Fezf1 is required for penetration of the basal lamina by olfactory axons to promote olfactory development

In the development of the olfactory system, olfactory receptor neurons (ORNs) project their axons from the olfactory epithelium (OE) to the olfactory bulb (OB). The surface of the OB is covered by the central nervous system (CNS) basal lamina. To establish this connection, pioneer axons of the ORNs penetrate the CNS basal lamina at embryonic day 12.5 in mice. The importance of this penetration is highlighted by the Kallmann syndrome. However, little has been known about the molecular mechanism underlying this penetration process. Fezf1 (also called as Fez, Zfp312‐like, and 3110069A13Rik) is a C2H2‐type zinc‐finger gene expressed in the OE and hypothalamic region in mice. In Fezf1‐deficient mice, ORN axons (olfactory axons) do not reach the OB. Here we demonstrate that Fezf1‐deficient olfactory axons do not penetrate the CNS basal lamina in vivo, and the penetration activity of the axons in Matrigel is impaired in vitro. Coculture experiments using the OE and OB reveal that axonal projection of ORNs is rescued in Fezf1‐deficient mice in which the meninges including the CNS basal lamina are removed from the mutant OB. These data indicate that Fezf1 is required for the penetration of olfactory axons through the CNS basal lamina before they innervate the OB. J. Comp. Neurol. 515:565–584, 2009. © 2009 Wiley‐Liss, Inc.     

The Dlx5 homeodomain gene is essential for olfactory development and connectivity in the mouse

The distalless-related homeogene Dlx5 is expressed in the olfactory placodes and derived tissues and in the anterior-basal forebrain. We investigated the role of Dlx5 in olfactory development. In Dlx5(-/-) mice, the olfactory bulbs (OBs) lack glomeruli, exhibit disorganized cellular layers, and show reduced numbers of TH- and GAD67-positive neurons. The olfactory epithelium in Dlx5(-/-) mice is composed of olfactory receptor neurons (ORNs) that appear identical to wild-type ORNs, but their axons fail to contact the OBs. We transplanted Dlx5(-/-) OBs into a wild-type newborn mouse; wild-type ORN axons enter the mutant OB and form glomeruli, but cannot rescue the lamination defect or the expression of TH and GAD67. Thus, the absence of Dlx5 in the OB does not per se prevent ORN axon ingrowth. In conclusion, Dlx5 plays major roles in the connectivity of ORN axons and in the differentiation of OB interneurons.     

Evidence for intrinsic development of olfactory structures in Pax-6 mutant mice

It has been reported that the arrival of primary olfactory axons is required to induce the development of the olfactory bulb (OB). On the other hand, the Sey(Neu)/Sey(Neu) mutant mouse (Small eye) has been previously described as a model for the absence of olfactory bulbs, owing to the lack of olfactory epithelium (OE). In the present report, we take advantage of this mutant and study a neural structure in the rostral pole of the telencephalon that phenotypically resembles the prospective OB. We named this formation olfactory bulb-like structure (OBLS). We also report the occurrence, in the mutants, of small epithelial vesicles in the malformed craneofacial pits, resembling an atrophic OE, although a mature olfactory nerve was not identified. Axonal tracing, birthdating, immunohistochemistry, and in situ hybridization using antibodies and probes expressed in the olfactory system, indicated that two distinct structures observed in the OBLS correspond to the main and accessory olfactory bulbs of the control mouse. We propose that the OBLS has developed independently of the external influences exerted by the olfactory nerve. The presence of a prospective OB in the mutants, without intervening olfactory fibers, suggests that intrinsic factors could define brain territories even in absence of the proper afferent innervation. The intrinsic mechanisms and environmental cues in the telencephalon could be sufficient to promote axonogenesis in the projection neurons of the OB and guide their axons in a lateral prospective tract, in the absence of olfactory axons.     

Inducible and conditional activation of ERK5 MAP kinase rescues mice from cadmium-induced olfactory memory deficits

Cadmium (Cd) is a heavy metal that is one of the most toxic environmental pollutants throughout the world. We previously reported that Cd exposure impairs olfactory memory in mice. However, the underlying mechanisms for its neurotoxicity for olfactory function are not well understood. Since adult Subventricular zone (SVZ) and Olfactory Bulb (OB) neurogenesis contributes to olfaction, olfactory memory defects caused by Cd may be due to inhibition of neurogenesis. In this study, using bromodeoxyuridine (BrdU) labeling and immunohistochemistry, we found that 0.6 mg/L Cd exposure through drinking water impaired adult SVZ/OB neurogenesis in C57BL/6 mice. To determine if the inhibition of olfactory memory by Cd can be reversed by stimulating adult neurogenesis, we utilized the transgenic caMEK5 mouse strain to conditional stimulate of adult neurogenesis by activating the endogenous ERK5 MAP kinase signaling pathway. This was accomplished by conditionally induced expression of active MEK5 (caMEK5) in adult neural stem/progenitor cells. The caMEK5 mice were exposed to 0.6 mg/L Cd for 38 weeks, and tamoxifen was administered to induce caMEK5 expression and stimulate adult SVZ/OB neurogenesis during Cd exposure. Short-term olfactory memory test and sand-digging based, odor-cued olfactory learning and memory test were conducted after Cd and tamoxifen treatments to examine their effects on olfaction. Here we report that Cd exposure impaired short-term olfactory memory and odor-cued associative learning and memory in mice. Furthermore, the Cd-impaired olfactory memory deficits were rescued by the tamoxifen-induction of caMEK5 expression. This suggests that Cd exposure impairs olfactory function by affecting adult SVZ/OB neurogenesis in mice.     

Changes in the connections of the main olfactory bulb after mitral cell selective neurodegeneration

The connections of the main olfactory bulb (OB) of the mouse were studied with iontophoretic injections of biotinylated dextran amine. To sort efferences from mitral cells and tufted cells, the Purkinje cell degeneration (PCD) mouse was used. This mutant animal undergoes a specific neurodegeneration of mitral cells, whereas tufted cells do not degenerate. The unilateral tracer injections used were small and confined largely to the OB of both PCD and control mice at P120. Seven days after tracer injection, the efferences from the OB and the centrifugal afferences from secondary olfactory structures to it were studied. Although there is a large overlap of their target fields, mitral cell axons innervated more caudal regions of the olfactory cortex than tufted cell axons, thus providing definitive evidence of the differential projections of olfactory output neurons. Additionally, an important increase in retrogradely-labeled neurons was detected in the ipsilateral anterior olfactory nucleus of the mutant animals. This was not observed in any other secondary olfactory structure, suggesting a strengthening of the centrifugal input to the OB from that central area after mitral cell loss. Moreover, we recorded a complete loss of bilaterality in the olfactory connections of the PCD mice due to degeneration of the anterior commissure. These results point to an important reorganization of this essential olfactory circuit between the anterior olfactory nucleus and the OB, and hint at a transsynaptic level of plasticity not considered previously in literature.     

Cellular and molecular characterization of oxidative stress in olfactory epithelium of Harlequin mutant mouse

Oxidative stress in the olfactory system is a major factor associated with age-related olfactory impairment, although the mechanisms by which this occurs are not completely understood. The Harlequin mutant mouse (Hq/Y), which carries an X-linked recessive mutation in the Aifm1 gene, is a model of progressive oxidative stress-induced neurodegeneration in the cerebellum and retina. To determine whether the Hq/Y mutant mouse is a suitable model of oxidative stress-associated olfactory aging, we investigated cellular and molecular changes in the olfactory epithelium (OE) and olfactory bulb (OB) of 6-month-old male Hq/Y mice compared to those in sex-matched littermate controls (+/Y) and in age- and sex-matched C57BL/6 mice. Immunoreactivity for apoptosis-inducing factor, the protein product of Aifm1, was localized in mature olfactory sensory neurons (mOSNs) in +/Y mice but was rarely detected in Hq/Y mice. Hq/Y mice also exhibited increased lipofuscin autofluorescence and increased immunoreactivity for an oxidative DNA/RNA damage marker in mOSNs and in mitral/tufted cells in the OB and an increased number of cleaved caspase-3 immunoreactive apoptotic cells in the OE. Microarray analysis demonstrated that Aifm1 expression was down-regulated by 80% in the OE of Hq/Y mice compared to that in +/Y mice. Most significantly, regulated genes were classified into functional categories of cell signaling/apoptosis/cell cycle, oxidative stress/aging, and cytoskeleton/extracellular matrix/transport-associated. Analysis with EASE software indicated that the functional categories significantly overrepresented in Hq/Y mice included up-regulated mitochondrial genes and down-regulated cytoskeletal organization- and neurogenesis-related genes. Our results strongly support the Hq/Y mutant mouse being a novel model for mechanistic studies of oxidative stress-associated olfactory aging.     

Cadherin expression in the developing mouse olfactory system

Although odor receptors have been implicated in establishing the topography of olfactory sensory neurons (OSNs) in the olfactory bulb (OB), it is likely other molecules are also involved. The cadherins (CDHs) are a large family of cell adhesion molecules that mediate cell:cell interactions elsewhere in the central nervous system. However, their distribution and role in the olfactory system have remained largely unexplored. We previously demonstrated that intracellular binding partners of cadherins, the catenins, have unique spatiotemporal patterns of expression in the developing olfactory system. To further our understanding of cadherin function within the developing olfactory system, we now report on the localization of 11 classical cadherins-CDH1, 2, 3, 4, 5, 6, 8, 10, 11, 13, and 15. We demonstrate the expression of all but CDH5 and CDH15 in neuronal and/or glial cells in primary olfactory structures. CDH1 and CDH2 are expressed by OSNs; CDH2 expression closely parallels that seen for gamma-catenin in OSN axons. CDH3 and CDH11 are expressed by olfactory ensheathing glia, which surround OSN axons in the outer OB. CDH2, CDH4, and CDH6 are expressed within neuropil. CDH2, CDH4, CDH6, CDH8, CDH10, CDH11, and CDH13 are expressed by projection neurons within the main and accessory OBs. We conclude that cadherin proteins in the developing olfactory system are positioned to underlie the formation of the odorant map and local circuits within the OB.     

Changes in reelin expression in the mouse olfactory bulb after chemical lesion to the olfactory epithelium

To explore the functional roles of Reelin in the adult olfactory system, we examined changes in the expression of reelin mRNA and Reelin protein in the olfactory bulb (OB) of adult mice after a chemical lesion to the olfactory epithelium. Following intranasal irrigation with 2% zinc sulphate solution, animals were perfused at various times between 5 and 40 days post-lesion. The expression of reelin mRNA in mitral cells in the OB was slightly reduced at 5 days post-lesion, completely abolished by 20 days, but restored almost to the normal level at 40 days post-lesion. Similarly, the expression of Reelin protein in mitral cells of the deafferented OB also recovered, although not to the normal level. No recovery of either reelin mRNA or Reelin immunoreactivity was seen in the periglomerular cells and external tufted cells. The expression profile of reelin mRNA and Reelin protein in the OB coincided with the time course of degeneration and regeneration of olfactory nerves, as indicated by anterograde labeling of olfactory nerves with WGA-HRP. These results suggest that expression of reelin mRNA in the adult OB is regulated by olfactory inputs.     

Changes in adult olfactory bulb neurogenesis in mice expressing the A30P mutant form of alpha-synuclein

In familial and sporadic forms of Parkinson's disease (PD), alpha-synuclein pathology is present in the brain stem nuclei and olfactory bulb (OB) long before Lewy bodies are detected in the substantia nigra. The OB is an active region of adult neurogenesis, where newly generated neurons physiologically integrate. While accumulation of wild-type alpha-synuclein is one of the pathogenic hallmarks of non-genetic forms of PD, the A30P alpha-synuclein mutation results in an earlier disease onset and a severe clinical phenotype. Here, we study the regulation of adult neurogenesis in the subventricular zone (SVZ)/OB system in a tetracycline-suppressive (tet-off) transgenic model of synucleinopathies, expressing human mutant A30P alpha-synuclein under the control of the calcium/calmodulin-dependent protein kinase II alpha (CaMK) promoter. In A30P transgenic mice alpha-synuclein was abundant at the site of integration in the glomerular cell layer of the OB. Without changes in proliferation in the SVZ, significantly fewer newly generated neurons were observed in the OB granule cell and glomerular layers of A30P transgenic mice than in controls, most probably due to increased cell death. By tetracycline-dependent abrogation of A30P alpha-synuclein expression, OB neurogenesis and programmed cell death was restored to control levels. Our results indicate that, using A30P conditional (tet-off) mice, A30P alpha-synuclein has a negative impact on olfactory neurogenesis and suppression of A30P alpha-synuclein enhances survival of newly generated neurons. This finding suggests that interfering with alpha-synuclein pathology can rescue newly generated neurons, possibly leading to new targets for therapeutic interventions in synucleinopathies.     

Extracellular matrix protein anosmin‐1 modulates olfactory ensheathing cell maturation in chick olfactory bulb development

Olfactory ensheathing cells (OECs) are a specialized class of glia, wrapping around olfactory sensory axons that target the olfactory bulb (OB) and cross the peripheral nervous system/central nervous system boundary during development and continue to do so post‐natally. OEC subpopulations perform distinct subtype‐specific functions dependent on their maturity status. Disrupted OEC development is thought to be associated with abnormal OB morphogenesis, leading to anosmia, a defining characteristic of Kallmann syndrome. Hence, anosmin‐1 encoded by Kallmann syndrome gene (KAL‐1) might modulate OEC differentiation/maturation in the OB. We performed in ovo electroporation of shRNA in the olfactory placode to knock‐down kal in chick embryos, resulting in abnormal OB morphogenesis and loss of olfactory sensory axonal innervation into OB. BLBP‐expressing OECs appeared to form a thinner and poorly organized outmost OB layer where SOX10 expressing OECs were completely absent with emergence of GFAP‐expressing OECs. Furthermore, in embryonic day 10 chick OB explant cultures, GFAP expression in OECs accumulating along the OB nerve layers was dramatically reduced by recombinant anosmin‐1. We then purified immature OECs from embryonic day 10 chick OB. These cells express GFAP after 7 days in vitro, exhibiting a multipolar morphology. Overexpression of chick anosmin, exogenous anosmin‐1 or FGF2 could inhibit GFAP expression with cells presenting elongated morphology, which was blocked by the FGF receptor inhibitor Su5402. These data demonstrate that anosmin‐1 functions via FGF signalling in regulating OEC maturation, thereby providing a permissive glial environment for axonal innervation into the OB during development.     

5T4 glycoprotein regulates the sensory input-dependent development of a specific subtype of newborn interneurons in the mouse olfactory bulb

Sensory input has been shown to regulate development in a variety of species and in various structures, including the retina, cortex, and olfactory bulb (OB). Within the mammalian OB specifically, the development of dendrites in mitral/tufted cells is well known to be odor-evoked activity dependent. However, little is known about the developmental role of sensory input in the other major OB population of the GABAgenic interneurons, such as granule cells and periglomerular cells. Here, we identified, with DNA microarray and in situ hybridization screenings, a trophoblast glycoprotein gene, 5T4, whose expression in a specific subtype of OB interneurons is dependent on sensory input. 5T4 is a type I membrane protein, whose extracellular domain contains seven leucine-rich repeats (LRR) flanked by characteristic LRR-N-flanking and C-flanking regions, and a cytoplasmic domain. 5T4 overexpression in the newborn OB interneurons facilitated their dendritic arborization even under the sensory input-deprived condition. By contrast, both 5T4 knockdown with RNAi and 5T4 knockout with mice resulted in a significant reduction in the dendritic arborization of 5T4(+) granule cells. Further, we identified the amino acid sequence in the 5T4 cytoplasmic domain that is necessary and sufficient for the sensory input-dependent dendritic shaping of specific neuronal subtypes in the OB. Thus, these results demonstrate that 5T4 glycoprotein contributes in the regulation of activity-dependent dendritic development of interneurons and the formation of functional neural circuitry in the OB.     

Illuminating the terminal nerve: Uncovering the link between GnRH-1 neuron and olfactory development

During embryonic development, the olfactory placode (OP) generates migratory neurons, including olfactory pioneer neurons, cells of the terminal nerve (TN), gonadotropin-releasing hormone-1 (GnRH-1) neurons, and other uncharacterized neurons. Pioneer neurons from the OP induce olfactory bulb (OB) morphogenesis. In mice, GnRH-1 neurons appear in the olfactory system around mid-gestation and migrate via the TN axons to different brain regions. The GnRH-1 neurons are crucial in controlling the hypothalamic-pituitary-gonadal axis. Kallmann syndrome is characterized by impaired olfactory system development, defective OBs, secretion of GnRH-1, and infertility. The precise mechanistic link between the olfactory system and GnRH-1 development remains unclear. Studies in humans and mice highlight the importance of the prokineticin-2/prokineticin-receptor-2 (Prokr2) signaling pathway in OB morphogenesis and GnRH-1 neuronal migration. Prokr2 loss-of-function mutations can cause Kallmann syndrome (KS), and hence the Prokr2 signaling pathway represents a unique model to decipher the olfactory/GnRH-1 connection. We discovered that Prokr2 is expressed in the TN neurons during the critical period of GnRH-1 neuron formation, migration, and induction of OB morphogenesis. Single-cell RNA sequencing identified that the TN is formed by neurons distinct from the olfactory neurons. The TN neurons express multiple genes associated with KS. Our study suggests that the aberrant development of pioneer/TN neurons might cause the KS spectrum.     

Semaphorin 3A-mediated axon guidance regulates convergence and targeting of P2 odorant receptor axons

Semaphorins are known to play an important role in axon guidance of vertebrate olfactory sensory neurons to their targets in specific glomeruli of the olfactory bulb (OB). However, it is not clear how semaphorin-mediated guidance contributes to a systematic hierarchy of cues that govern the organization of this system. Because of the putative role that odorant receptor molecules such as P2 could play in establishing appropriate glomerular destinations for growing olfactory axons, we have also determined the spatial organization of P2 glomeruli in semaphorin 3A (Sema3A) mutant mice. First, in the postnatal OB of control and Sema3A(-/-) mice, we analysed the trajectories of olfactory axons that express the Sema3A receptor, neuropilin-1 (npn-1) and the positions of npn-1(+) glomeruli. Sema3A at the ventral OB midline guides npn-1(+) axons to targets in the lateral and medial OB. Absence of Sema3A permits many npn-1 axons to terminate aberrantly in the rostral and ventral OB. Second, in Sema3A(-/-) mice, many P2 axons are abnormally distributed throughout the ventral OB nerve layer and converge in atypical locations compared with littermate controls where P2 axons converge on stereotypically located lateral and medial glomeruli. In addition to their radically altered spatial distribution, P2 glomeruli in Sema3A(-/-) mice are significantly smaller and more numerous than in heterozygote littermates. These data show that Sema3A is an important repulsive olfactory guidance cue that establishes restricted npn-1(+) subcompartments in the olfactory bulb. Furthermore, Sema3A plays a key role in the convergence of axons expressing the odorant receptor P2 onto their appropriate targets.     

Caspase-3 and caspase-9 mediate developmental apoptosis in the mouse olfactory system

Neuronal apoptosis is a key component in the sculpting of tissues during embryonic and postnatal development and is driven largely by the action of caspases. In the mouse olfactory system, caspase-3 and -9 are expressed in olfactory receptor neurons (ORNs) of adult mice, and their selective retrograde activation drives ORN apoptosis following ablation of their target, the olfactory bulb (OB). Here, we show that both of these caspases are expressed at the earliest stages of ORN embryonic development, and their expression is concentrated in outgrowing ORN axons. The retention, in null mice for both caspases, of a population of ORNs that would normally undergo developmental apoptosis beginning at E13 of development, results in a permanently expanded population of ORNs. In turn, in some caspase-3 null mice, the ORN target organ, the OB, also develops abnormally, resulting in the formation of secondary, apparently functional, extracranial ectopic OBs.     

BMP mRNA and protein expression in the developing mouse olfactory system

The bone morphogenetic proteins (BMPs) play fundamental roles during the organization of the central nervous system. The presence of these proteins has also been demonstrated in regions of the adult brain that are characterized by neural plasticity. In this study, we examined the expression of BMP4, 6, and 7 mRNAs and proteins in the murine olfactory system. The olfactory system is a useful model for studying cell proliferation and neural differentiation because both of these processes persist throughout life in the olfactory epithelium (OE) and olfactory bulb (OB). Our results demonstrate a differential expression of BMP4, 6, and 7 in the embryonic, postnatal, and adult olfactory system. In particular, BMP4 and BMP7 showed similar immunostaining patterns, being expressed in the olfactory region from the earliest stages studied (embryonic day 15.5) to adulthood. During development BMPs were expressed in the OE, olfactory bulb nerve layer, glomerular layer (GL), mitral cell layer (MCL), and subventricular zone. During the first postnatal week of life, BMP4 and 7 immunoreactivity (-ir) was particularly evident in the GL, MCL, and in the subependymal layer (SEL), which originates postnatally from the subventricular zone. In adults, BMP4 and 7 immunostaining was present in the GL and SEL. Within the SEL, BMP4 and 7 proteins were expressed primarily in association with the astrocytic glial compartment. BMP6-ir was always found in mature olfactory receptor neurons and their axonal projections to the OB. In summary, these data support the hypothesis that BMPs play a role in the morphogenesis of the olfactory system during development and in its plasticity during adulthood.     

Distribution of EphA5 receptor protein in the developing and adult mouse nervous system

The EphA5 receptor tyrosine kinase plays key roles in axon guidance during development. However, the presence of EphA5 protein in the nervous system has not been fully characterized. To examine EphA5 localization better, mutant mice, in which the EphA5 cytoplasmic domain was replaced with β‐galactosidase, were analyzed for both temporal and regional changes in the distribution of EphA5 protein in the developing and adult nervous system. During embryonic development, high levels of EphA5 protein were found in the retina, olfactory bulb, cerebral neocortex, hippocampus, pretectum, tectum, cranial nerve nuclei, and spinal cord. Variations in intensity were observed as development proceeded. Staining of pretectal nuclei, tectal nuclei, and other areas of the mesencephalon became more diffuse after maturity, whereas the cerebral neocortex gained more robust intensity. In the adult, receptor protein continued to be detected in many areas including the olfactory nuclei, neocortex, piriform cortex, induseum griseum, hippocampus, thalamus, amygdala, hypothalamus, and septum. In addition, EphA5 protein was found in the claustrum, stria terminalis, barrel cortex, and striatal patches, and along discrete axon tracts within the corpus callosum of the adult. We conclude that EphA5 function is not limited to the developing mouse brain and may play a role in synaptic plasticity in the adult. J. Comp. Neurol. 514:310–328, 2009. © 2009 Wiley‐Liss, Inc.     

Comparative gene expression profiling of olfactory ensheathing cells from olfactory bulb and olfactory mucosa

Olfactory ensheathing cells (OEC) have the ability to promote regeneration in the nervous system. Hence, they hold promise for cell therapy. Most of the experimental studies have investigated the role of OECs taken from olfactory bulb (OB). However, for a clinical human application, olfactory mucosa (OM) seems to be the only acceptable source for OECs. Many studies have compared the distinct ability of OECs from OB and OM to improve functional nerve regeneration after lesion of the nervous system. Nevertheless, the two populations of OECs may differ in several points, which might affect all fate after transplantation in vivo. We report here the first study which compares gene expression profiling between these two populations of OECs. It appears that OB‐OECs and OM‐OECs display distinct gene expression pattern, which suggest that they may be implicated in different physiological processes. Notably, OM‐OECs overexpress genes characteristic of wound healing and regulation of extra cellular matrix. In contrast, OB‐OECs gene profile suggests a prominent role in nervous system development. Hence, OB‐OECs and OM‐OECs fundamentally differ in their gene expression pattern, which may represent a crucial point for future clinical application. © 2010 Wiley‐Liss, Inc.     

Efferent and afferent connections of the olfactory bulb and prepiriform cortex in the pigeon (Columba livia)

Although olfaction in birds is known to be involved in a variety of behaviors, there is comparatively little detailed information on the olfactory brain. In the pigeon brain, the olfactory bulb (OB) is known to project to the prepiriform cortex (CPP), piriform cortex (CPi), and dorsolateral corticoid area (CDL), which together are called the olfactory pallium, but centrifugal pathways to the OB have not been fully explored. Fiber connections of CPi and CDL have been reported, but those of other olfactory pallial nuclei remain unknown. The present study examines the fiber connections of OB and CPP in pigeons to provide a more detailed picture of their connections using tract‐tracing methods. When anterograde and retrograde tracers were injected in OB, projections to a more extensive olfactory pallium were revealed, including the anterior olfactory nucleus, CPP, densocellular part of the hyperpallium, tenia tecta, hippocampal continuation, CPi, and CDL. OB projected commissural fibers to the contralateral OB but did not receive afferents from the contralateral olfactory pallium. When tracers were injected in CPP, reciprocal ipsilateral connections with OB and nuclei of the olfactory pallium were observed, and CPP projected to the caudolateral nidopallium and the limbic system, including the hippocampal formation, septum, lateral hypothalamic nucleus, and lateral mammillary nucleus. These results show that the connections of OB have a wider distribution throughout the olfactory pallium than previously thought and that CPP provides a centrifugal projection to the OB and acts as a relay station to the limbic system. J. Comp. Neurol. 522:1728–1752, 2014. © 2013 Wiley Periodicals, Inc.     

Dibutyryl Cyclic AMP Inhibits the Progression of Experimental Autoimmune Encephalomyelitis and Potentiates Recruitment of Endogenous Neural Stem Cells

Multiple sclerosis is a chronic inflammatory demyelinating disease of the central nervous system. Cyclic AMP and its analogs enhance regeneration of adult mammalian central nervous system (CNS). Endogenous neural stem cells (NSCs) play a pivotal role in CNS regeneration, producing new neuron and glial cells. Here, we examined the effect of dibutyryl cyclic AMP (dbcAMP) on experimental autoimmune encephalomyelitis (EAE) symptoms, endogenous remyelination, and recruitment of NSCs. EAE was induced by immunizing mice using myelin oligodendrocyte glycoprotein peptide and pertussis toxin. Proliferative cells within CNS were labeled using repetitive systemic injections of 5-bromo-2-deoxyuridine (BrdU) before EAE induction. Myelin staining was performed using Luxol fast blue. The number of nestin⁺ and BrdU⁺ cells in subventricular zone (SVZ) and olfactory bulb (OB) was evaluated using immunohistochemistry. dbcAMP suppressed EAE progression and decreased the extent of demyelinated plaques in the lumbar spinal cord. EAE induction reduced the number of proliferative cells in SVZ and increased their population in OB. EAE also increased the number of nestin⁺ cells in OB. We also found that dbcAMP increased the recruitment of NSCs into the OB and brain parenchyma of EAE mice. Our results suggest dbcAMP as a potential therapy for inducing myelin repair in the context of demyelinating diseases like multiple sclerosis. Its positive effect seems to be mediated, at least partially, by endogenous neural stem cells and their increased recruitment.