α-Synuclein Pathology and Reduced Neurogenesis in the Olfactory System Affect Olfaction in a Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) is characterized by multiple symptoms including olfactory dysfunction, whose underlying mechanisms remain unclear. Here, we explored pathologic changes in the olfactory pathway of transgenic (Tg) mice of both sexes expressing the human A30P mutant α-synuclein (α-syn; α-syn-Tg mice) at 6-7 and 12-14 months of age, representing early and late-stages of motor progression, respectively. α-Syn-Tg mice at late stages exhibited olfactory behavioral deficits, which correlated with severe α-syn pathology in projection neurons (PNs) of the olfactory pathway. In parallel, olfactory bulb (OB) neurogenesis in α-syn-Tg mice was reduced in the OB granule cells at six to seven months and OB periglomerular cells at 12-14 months, respectively, both of which could contribute to olfactory dysfunction. Proteomic analyses showed a disruption in endocytic and exocytic pathways in the OB during the early stages which appeared exacerbated at the synaptic terminals when the mice developed olfactory deficits at 12-14 months. Our data suggest that (1) the α-syn-Tg mice recapitulate the olfactory functional deficits seen in PD; (2) olfactory structures exhibit spatiotemporal disparities for vulnerability to α-syn pathology; (3) α-syn pathology is restricted to projection neurons in the olfactory pathway; (4) neurogenesis in adult α-syn-Tg mice is reduced in the OB; and (5) synaptic endocytosis and exocytosis defects in the OB may further explain olfactory deficits.SIGNIFICANCE STATEMENT Olfactory dysfunction is a characteristic symptom of Parkinson's disease (PD). Using the human A30P mutant α-synuclein (α-syn)-expressing mouse model, we demonstrated the appearance of olfactory deficits at late stages of the disease, which was accompanied by the accumulation of α-syn pathology in projection neurons (PNs) of the olfactory system. This dysfunction included a reduction in olfactory bulb (OB) neurogenesis as well as changes in synaptic vesicular transport affecting synaptic function, both of which are likely contributing to olfactory behavioral deficits.

Microglia Maintain Homeostatic Conditions in the Developing Rostral Migratory Stream.

Microglia invade the neuroblast migratory corridor of the rostral migratory stream (RMS) early in development. The early postnatal RMS does not yet have the dense astrocyte and vascular scaffold that helps propel forward migrating neuroblasts, which led us to consider whether microglia help regulate conditions permissive to neuroblast migration in the RMS. GFP-labeled microglia in CX3CR-1GFP/+ mice assemble primarily along the outer borders of the RMS during the first postnatal week, where they exhibit predominantly an ameboid morphology and associate with migrating neuroblasts. Microglia ablation for 3 d postnatally does not impact the density of pulse labeled BrdU+ neuroblasts nor the distance migrated by tdTomato electroporated neuroblasts in the RMS. However, microglia wrap DsRed-labeled neuroblasts in the RMS of P7 CX3CR-1GFP/+;DCXDsRed/+ mice and express the markers CD68, CLEC7A, MERTK, and IGF-1, suggesting active regulation in the developing RMS. Microglia depletion for 14 d postnatally further induced an accumulation of CC3+ DCX+ apoptotic neuroblasts in the RMS, a wider RMS and extended patency of the lateral ventricle extension in the olfactory bulb. These findings illustrate the importance of microglia in maintaining a healthy neuroblast population and an environment permissive to neuroblast migration in the early postnatal RMS.

Sequential Maturation of Olfactory Sensory Neurons in the Mature Olfactory Epithelium.

The formation of the olfactory nerve and olfactory bulb (OB) glomeruli begins embryonically in mice. However, the development of the olfactory system continues throughout life with the addition of new olfactory sensory neurons (OSNs) in the olfactory epithelium (OE). Much attention has been given to the perinatal innervation of the OB by OSN axons, but in the young adult the process of OSN maturation and axon targeting to the OB remains controversial. To address this gap in understanding, we used BrdU to label late-born OSNs in young adult mice at postnatal day 25 (P25-born OSNs) and timed their molecular maturation following basal cell division. We show that OSNs in young adults undergo a sequential molecular development with the expression of GAP 43 (growth-associated protein 43) > AC3 (adenylyl cyclase 3) > OMP (olfactory marker protein), consecutively, in a time frame of ∼8 d. To assess OSN axon development, we implemented an in vivo fate-mapping strategy to label P25-born OSNs with ZsGreen. Using sampling intervals of 24 h, we demonstrate the progressive extension of OSN axons in the OE, through the foramen of the cribriform plate, and onto the surface of the OB. OSN axons reached the OB and began to target and robustly innervate specific glomeruli ∼10 d following basal cell division, a time point at which OMP expression becomes evident. Our data demonstrate a sequential process of correlated axon extension and molecular maturation that is similar to that seen in the neonate, but on a slightly longer timescale and with regional differences in the OE.

Embryonic and postnatal development of mouse olfactory tubercle.

The olfactory tubercle (OT) is located in the ventral-medial region of the brain where it receives primary input from olfactory bulb (OB) projection neurons and processes olfactory behaviors related to motivation, hedonics of smell and sexual encounters. The OT is part of the dopamine reward system that shares characteristics with the striatum. Together with the nucleus accumbens, the OT has been referred to as the "ventral striatum". However, despite its functional importance little is known about the embryonic development of the OT and the phenotypic properties of the OT cells. Here, using thymidine analogs, we establish that mouse OT neurogenesis occurs predominantly between E11-E15 in a lateral-to-medial gradient. Then, using a piggyBac multicolor technique we characterized the migratory route of OT neuroblasts from their embryonic point of origin. Following neurogenesis in the ventral lateral ganglionic eminence (vLGE), neuroblasts destined for the OT followed a dorsal-ventral pathway we named "ventral migratory course" (VMC). Upon reaching the nascent OT, neurons established a prototypical laminar distribution that was determined, in part, by the progenitor cell of origin. A phenotypic analysis of OT neuroblasts using a single-color piggyBac technique, showed that OT shared the molecular specification of striatal neurons. In addition to primary afferent input from the OB, the OT also receives a robust dopaminergic input from ventral tegmentum (Ikemoto, 2007). We used tyrosine hydroxylase (TH) expression as a proxy for dopaminergic innervation and showed that TH onset occurs at E13 and progressively increased until postnatal stages following an 'inside-out' pattern. Postnatally, we established the myelination in the OT occurring between P7 and P14, as shown with CNPase staining, and we characterized the cellular phenotypes populating the OT by immunohistochemistry. Collectively, this work provides the first detailed analysis of the developmental and maturation processes occurring in mouse OT, and demonstrates the striatal nature of the OT as part of the ventral striatum (vST).

Cranial Pair I: The Olfactory Nerve.

The olfactory nerve constitutes the first cranial pair. Compared with other cranial nerves, it depicts some atypical features. First, the olfactory nerve does not form a unique bundle. The olfactory axons join other axons and form several small bundles or fascicles: the fila olfactoria. These fascicles leave the nasal cavity, pass through the lamina cribrosa of the ethmoid bone and enter the brain. The whole of these fascicles is what is known as the olfactory nerve. Second, the olfactory sensory neurons, whose axons integrate the olfactory nerve, connect the nasal cavity and the brain without any relay. Third, the olfactory nerve is composed by unmyelinated axons. Fourth, the olfactory nerve contains neither Schwann cells nor oligodendrocytes wrapping its axons. But it contains olfactory ensheathing glia, which is a type of glia unique to this nerve. Fifth, the olfactory axons participate in the circuitry of certain spherical structures of neuropil that are unique in the brain: the olfactory glomeruli. Sixth, the axons of the olfactory nerve are continuously replaced and their connections in the central nervous system are remodeled continuously. Therefore, the olfactory nerve is subject to lifelong plasticity. Finally seventh, the olfactory nerve can be a gateway for the direct entrance of viruses, neurotoxins and other xenobiotics to the brain. In the same way, it can be used as a portal of entry to the brain for therapeutic substances, bypassing the blood-brain barrier. In this article, we analyze some features of the anatomy and physiology of the first cranial pair. Anat Rec, 302:405-427, 2019. © 2018 Wiley Periodicals, Inc.

Synaptic connectivity of the cholinergic axons in the olfactory bulb of the cynomolgus monkey.

The olfactory bulb (OB) of mammals receives cholinergic afferents from the horizontal limb of the diagonal band of Broca (HDB). At present, the synaptic connectivity of the cholinergic axons on the circuits of the OB has only been investigated in the rat. In this report, we analyze the synaptic connectivity of the cholinergic axons in the OB of the cynomolgus monkey (Macaca fascicularis). Our aim is to investigate whether the cholinergic innervation of the bulbar circuits is phylogenetically conserved between macrosmatic and microsmatic mammals. Our results demonstrate that the cholinergic axons form synaptic contacts on interneurons. In the glomerular layer, their main targets are the periglomerular cells, which receive axo-somatic and axo-dendritic synapses. In the inframitral region, their main targets are the granule cells, which receive synaptic contacts on their dendritic shafts and spines. Although the cholinergic boutons were frequently found in close vicinity of the dendrites of principal cells, we have not found synaptic contacts on them. From a comparative perspective, our data indicate that the synaptic connectivity of the cholinergic circuits is highly preserved in the OB of macrosmatic and microsmatic mammals.

Olfactory and cortical projections to bulbar and hippocampal adult-born neurons.

New neurons are continually generated in the subependymal layer of the lateral ventricles and the subgranular zone of dentate gyrus during adulthood. In the subventricular zone, neuroblasts migrate a long distance to the olfactory bulb where they differentiate into granule or periglomerular interneurons. In the hippocampus, neuroblasts migrate a short distance from the subgranular zone to the granule cell layer of the dentate gyrus to become granule neurons. In addition to the short-distance inputs, bulbar interneurons receive long-distance centrifugal afferents from olfactory-recipient structures. Similarly, dentate granule cells receive differential inputs from the medial and lateral entorhinal cortices through the perforant pathway. Little is known concerning these new inputs on the adult-born cells. In this work, we have characterized afferent inputs to 21-day old newly-born neurons. Mice were intraperitoneally injected with bromodeoxyuridine. Two weeks later, rhodamine-labeled dextran-amine was injected into the anterior olfactory nucleus, olfactory tubercle, piriform cortex and lateral and medial entorhinal cortices. One week later, animals were perfused and immunofluorescences were carried out. The data show that projection neurons from the mentioned structures, establish putative synaptic contacts onto 21-day-old neurons in the olfactory bulb and dentate gyrus, in some cases even before they start to express specific subpopulation proteins. Long-distance afferents reach middle and outer one-third portions of the molecular layer of the dentate gyrus and granule and, interestingly, periglomerular layers of the olfactory bulb. In the olfactory bulb, these fibers appear to establish presumptive axo-somatic contacts onto newly-born granule and periglomerular cells.

The dendritic spines of interneurons are dynamic structures influenced by PSA-NCAM expression.

Excitatory neurons undergo dendritic spine remodeling in response to different stimuli. However, there is scarce information about this type of plasticity in interneurons. The polysialylated form of the neural cell adhesion molecule (PSA-NCAM) is a good candidate to mediate this plasticity as it participates in neuronal remodeling and is expressed by some mature cortical interneurons, which have reduced dendritic arborization, spine density, and synaptic input. To study the connectivity of the dendritic spines of interneurons and the influence of PSA-NCAM on their dynamics, we have analyzed these structures in a subpopulation of fluorescent spiny interneurons in the hippocampus of glutamic acid decarboxylase-enhanced green fluorescent protein transgenic mice. Our results show that these spines receive excitatory synapses. The depletion of PSA in vivo using the enzyme Endo-Neuraminidase-N (Endo-N) increases spine density when analyzed 2 days after, but decreases it 7 days after. The dendritic spine turnover was also analyzed in real time using organotypic hippocampal cultures: 24 h after the addition of EndoN, we observed an increase in the apparition rate of spines. These results indicate that dendritic spines are important structures in the control of the synaptic input of hippocampal interneurons and suggest that PSA-NCAM is relevant in the regulation of their morphology and connectivity.

The circuits of the olfactory bulb. The exception as a rule.

The connectivity of the neurons of the olfactory bulb is highly idiosyncratic and constitutes an exception to the general plan of how neurons, and especially cortical neurons, construct circuits. The majority of synaptic contacts in the circuits of the cortex are axo-dendritic. In these contacts, the axon is the presynaptic element, which transmits the signal, and the dendrite is the postsynaptic element, which receives the signal. However, the majority of synaptic contacts in the circuits of the olfactory bulb are dendro-dendritic. In fact, most of the neurons of the olfactory bulb lack an axon. Moreover, a high percentage of the dendro-dendritic synapses are reciprocal. This means that the roles of presynaptic and postsynaptic element are not clearly defined, in clear contrast with the universality of unidirectional synaptic transmission in the cortex and elsewhere in the central nervous system. In this review, we analyze and discuss some peculiarities of the circuits of the olfactory bulb.

Two types of periglomerular cells in the olfactory bulb of the macaque monkey (Macaca fascicularis).

The olfactory bulb (OB) of mammals is the brain region that receives the sensory information coming from the olfactory epithelium. The entrance of the olfactory information occurs in spherical structures of neuropil named olfactory glomeruli and is modulated by a population of interneurons known as periglomerular cells (PG). It has been demonstrated that there are two types of PG in the OB of some macrosmatic mammals, including rats and mice. Type 1 PG (PG-1) receive synapses from the olfactory nerve, whereas type 2 PG (PG-2) do not receive synapses from the olfactory axons. To date, the presence of the two types of PG has not been investigated in microsmatic mammals. In this context, we analyze the presence of PG-1 and PG-2 in the OB of the long-tailed macaque (Macaca fascicularis). For that, we used the enzyme tyrosine hydroxylase, the neuronal isoform of the enzyme nitric oxide synthase and the calcium-binding proteins calbindin D-28k and calretinin as neurochemical markers. Our results demonstrate that the OB of the macaque contains PG-1 and PG-2. A subpopulation of PG-1 expresses tyrosine hydroxylase and another expresses the neuronal isoform of nitric oxide synthase. In addition, a subpopulation of PG-2 expresses calbindin D-28k and another expresses calretinin. Double immunofluorescence demonstrates that there is no colocalization of two markers in the same PG. These results mimic those found in macrosmatic animals. The presence of two types of PG in the glomerular circuits seems to be a key principle for the organization of the OB of mammals.