficial glomerular layer. In the present work, we demonstrate that the bHLH transcription factor NeuroD6 is specifically and transitorily expressed in the dorsal neurogenic lineage that generates glutamatergic juxtaglomerular cells (JGCs) for the OB. Using lineage tracing combined with whole brain clearing, we provide new insight into timing of generation, morphology, and connectivity of glutamatergic JGCs. Specifically, we show that all glutamatergic JGCs send complex axons with varying projection patterns into different layers of the OB. Moreover, we find that, contrary to GABAergic OB interneurons, glutamatergic JGCs survive under sensory deprivation, indicating that inhibitory and excitatory populations are differentially susceptible to environmental stimulation.

Neuronal integration in the adult mouse olfactory bulb is a non-selective addition process.

Adult neurogenesis in the olfactory bulb (OB) is considered as a competition in which neurons scramble during a critical selection period for integration and survival. Moreover, newborn neurons are thought to replace pre-existing ones that die. Despite indirect evidence supporting this model, systematic in vivo observations are still scarce. We used two-photon in vivo imaging to study neuronal integration and survival. We show that loss of new neurons in the OB after arrival at terminal positions occurs only at low levels. Moreover, long-term observations showed that no substantial cell death occurred at later stages. Neuronal death was induced by standard doses of thymidine analogs, but disappeared when low doses were used. Finally, we demonstrate that the OB grows throughout life. This shows that neuronal selection during OB-neurogenesis does not occur after neurons reached stable positions. Moreover, this suggests that OB neurogenesis does not represent neuronal turnover but lifelong neuronal addition.

Control of neuroblast production and migration by converging GABA and glutamate signals in the postnatal forebrain.

The production of adult-born neurons is an ongoing process accounting for > 10,000 immature neurons migrating to the olfactory bulb every day. This high turnover rate necessitates profound control mechanisms converging onto neural stem cells and neuroblasts to achieve adequate adult-born neuron production. Here, we elaborate on a novel epigenetic control of adult neurogenesis via highly coordinated, non-synaptic, intercellular signalling. This communication engages the neurotransmitters GABA and glutamate, whose extracellular concentrations depend on neuroblast number and high affinity uptake systems in stem cells. Previous studies show that neuroblasts release GABA providing a negative feedback control of stem cell proliferation. Recent findings show an unexpected mosaic expression of glutamate receptors leading to calcium elevations in migrating neuroblasts. We speculate that stem cells release glutamate that activates glutamate receptors on migrating neuroblasts providing them with migratory and survival cues. In addition, we propose that the timing of neurotransmitter release and their spatial diffusion will determine the convergent coactivation of neuroblasts and stem cells, and provide a steady-state level of neuroblast production. Upon external impact or injury this signalling may adjust to a new steady-state level, thus providing non-synaptic scaling of neuroblast production.

Tonic activation of GLUK5 kainate receptors decreases neuroblast migration in whole-mounts of the subventricular zone.

In the postnatal subventricular zone (SVZ), neuroblasts migrate in chains along the lateral ventricle towards the olfactory bulb. AMPA/kainate receptors as well as metabotropic glutamate receptors subtype 5 (mGluR5) are expressed in SVZ cells. However, the cells expressing these receptors and the function of these receptors remain unexplored. We thus examined whether SVZ neuroblasts express mGluR5 and Ca(2+)-permeable kainate receptors in mouse slices. Doublecortin (DCX)-immunopositive cells (i.e. neuroblasts) immunostained positive for mGluR5 and GLU(K5-7)-containing kainate receptors. RT-PCR from approximately 10 GFP-fluorescent cell aspirates obtained in acute slices from transgenic mice expressing green fluorescent protein (GFP) under the DCX promoter showed mGluR5 and GLU(K5) receptor mRNA in SVZ neuroblasts. Patch-clamp data suggest that approximately 60% of neuroblasts express functional GLU(K5)-containing receptors. Activation of mGluR5 and GLU(K5)-containing receptors induced Ca(2+) increases in 50% and 60% of SVZ neuroblasts, respectively, while most neuroblasts displayed GABA(A)-mediated Ca(2+) responses. To examine the effects of these receptors on the speed of neuroblast migration, we developed a whole-mount preparation of the entire lateral ventricle from postnatal day (P) 20-25 DCX-GFP mice. The GABA(A) receptor (GABA(A)R) antagonist bicuculline increased the speed of neuroblast migration by 27%, as previously reported in acute slices. While the mGluR5 antagonist MPEP did not affect the speed of neuroblast migration, the homomeric and heteromeric GLU(K5) receptor antagonists, NS3763 and UB302, respectively, increased the migration speed by 38%. These data show that although both GLU(K5) receptor and mGluR5 activations increase Ca(2+) in neuroblasts, only GLU(K5) receptors tonically reduce the speed of neuroblast migration along the lateral ventricle.

GABA and glutamate signaling: homeostatic control of adult forebrain neurogenesis.

The neurotransmitter GABA exerts a strong negative influence on the production of adult-born olfactory bulb interneurons via tightly regulated, non-synaptic GABAergic signaling. After discussing some findings on GABAergic signaling in the neurogenic subventricular zone (SVZ), we provide data suggesting ambient GABA clearance via two GABA transporter subtypes and further support for a non-vesicular mechanism of GABA release from neuroblasts. While GABA works in cooperation with the neurotransmitter glutamate during embryonic cortical development, the role of glutamate in adult forebrain neurogenesis remains obscure. Only one of the eight metabotropic glutamate receptors (mGluRs), mGluR5, has been reported to tonically increase the number of proliferative SVZ cells in vivo, suggesting a local source of glutamate in the SVZ. We show here that glutamate antibodies strongly label subventricular zone (SVZ) astrocytes, some of which are stem cells. We also show that some SVZ neuroblasts express one of the ionotropic glutamate receptors, AMPA/kainate receptors, earlier than previously thought. Collectively, these findings suggest that neuroblast-to-astrocyte GABAergic signaling may cooperate with astrocyte-to-neuroblast glutamatergic signaling to provide strong homeostatic control on the production of adult-born olfactory bulb interneurons.

Activation of GABAA receptors controls mesiotemporal lobe epilepsy despite changes in chloride transporters expression: In vivo and in silico approach.

Mesiotemporal lobe Epilepsy (MTLE), the most frequent form of focal epilepsy, is often drug-resistant. Enriching the epileptic focus with GABA-releasing engineered cells has been proposed as a strategy to prevent seizures. However, ex vivo data from animal models and MTLE patients suggest that, due to changes in chloride homeostasis, GABAA receptor activation is depolarizing and partly responsible for focal interictal discharges and seizure initiation. To understand how these two contradictory aspects of GABAergic neurotransmission coexist in MTLE, we used an established mouse model of MTLE presenting hippocampal sclerosis and recurrent hippocampal paroxysmal discharges (HPDs) 30-40days after a unilateral injection of kainate in the dorsal hippocampus. We first showed that injections of GABAA receptor agonists either systemically or directly into hippocampus suppressed HPDs. Western-blotting and immunostaining revealed that levels of α1, α3 and γ2 GABAA receptor subunits were increased in epileptic mice, compared to saline controls, while levels of R1 and R2 GABAB receptor subunits but also NR1, NR2A and NR2B NMDA receptor subunits and GluR1 and GluR2 AMPA receptor subunits were decreased. In addition, we showed that the expression of the transporter NKCC1, which load neurons with chloride, was increased, whereas KCC2, a chloride extruder, was decreased and that HPDs were suppressed by injection of blockers of NKCC1. These different changes were integrated in a numerical model, and in silico simulations supported the notion that chloride imbalance impair local inhibitory control of pyramidal neurons' activity in this model of MTLE. However, our numerical model also suggested that lasting activation of these receptors restore physiological intracellular chloride concentrations and suppress HPDs. Overall, our study suggests that activation of GABAA receptor remains an effective antiepileptic strategy to suppress focal seizures in MTLE, and demonstrates that modeling and simulation studies provide new insights about the cellular and synaptic mechanisms of this disease.

Early expression of sodium channel transcripts and sodium current by cajal-retzius cells in the preplate of the embryonic mouse neocortex.

In mouse, the first neurons are generated at embryonic day (E) 12 and form the preplate (PP), which contains a mix of future marginal zone cells, including Cajal-Retzius cells, and subplate cells. To detect developmental changes in channel populations in these earliest-generated neurons of the cerebral cortex, we studied the electrophysiological properties of proliferative cells of the ventricular zone and postmitotic neurons of the PP at E12 and E13, using whole-cell patch-clamp recordings. We found an inward sodium current in 55% of PP cells. To determine whether sodium currents occur in a specific cell type, we stained recorded cells with an antibody for calretinin, a calcium-binding protein found specifically in Cajal-Retzius cells. All calretinin-positive cells had sodium currents, although so did some calretinin-negative cells. To correlate the Na current expression to Na channel gene expression with the Cajal-Retzius cell phenotype, we performed single-cell reverse transcription-PCR on patch-clamp recorded cells to detect expression of the Cajal-Retzius cell marker reelin and the Na channel isoforms SCN 1, 2, and 3. These results showed that virtually all Cajal-Retzius cells (97%), as judged by reelin expression, express the SCN transcript identified as the SCN3 isoform. Of these, 41% presented a functional Na current. There is, however, a substantial SCN-positive population in the PP (27% of SCN-positive cells) that does not express reelin. These results raise the possibility that populations of pioneer neurons of the PP, including Cajal-Retzius cells, gain neuronal physiological properties early in development via expression of the Na(v)1.3 (SCN3) Na channel isoform.

GABAergic striatal neurons project dendrites and axons into the postnatal subventricular zone leading to calcium activity.

GABA regulates the behavior of neuroblasts and neural progenitor cells in the postnatal neurogenic subventricular zone (SVZ) through GABAA receptor (GABAAR)-mediated calcium increases. However, the source of GABA necessary for sufficient GABAAR-mediated depolarization and calcium increase has remained speculative. Here, we explored whether GABAergic striatal neurons functionally connect with SVZ cells. Using patch clamp recordings or single cell electroporation, striatal neurons along the SVZ were filled with a fluorescent dye revealing that they send both dendrites and axons into the SVZ. About 93% of the recorded neurons were medium spiny or aspiny GABAergic neurons and each neuron sent 3-4 processes into the SVZ covering ~56 µm. Using calcium imaging, we found that depolarization of striatal neurons led to increased calcium activity in SVZ cells that were mediated by GABAAR activation. Collectively, these findings undercover a novel mode of signaling in the SVZ providing a mechanism of brain activity-mediated regulation of postnatal neurogenesis through GABAergic striatal activity.

Preparation of acute subventricular zone slices for calcium imaging.

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells. Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis. Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

Single-cell Tsc1 knockout during corticogenesis generates tuber-like lesions and reduces seizure threshold in mice.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by mutations in Tsc1 or Tsc2 that lead to mammalian target of rapamycin (mTOR) hyperactivity. Patients with TSC suffer from intractable seizures resulting from cortical malformations known as tubers, but research into how these tubers form has been limited because of the lack of an animal model. To address this limitation, we used in utero electroporation to knock out Tsc1 in selected neuronal populations in mice heterozygous for a mutant Tsc1 allele that eliminates the Tsc1 gene product at a precise developmental time point. Knockout of Tsc1 in single cells led to increased mTOR activity and soma size in the affected neurons. The mice exhibited white matter heterotopic nodules and discrete cortical tuber-like lesions containing cytomegalic and multinucleated neurons with abnormal dendritic trees resembling giant cells. Cortical tubers in the mutant mice did not exhibit signs of gliosis. Furthermore, phospho-S6 immunoreactivity was not upregulated in Tsc1-null astrocytes despite a lower seizure threshold. Collectively, these data suggest that a double-hit strategy to eliminate Tsc1 in discrete neuronal populations generates TSC-associated cortical lesions, providing a model to uncover the mechanisms of lesion formation and cortical hyperexcitability. In addition, the absence of glial reactivity argues against a contribution of astrocytes to lesion-associated hyperexcitability.