Developmental Profile of Brain Neprilysin Expression Correlates with Olfactory Behaviour of Rats.

A neuropeptidase, neprilysin (NEP), is a major amyloid (Aß)-degrading enzyme involved in the pathogenesis of Alzheimer's disease (AD). The olfactory system is affected early in AD with characteristic Aß accumulation, but data on the dynamics of NEP expression in the olfactory system are absent. Our study demonstrates that NEP mRNA expression in rat olfactory bulbs (OB), entorhinal cortex (ECx), hippocampus (Hip), parietal cortex (PCx) and striatum (Str) increases during the first postnatal month being the highest in the OB and Str. By 3 months, NEP mRNA levels sharply decrease in the ECx, Hip and PCx and by 9 months in the OB, but not in the Str, which correlates with declining olfaction in aged rats tested in the food search paradigm. One-month-old rats subjected to prenatal hypoxia on E14 had lower NEP mRNA levels in the ECx, Hip and PCx (but not in the OB and Str) compared with the control offspring and demonstrated impaired olfaction in the odour preference and food search paradigms. Administration to these rats of a histone deacetylase inhibitor, sodium valproate, restored NEP expression in the ECx, Hip and PCx and improved olfaction. Our data support NEP involvement in olfactory function.

Timely Inhibitory Circuit Formation Controlled by Abl1 Regulates Innate Olfactory Behaviors in Mouse.

More than one-half of the interneurons in a mouse olfactory bulb (OB) develop during the first week after birth and predominantly connect to excitatory tufted cells near the superficial granule cell layer (sGCL), unlike late-born interneurons. However, the molecular mechanisms underlying the temporal specification are yet to be identified. In this study, we determined the role of Abelson tyrosine-protein kinase 1 (Abl1) in the temporal development of early-born OB interneurons. Lentiviral knockdown of Abl1 disrupts the sGCL circuit of early-born interneurons through defects in function and circuit integration, resulting in olfactory hyper-sensitivity. We show that doublecortin (Dcx) is phosphorylated by Abl1, which contributes to the stabilization of Dcx, thereby regulating microtubule dynamics. Finally, Dcx overexpression rescues Abl1 knockdown-induced anatomic or functional defects. In summary, specific signaling by Abl1-Dcx in early-born interneurons facilitates the temporal development of the sGCL circuit to regulate innate olfactory functions, such as detection and sensitivity.

Gli3 Regulates Vomeronasal Neurogenesis, Olfactory Ensheathing Cell Formation, and GnRH-1 Neuronal Migration.

During mammalian development, gonadotropin-releasing-hormone-1 neurons (GnRH-1ns) migrate from the developing vomeronasal organ (VNO) into the brain asserting control of pubertal onset and fertility. Recent data suggest that correct development of the olfactory ensheathing cells (OEC) is imperative for normal GnRH-1 neuronal migration. However, the full ensemble of molecular pathways that regulate OEC development remains to be fully deciphered. Loss-of-function of the transcription factor Gli3 is known to disrupt olfactory development, however, if Gli3 plays a role in GnRH-1 neuronal development is unclear. By analyzing Gli3 extra-toe mutants (Gli3Xt/Xt), we found that Gli3 loss-of-function compromises the onset of achaete-scute family bHLH transcription factor 1 (Ascl-1)+ vomeronasal progenitors and the formation of OEC in the nasal mucosa. Surprisingly, GnRH-1 neurogenesis was intact in Gli3Xt/Xt mice but they displayed significant defects in GnRH-1 neuronal migration. In contrast, Ascl-1null mutants showed reduced neurogenesis for both vomeronasal and GnRH-1ns but less severe defects in OEC development. These observations suggest that Gli3 is critical for OEC development in the nasal mucosa and subsequent GnRH-1 neuronal migration. However, the nonoverlapping phenotypes between Ascl-1 and Gli3 mutants indicate that Ascl-1, while crucial for GnRH-1 neurogenesis, is not required for normal OEC development. Because Kallmann syndrome (KS) is characterized by abnormal GnRH-1ns migration, we examined whole-exome sequencing data from KS subjects. We identified and validated a GLI3 loss-of-function variant in a KS individual. These findings provide new insights into GnRH-1 and OECs development and demonstrate that human GLI3 mutations contribute to KS etiology.SIGNIFICANCE STATEMENT The transcription factor Gli3 is necessary for correct development of the olfactory system. However, if Gli3 plays a role in controlling GnRH-1 neuronal development has not been addressed. We found that Gli3 loss-of-function compromises the onset of Ascl-1+ vomeronasal progenitors, formation of olfactory ensheathing cells in the nasal mucosa, and impairs GnRH-1 neuronal migration to the brain. By analyzing Ascl-1null mutants we dissociated the neurogenic defects observed in Gli3 mutants from lack of olfactory ensheathing cells in the nasal mucosa, moreover, we discovered that Ascl-1 is necessary for GnRH-1 ontogeny. Analyzing human whole-exome sequencing data, we identified a GLI3 loss-of-function variant in a KS individual. Our data suggest that GLI3 is a candidate gene contributing to KS etiology.

Detachment of Chain-Forming Neuroblasts by Fyn-Mediated Control of cell-cell Adhesion in the Postnatal Brain.

In the rodent olfactory system, neuroblasts produced in the ventricular-subventricular zone of the postnatal brain migrate tangentially in chain-like cell aggregates toward the olfactory bulb (OB) through the rostral migratory stream (RMS). After reaching the OB, the chains are dissociated and the neuroblasts migrate individually and radially toward their final destination. The cellular and molecular mechanisms controlling cell-cell adhesion during this detachment remain unclear. Here we report that Fyn, a nonreceptor tyrosine kinase, regulates the detachment of neuroblasts from chains in the male and female mouse OB. By performing chemical screening and in vivo loss-of-function and gain-of-function experiments, we found that Fyn promotes somal disengagement from the chains and is involved in neuronal migration from the RMS into the granule cell layer of the OB. Fyn knockdown or Dab1 (disabled-1) deficiency caused p120-catenin to accumulate and adherens junction-like structures to be sustained at the contact sites between neuroblasts. Moreover, a Fyn and N-cadherin double-knockdown experiment indicated that Fyn regulates the N-cadherin-mediated cell adhesion between neuroblasts. These results suggest that the Fyn-mediated control of cell-cell adhesion is critical for the detachment of chain-forming neuroblasts in the postnatal OB.SIGNIFICANCE STATEMENT In the postnatal brain, newly born neurons (neuroblasts) migrate in chain-like cell aggregates toward their destination, where they are dissociated into individual cells and mature. The cellular and molecular mechanisms controlling the detachment of neuroblasts from chains are not understood. Here we show that Fyn, a nonreceptor tyrosine kinase, promotes the somal detachment of neuroblasts from chains, and that this regulation is critical for the efficient migration of neuroblasts to their destination. We further show that Fyn and Dab1 (disabled-1) decrease the cell-cell adhesion between chain-forming neuroblasts, which involves adherens junction-like structures. Our results suggest that Fyn-mediated regulation of the cell-cell adhesion of neuroblasts is critical for their detachment from chains in the postnatal brain.

Haploinsufficiency of SIX3 Abolishes Male Reproductive Behavior Through Disrupted Olfactory Development, and Impairs Female Fertility Through Disrupted GnRH Neuron Migration.

Mating behavior in males and females is dependent on olfactory cues processed through both the main olfactory epithelium (MOE) and the vomeronasal organ (VNO). Signaling through the MOE is critical for the initiation of male mating behavior, and the loss of MOE signaling severely compromises this comportment. Here, we demonstrate that dosage of the homeodomain gene Six3 affects the degree of development of MOE but not the VNO. Anomalous MOE development in Six3 heterozygote mice leads to hyposmia, specifically disrupting male mounting behavior by impairing detection of volatile female estrus pheromones. Six3 is highly expressed in the MOE, main olfactory bulb (MOB), and hypothalamus; all regions essential in the proper migration of the gonadotropin-releasing hormone (GnRH) neurons, a key reproductive neuronal population that migrates along olfactory axons from the developing nose into the brain. Interestingly, we find that the reduction in Six3 expression in Six3 heterozygote mice compromises development of the MOE and MOB, resulting in mis-migration of GnRH neurons due to improper olfactory axon targeting. This reduction in the hypothalamic GnRH neuron population, by 45% in adulthood, leads to female subfertility, but does not impact male hormone levels, suggesting that male infertility is not related to GnRH neuron numbers, but exclusively linked to abnormal olfaction. We here determine that Six3 is haploinsufficient for MOE development, GnRH neuron migration, and fertility, and represents a novel candidate gene for Kallmann syndrome, a form of inherited infertility.

Behavioral and molecular analyses of olfaction-mediated avoidance responses of Rana (Lithobates) catesbeiana tadpoles: Sensitivity to thyroid hormones, estrogen, and treated municipal wastewater effluent.

Olfaction is critical for survival, facilitating predator avoidance and food location. The nature of the olfactory system changes during amphibian metamorphosis as the aquatic herbivorous tadpole transitions to a terrestrial, carnivorous frog. Metamorphosis is principally dependent on the action of thyroid hormones (THs), l-thyroxine (T4) and 3,5,3'-triiodothyronine (T3), yet little is known about their influence on olfaction during this phase of postembryonic development. We exposed Taylor Kollros stage I-XIII Rana (Lithobates) catesbeiana tadpoles to physiological concentrations of T4, T3, or 17-beta-estradiol (E2) for 48h and evaluated a predator cue avoidance response. The avoidance response in T3-exposed tadpoles was abolished while T4- or E2-exposed tadpoles were unaffected compared to control tadpoles. qPCR analyses on classic TH-response gene transcripts (thra, thrb, and thibz) in the olfactory epithelium demonstrated that, while both THs produced molecular responses, T3 elicited greater responses than T4. Municipal wastewater feed stock was spiked with a defined pharmaceutical and personal care product (PPCP) cocktail and treated with an anaerobic membrane bioreactor (AnMBR). Despite substantially reduced PPCP levels, exposure to this effluent abolished avoidance behavior relative to AnMBR effluent whose feed stock was spiked with vehicle. Thibz transcript levels increased upon exposure to either effluent indicating TH mimic activity. The present work is the first to demonstrate differential TH responsiveness of the frog tadpole olfactory system with both behavioral and molecular alterations. A systems-based analysis is warranted to further elucidate the mechanism of action on the olfactory epithelium and identify further molecular bioindicators linked to behavioral response disruption.

Rostro-caudal maturation of glial cells in the accessory olfactory system during development: involvement in outgrowth of GnRH neurites.

During mammalian embryonic development, GnRH neurones differentiate from the nasal placode and migrate through the nasal septum towards the forebrain. We previously showed that a category of glial cells, the olfactory ensheathing cells (OEC), forms the microenvironment of migrating GnRH neurones. Here, to characterize the quantitative and qualitative importance of this glial, we investigated the spatiotemporal maturation of glial cells in situ and the role of maturing glia in GnRH neurones development ex vivo. More than 90% of migrating GnRH neurones were found to be associated with glial cells. There was no change in the cellular microenvironment of GnRH neurones in the regions crossed during embryonic development as glial cells formed the main microenvironment of these neurones (53.4%). However, the phenotype of OEC associated with GnRH neurones changed across regions. The OEC progenitors immunoreactive to brain lipid binding protein formed the microenvironment of migrating GnRH neurones from the vomeronasal organ to the telencephalon and were also present in the diencephalon. However, during GnRH neurone migration, maturation of OEC to [GFAP+] state (glial fibrillary acid protein) was only observed in the nasal septum. Inducing depletion of OEC in maturation, using transgenic mice expressing herpes simplex virus thymidine kinase driven by the GFAP promoter, had no impact on neurogenesis or on triggering GnRH neurones migration in nasal explant culture. Nevertheless, depletion of [GFAP+] cells decreased GnRH neurites outgrowth by 57.4%. This study suggests that specific maturation of OEC in the nasal septum plays a role in morphological differentiation of GnRH neurones.

The temporal expression profile of a Nos3-related natural antisense RNA in the brain suggests a possible role in neurogenesis.

Experimental work over the past several years has revealed an unexpected abundance of long natural antisense transcripts (NATs) in eukaryotic species. In light of the proposed role of such RNA molecules in the regulation of gene expression in the brain, attention is now focused on specific examples of neuronal NATs. Of particular interest are NATs that are complementary to mRNAs encoding nitric oxide synthase (NOS), the enzyme responsible for production of the important gaseous neurotransmitter nitric oxide (NO). Here we study the temporal expression profile of murine Nos3as NAT in the brain. Notably, Nos3as NAT is known to act as a negative regulator of Nos3 gene expression. The results of our quantitative analysis reveal differential expression of Nos3as NAT during embryonic and post-embryonic stages of development of the brain. Also, they show that the low levels of Nos3as NAT coincides with active neurogenesis. In addition we report on an inverse correlation between the relative expression level of Nos3as NAT and the level of Nos3 protein. Thus our data raise the hypothesis that the Nos3as NAT regulates neurogenesis through suppression of Nos3 gene activity. This idea is further supported by experiments conducted on the olfactory bulbs and cultured neuroblastoma cells.

Hyperpolarization-Activated Currents and Subthreshold Resonance in Granule Cells of the Olfactory Bulb.

An important contribution to neural circuit oscillatory dynamics is the ongoing activation and inactivation of hyperpolarization-activated currents (Ih). Network synchrony dynamics play an important role in the initial processing of odor signals by the main olfactory bulb (MOB) and accessory olfactory bulb (AOB). In the mouse olfactory bulb, we show that Ih is present in granule cells (GCs), the most prominent inhibitory neuron in the olfactory bulb, and that Ih underlies subthreshold resonance in GCs. In accord with the properties of Ih, the currents exhibited sensitivity to changes in extracellular K+ concentration and ZD7288 (4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidin chloride), a blocker of Ih. ZD7288 also caused GCs to hyperpolarize and increase their input resistance, suggesting that Ih is active at rest in GCs. The inclusion of cAMP in the intracellular solution shifted the activation of Ih to less negative potentials in the MOB, but not in the AOB, suggesting that channels with different subunit composition mediate Ih in these regions. Furthermore, we show that mature GCs exhibit Ih-dependent subthreshold resonance in the theta frequency range (4-12 Hz). Another inhibitory subtype in the MOB, the periglomerular cells, exhibited Ih-dependent subthreshold resonance in the delta range (1-4 Hz), while principal neurons, the mitral cells, do not exhibit Ih-dependent subthreshold resonance. Importantly, Ih size, as well as the strength and frequency of resonance in GCs, exhibited a postnatal developmental progression, suggesting that this development of Ih in GCs may differentially contribute to their integration of sensory input and contribution to oscillatory circuit dynamics.

Amyloid ß precursor protein regulates neuron survival and maturation in the adult mouse brain.

The amyloid-ß precursor protein (APP) is a transmembrane protein that is widely expressed within the central nervous system (CNS). While the pathogenic dysfunction of this protein has been extensively studied in the context of Alzheimer's disease, its normal function is poorly understood, and reports have often appeared contradictory. In this study we have examined the role of APP in regulating neurogenesis in the adult mouse brain by comparing neural stem cell proliferation, as well as new neuron number and morphology between APP knockout mice and C57bl6 controls. Short-term EdU administration revealed that the number of proliferating EdU+ neural progenitor cells and the number of PSA-NCAM+ neuroblasts produced in the SVZ and dentate gyrus were not affected by the life-long absence of APP. However, by labelling newborn cells with EdU and then following their fate over-time, we determined that ~48% more newly generated EdU+ NeuN+ neurons accumulated in the granule cell layer of the olfactory bulb and ~57% more in the dentate gyrus of young adult APP knockout mice relative to C57bl6 controls. Furthermore, proportionally fewer of the adult-born olfactory bulb granule neurons were calretinin+. To determine whether APP was having an effect on neuronal maturation, we administered tamoxifen to young adult Nestin-CreERT2::Rosa26-YFP and Nestin-CreERT2::Rosa26-YFP::APP-knockout mice, fluorescently labelling ~80% of newborn (EdU+) NeuN+ dentate granule neurons formed between P75 and P105. Our analysis of their morphology revealed that neurons added to the hippocampus of APP knockout mice have shorter dendritic arbors and only half the number of branch points as those generated in C57bl6 mice. We conclude that APP reduces the survival of newborn neurons in the olfactory bulb and hippocampus, but that it does not influence all neuronal subtypes equally. Additionally, APP influences dentate granule neuron maturation, acting as a robust regulator of dendritic extension and arborisation.

miR-200 family controls late steps of postnatal forebrain neurogenesis via Zeb2 inhibition.

During neurogenesis, generation, migration and integration of the correct numbers of each neuron sub-type depends on complex molecular interactions in space and time. MicroRNAs represent a key control level allowing the flexibility and stability needed for this process. Insight into the role of this regulatory pathway in the brain is still limited. We performed a sequential experimental approach using postnatal olfactory bulb neurogenesis in mice, starting from global expression analyses to the investigation of functional interactions between defined microRNAs and their targets. Deep sequencing of small RNAs extracted from defined compartments of the postnatal neurogenic system demonstrated that the miR-200 family is specifically induced during late neuronal differentiation stages. Using in vivo strategies we interfered with the entire miR-200 family in loss- and gain-of-function settings, showing a role of miR-200 in neuronal maturation. This function is mediated by targeting the transcription factor Zeb2. Interestingly, so far functional interaction between miR-200 and Zeb2 has been exclusively reported in cancer or cultured stem cells. Our data demonstrate that this regulatory interaction is also active during normal neurogenesis.

Lgl1 Is Required for Olfaction and Development of Olfactory Bulb in Mice.

Lethal giant larvae 1 (Lgl1) was initially identified as a tumor suppressor in Drosophila and functioned as a key regulator of epithelial polarity and asymmetric cell division. In this study, we generated Lgl1 conditional knockout mice mediated by Pax2-Cre, which is expressed in olfactory bulb (OB). Next, we examined the effects of Lgl1 loss in the OB. First, we determined the expression patterns of Lgl1 in the neurogenic regions of the embryonic dorsal region of the LGE (dLGE) and postnatal OB. Furthermore, the Lgl1 conditional mutants exhibited abnormal morphological characteristics of the OB. Our behavioral analysis exhibited greatly impaired olfaction in Lgl1 mutant mice. To elucidate the possible mechanisms of impaired olfaction in Lgl1 mutant mice, we investigated the development of the OB. Interestingly, reduced thickness of the MCL and decreased density of mitral cells (MCs) were observed in Lgl1 mutant mice. Additionally, we observed a dramatic loss in SP8+ interneurons (e.g. calretinin and GABAergic/non-dopaminergic interneurons) in the GL of the OB. Our results demonstrate that Lgl1 is required for the development of the OB and the deletion of Lgl1 results in impaired olfaction in mice.

Characterization of a new Gsx2-cre line in the developing mouse telencephalon.

In this study, we generated a transgenic mouse line driving Cre and EGFP expression with two putative cis-regulatory modules (CRMs) (i.e., hs687 and hs678) upstream of the homeobox gene Gsx2 (formerly Gsh2), a critical gene for establishing lateral ganglionic eminence (LGE) identity. The combination of these two CRMs drives transgene expression within the endogenous Gsx2 expression domains along the anterior-posterior neuraxis. By crossing this transgenic line with the RosatdTomato (Ai14) reporter mouse line, we observed a unique recombination pattern in the lateral ventral telencephalon, namely the LGE and the dorsal half of the medial GE (MGE), but not in the septum. We found robust recombination in many cell types derived from these embryonic regions, including olfactory bulb and amygdala interneurons and striatal projection neurons from the LGE, as well as cortical interneurons from the MGE and caudal GE (CGE). In summary, this transgenic mouse line represents a new tool for genetic manipulation in the LGE/CGE and the dorsal half of MGE.

Expression patterns of homeobox genes in the mouse vomeronasal organ at postnatal stages.

Homeodomain proteins are encoded by homeobox genes and regulate development and differentiation in many neuronal systems. The mouse vomeronasal organ (VNO) generates in situ mature chemosensory neurons from stem cells. The roles of homeodomain proteins in neuronal differentiation in the VNO are poorly understood. Here we have characterized the expression patterns of 28 homeobox genes in the VNO of C57BL/6 mice at postnatal stages using multicolor fluorescent in situ hybridization. We identified 11 homeobox genes (Dlx3, Dlx4, Emx2, Lhx2, Meis1, Pbx3, Pknox2, Pou6f1, Tshz2, Zhx1, Zhx3) that were expressed exclusively in neurons; 4 homeobox genes (Pax6, Six1, Tgif1, Zfhx3) that were expressed in all non-neuronal cell populations, with Pax6, Six1 and Tgif1 also expressed in some neuronal progenitors and precursors; 12 homeobox genes (Adnp, Cux1, Dlx5, Dlx6, Meis2, Pbx2, Pknox1, Pou2f1, Satb1, Tshz1, Tshz3, Zhx2) with expression in both neuronal and non-neuronal cell populations; and one homeobox gene (Hopx) that was exclusively expressed in the non-sensory epithelium. We studied further in detail the expression of Emx2, Lhx2, Meis1, and Meis2. We found that expression of Emx2 and Lhx2 initiated between neuronal progenitor and neuronal precursor stages. As far as the sensory neurons of the VNO are concerned, Meis1 and Meis2 were only expressed in the apical layer, together with Gnai2, but not in the basal layer.

Attractant and repellent cues cooperate in guiding a subset of olfactory sensory axons to a well-defined protoglomerular target.

Olfactory sensory axons target well-defined intermediate targets in the zebrafish olfactory bulb called protoglomeruli well before they form odorant receptor-specific glomeruli. A subset of olfactory sensory neurons are labeled by expression of the or111-7:IRES:GAL4 transgene whose axons terminate in the central zone (CZ) protoglomerulus. Previous work has shown that some of these axons misproject to the more dorsal and anterior dorsal zone (DZ) protoglomerulus in the absence of Netrin 1/Dcc signaling. In search of additional cues that guide these axons to the CZ, we found that Semaphorin 3D (Sema3D) is expressed in the anterior bulb and acts as a repellent that pushes them towards the CZ. Further analysis indicates that Sema3D signaling is mediated through Nrp1a, while Nrp2b also promotes CZ targeting but in a Sema3D-independent manner. nrp1a, nrp2b and dcc transcripts are detected in or111-7 transgene-expressing neurons early in development and both Nrp1a and Dcc act cell-autonomously in sensory neurons to promote accurate targeting to the CZ. dcc and nrp1a double mutants have significantly more DZ misprojections than either single mutant, suggesting that the two signaling systems act independently and in parallel to direct a specific subset of sensory axons to their initial protoglomerular target.

Developmental Markers Expressed in Neocortical Layers Are Differentially Exhibited in Olfactory Cortex.

Neurons in the cerebral cortex stratify on the basis of their time of origin, axonal terminations and the molecular identities assigned during early development. Olfactory cortices share many feature with the neocortex, including clear lamination and similar cell types. The present study demonstrates that the markers differentially expressed in the projection neurons of the cerebral cortex are also found in olfactory areas. Three of the four regions examined (pars principalis of the anterior olfactory nucleus: AONpP, anterior and posterior piriform cortices: APC, PPC, and the olfactory tubercle) expressed transcription factors found in deep or superficial neurons in the developing neocortex, though large differences were found between areas. For example, while the AONpP, APC and PPC all broadly expressed the deep cortical marker CTIP2, NOR1 (NR4a3) levels were higher in AONpP and DAARP-32 was more prevalent in the APC and PPC. Similar findings were encountered for superficial cortical markers: all three regions broadly expressed CUX1, but CART was only observed in the APC and PPC. Furthermore, regional variations were observed even within single structures (e.g., NOR1 was found primarily in in the dorsal region of AONpP and CART expression was observed in a discrete band in the middle of layer 2 of both the APC and PPC). Experiments using the mitotic marker EDU verified that the olfactory cortices and neocortex share similar patterns of neuronal production: olfactory cells that express markers found in the deep neocortex are produced earlier than those that express superficial makers. Projection neurons were filled by retrograde tracers injected into the olfactory bulb to see if olfactory neurons with deep and superficial markers had different axonal targets. Unlike the cerebral cortex, no specificity was observed: neurons with each of the transcription factors examined were found to be labelled. Together the results indicate that olfactory cortices are complex: they differ from each other and each is formed from a variable mosaic of neurons. The results suggest that the olfactory cortices are not merely a remnant architype of the primordial forebrain but varied and independent regions.

A Dorsal SHH-Dependent Domain in the V-SVZ Produces Large Numbers of Oligodendroglial Lineage Cells in the Postnatal Brain.

Neural stem cells in different locations of the postnatal mouse ventricular-subventricular zone (V-SVZ) generate different subtypes of olfactory bulb (OB) interneurons. High Sonic hedgehog (SHH) signaling in the ventral V-SVZ regulates the production of specific subtypes of neurons destined for the OB. Here we found a transient territory of high SHH signaling in the dorsal V-SVZ beneath the corpus callosum (CC). Using intersectional lineage tracing in neonates to label dorsal radial glial cells (RGCs) expressing the SHH target gene Gli1, we demonstrate that this region produces many CC cells in the oligodendroglial lineage and specific subtypes of neurons in the OB. The number of oligodendroglial cells generated correlated with the levels of SHH signaling. This work identifies a dorsal domain of SHH signaling, which is an important source of oligodendroglial cells for the postnatal mammalian forebrain.

Olfactory ensheathing cells are the main phagocytic cells that remove axon debris during early development of the olfactory system.

During development of the primary olfactory system, axon targeting is inaccurate and axons inappropriately project within the target layer or overproject into the deeper layers of the olfactory bulb. As a consequence there is considerable apoptosis of primary olfactory neurons during embryonic and postnatal development and axons of the degraded neurons need to be removed. Olfactory ensheathing cells (OECs) are the glia of the primary olfactory nerve and are known to phagocytose axon debris in the adult and postnatal animal. However, it is unclear when phagocytosis by OECs first commences. We investigated the onset of phagocytosis by OECs in the developing mouse olfactory system by utilizing two transgenic reporter lines: OMP-ZsGreen mice which express bright green fluorescent protein in primary olfactory neurons, and S100ß-DsRed mice which express red fluorescent protein in OECs. In crosses of these mice, the fate of the degraded axon debris is easily visualized. We found evidence of axon degradation at embryonic day (E)13.5. Phagocytosis of the primary olfactory axon debris by OECs was first detected at E14.5. Phagocytosis of axon debris continued into the postnatal animal during the period when there was extensive mistargeting of olfactory axons. Macrophages were often present in close proximity to OECs but they contributed only a minor role to clearing the axon debris, even after widespread degeneration of olfactory neurons by unilateral bulbectomy and methimazole treatment. These results demonstrate that from early in embryonic development OECs are the primary phagocytic cells of the primary olfactory nerve.

PACAP modulation of calcium ion activity in developing granule cells of the neonatal mouse olfactory bulb.

Ca(2+) activity in the CNS is critical for the establishment of developing neuronal circuitry prior to and during early sensory input. In developing olfactory bulb (OB), the neuromodulators that enhance network activity are largely unknown. Here we provide evidence that pituitary adenylate cyclase-activating peptide (PACAP)-specific PAC1 receptors (PAC1Rs) expressed in postnatal day (P)2-P5 mouse OB are functional and enhance network activity as measured by increases in calcium in genetically identified granule cells (GCs). We used confocal Ca(2+) imaging of OB slices from Dlx2-tdTomato mice to visualize GABAergic GCs. To address whether the PACAP-induced Ca(2+) oscillations were direct or indirect effects of PAC1R activation, we used antagonists for the GABA receptors (GABARs) and/or glutamate receptors (GluRs) in the presence and absence of PACAP. Combined block of GABARs and GluRs yielded a 66% decrease in the numbers of PACAP-responsive cells, suggesting that 34% of OB neurons are directly activated by PACAP. Similarly, immunocytochemistry using anti-PAC1 antibody showed that 34% of OB neurons express PAC1R. Blocking either GluRs or GABARs alone indirectly showed that PACAP stimulates release of both glutamate and GABA, which activate GCs. The appearance of PACAP-induced Ca(2+) activity in immature GCs suggests a role for PACAP in GC maturation. To conclude, we find that PACAP has both direct and indirect effects on neonatal OB GABAergic cells and may enhance network activity by promoting glutamate and GABA release. Furthermore, the numbers of PACAP-responsive GCs significantly increased between P2 and P5, suggesting that PACAP-induced Ca(2+) activity contributes to neonatal OB development.

Npas4 regulates Mdm2 and thus Dcx in experience-dependent dendritic spine development of newborn olfactory bulb interneurons.

Sensory experience regulates the development of various brain structures, including the cortex, hippocampus, and olfactory bulb (OB). Little is known about how sensory experience regulates the dendritic spine development of OB interneurons, such as granule cells (GCs), although it is well studied in mitral/tufted cells. Here, we identify a transcription factor, Npas4, which is expressed in OB GCs immediately after sensory input and is required for dendritic spine formation. Npas4 overexpression in OB GCs increases dendritic spine density, even under sensory deprivation, and rescues reduction of dendrite spine density in the Npas4 knockout OB. Furthermore, loss of Npas4 upregulates expression of the E3-ubiquitin ligase Mdm2, which ubiquitinates a microtubule-associated protein Dcx. This leads to reduction in the dendritic spine density of OB GCs. Together, these findings suggest that Npas4 regulates Mdm2 expression to ubiquitinate and degrade Dcx during dendritic spine development in newborn OB GCs after sensory experience.