Structural Basis for Histaminergic Regulation of Neural Circuits in the Mouse Olfactory Bulb.

Odor information is modulated by centrifugal inputs from other brain regions to the olfactory bulb (OB). Neurons containing monoamines, such as serotonin, acetylcholine, and noradrenaline, are well known as centrifugal inputs; however, the role of histamine, which is also present in the OB, is not well understood. In this study, we examined the histaminergic neurons projecting from the hypothalamus to the OB. We used an antibody against histidine decarboxylase (HDC), a synthesizing enzyme of histamine, to identify histaminergic neurons and assess their localization within the OB and the ultrastructure of their fibers and synapses using multiple immunostaining laser microscopy, ultra-high voltage electron microscopy (EM), and EM to confirm their relationships with other neurons. To further identify the origin nucleus of the histaminergic neurons projecting to the OB, we injected the retrograde tracer FluoroGold and analyzed the pathway to the OB anterogradely. HDC-immunoreactive (-ir) fibers were abundant in the olfactory nerve (ON) layer compared to other monoamines. HDC-ir neurons received asymmetrical synapses from ONs and formed synapses containing pleomorphic vesicles with variable postsynaptic densities to non-ON elements, thus forming serial synapses. We also confirmed that histaminergic neurons project from the rostral ventral tuberomammillary nucleus to the granule cell layer of the OB and, for the first time, successfully visualized their axons from the hypothalamus to the OB. These findings indicate that histamine may regulate odor discrimination in the OB, suggesting a regulatory relationship between hypothalamic function and olfaction. We thus elucidate morphological mechanisms with tuberomammillary nucleus-derived histaminergic neurons involved in olfactory information.

In-depth histological, lectin-histochemical, immunohistochemical and ultrastructural description of the olfactory rosettes and olfactory bulbs of turbot (Scophthalmus maximus).

Chemical communication through olfaction is crucial for fish behaviours, mediating in socio-sexual behaviours as reproduction. Turbot, a flatfish with significant aquaculture production, possesses a well-developed olfactory system from early developmental stages. After metamorphosis, flatfish acquire their characteristic bilateral asymmetry with an ocular side facing the open water column, housing the dorsal olfactory rosette, and a blind side in contact with the sea bottom where the ventral rosette is located. This study aimed to address the existing gap in specific histological, ultrastructural, lectin-histochemical and immunohistochemical studies of the turbot olfactory rosettes and olfactory bulbs. We examined microdissected olfactory organs of adult turbots and premetamorphic larvae by using routine histological staining techniques, and a wide array of lectins and primary antibodies against G-proteins and calcium-binding proteins. We observed no discernible structural variations in the olfactory epithelium between rosettes, except for the dorsal rosette being larger in size compared to the ventral rosette. Additionally, the use of transmission electron microscopy significantly improved the characterization of the adult olfactory epithelium, exhibiting high cell density, small cell size, and a wide diversity of cell types. Moreover, specific immunopositivity in sensory and non-sensory cells provided us of essential information regarding their olfactory roles. The results obtained significantly enriched the scarce morphological and neurochemical information available on the turbot olfactory system, revealing a highly complex olfactory epithelium with distinct features compared to other teleost species, especially with regard to olfactory cell distribution and immunolabelling patterns.

Effects of intranasal instillation of nanoparticulate matter in the olfactory bulb.

Nanoparticulate matter activates the aryl hydrocarbon receptor (AhR) pathway in the respiratory system in a process involving the AhR nuclear translocator (ARNT) and cytochrome P450 family 1, member A1 (CYP1A1). We examined changes in AhR-related pathways following intranasal instillation of nanoparticulate matter in the olfactory bulb and cerebral cortex. Twice a day for 5 days per week for 1 week or 2 weeks, 8-week-old Sprague-Dawley rats were intranasally instilled with 10 µL nanoparticulate matter (nano group; n = 36). An equal volume of saline was intranasally instilled in control rats (n = 36). One week after intranasal instillation, olfactory function and Y-maze tests were performed. The expression levels of AhR in the olfactory bulb and temporal cortex were analyzed using western blotting and immunofluorescence assays. The expression levels of AhR, CYP1A1, inducible nitric oxide synthase (iNOS), and five genes encoding cation transporters (ARNT, ATP7B, ATPB1, OCT1, and OCT2) in the olfactory bulb were analyzed using quantitative reverse transcription. The olfactory discrimination capability was reduced in the nano group compared with the control group. Proportional changes in the Y-maze test were not significantly different between the nano and control groups. AhR mRNA and protein expression in the olfactory bulb increased 1.71-fold (P < 0.001) and 1.60-fold (P = 0.008), respectively. However, no significant changes were observed in the temporal cortex. In the olfactory bulb, the expression of ARNT, ATP7B, ATPB1, and OCT2 was downregulated. CYP1A1 and iNOS expression in the olfactory bulb was upregulated compared with that in the temporal cortex. The intranasal instillation of nanoparticulate matter decreased the olfactory discrimination ability, which was accompanied by upregulation of AhR expression and downregulation of cation transporters in the olfactory bulb.

Structural basis for noradrenergic regulation of neural circuits in the mouse olfactory bulb.

Olfactory input is processed in the glomerulus of the main olfactory bulb (OB) and relayed to higher centers in the brain by projection neurons. Conversely, centrifugal inputs from other brain regions project to the OB. We have previously analyzed centrifugal inputs into the OB from several brain regions using single-neuron labeling. In this study, we analyzed the centrifugal noradrenergic (NA) fibers derived from the locus coeruleus (LC), because their projection pathways and synaptic connections in the OB have not been clarified in detail. We analyzed the NA centrifugal projections by single-neuron labeling and immunoelectron microscopy. Individual NA neurons labeled by viral infection were three-dimensionally traced using Neurolucida software to visualize the projection pathway from the LC to the OB. Also, centrifugal NA fibers were visualized using an antibody for noradrenaline transporter (NET). NET immunoreactive (-ir) fibers contained many varicosities and synaptic vesicles. Furthermore, electron tomography demonstrated that NET-ir fibers formed asymmetrical synapses of varied morphology. Although these synapses were present at varicosities, the density of synapses was relatively low throughout the OB. The maximal density of synapses was found in the external plexiform layer; about 17% of all observed varicosities contained synapses. These results strongly suggest that NA-containing fibers in the OB release NA from both varicosities and synapses to influence the activities of OB neurons. The present study provides a morphological basis for olfactory modulation by centrifugal NA fibers derived from the LC.

Damage to Olfactory Organs of Adult Zebrafish Induced by Diesel Particulate Matter.

Particulate matter (PM) is an environmental hazard that is associated with various human health risks. The olfactory system is directly exposed to PM; therefore, the influence of PM exposure on olfactory function must be investigated. In this study, we propose a zebrafish olfactory model to evaluate the effects of exposure to diesel particulate matter (DPM), which was labeled Korean diesel particulate matter (KDP20). KDP20 comprises heavy metals and polycyclic aromatic hydrocarbons (PAHs). KDP20 exposed olfactory organs exhibited reduced cilia and damaged epithelium. Olfactory dysfunction was confirmed using an odor-mediated behavior test. Furthermore, the olfactory damage was analyzed using Alcian blue and anti-calretinin staining. KDP20 exposed olfactory organs exhibited histological damages, such as increased goblet cells, decreased cell density, and calretinin level. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed that PAHs exposure related genes (AHR2 and CYP1A) were upregulated. Reactive oxidation stress (ROS) (CAT) and inflammation (IL-1B) related genes were upregulated. Furthermore, olfactory sensory neuron (OSN) related genes (OMP and S100) were downregulated. In conclusion, KDP20 exposure induced dysfunction of the olfactory system. Additionally, the zebrafish olfactory system exhibited a regenerative capacity with recovery conditions. Thus, this model may be used in future investigating PM-related diseases.

Convergence of Olfactory Inputs within the Central Nervous System of a Cartilaginous and a Bony Fish: An Anatomical Indicator of Olfactory Sensitivity.

The volume of the olfactory bulbs (OBs) relative to the brain has been used previously as a proxy for olfactory capabilities in many vertebrate taxa, including fishes. Although this gross approach has predictive power, a more accurate assessment of the number of afferent olfactory inputs and the convergence of this information at the level of the telencephalon is critical to our understanding of the role of olfaction in the behaviour of fishes. In this study, we used transmission electron microscopy to assess the number of first-order axons within the olfactory nerve (ON) and the number of second-order axons in the olfactory peduncle (OP) in established model species within cartilaginous (brownbanded bamboo shark, Chiloscyllium punctatum [CP]) and bony (common goldfish, Carassius auratus [CA]) fishes. The total number of axons varied from a mean of 18.12 ± 7.50 million in the ON to a mean of 0.38 ± 0.21 million in the OP of CP, versus 0.48 ± 0.16 million in the ON and 0.09 ± 0.02 million in the OP of CA. This resulted in a convergence ratio of approximately 50:1 and 5:1, respectively, for these two species. Based on astroglial ensheathing, axon type (unmyelinated [UM] and myelinated [M]) and axon size, we found no differentiated tracts in the OP of CP, whereas a lateral and a medial tract (both of which could be subdivided into two bundles or areas) were identified for CA, as previously described. Linear regression analyses revealed significant differences not only in axon density between species and locations (nerves and peduncles), but also in axon type and axon diameter (p < 0.05). However, UM axon diameter was larger in the OPs than in the nerve in both species (p = 0.005), with no significant differences in UM axon diameter in the ON (p = 0.06) between species. This study provides an in-depth analysis of the neuroanatomical organisation of the ascending olfactory pathway in two fish taxa and a quantitative anatomical comparison of the summation of olfactory information. Our results support the assertion that relative OB volume is a good indicator of the level of olfactory input and thereby a proxy for olfactory capabilities.

Variations in GABA immunoreactivity among granule cells of the mouse olfactory bulb, as revealed by high-voltage electron microscopy.

Odor information is processed in the olfactory bulb (OB), which is organized into olfactory inputs, interneurons, projection neurons, and centrifugal inputs, and these various structures regulate olfactory information processing. Similar to other brain regions, the OB structures include many types of interneurons, including γ-aminobutyric acid (GABA)ergic interneurons. Many interneurons are granule cells that are found in the granule cell layer (GCL), which is a deep layer of the OB. Interestingly, these interneurons exhibit variations in GABA immunoreactivity, and previous studies have observed differing intensities among morphologically and chemically similar neuronal populations. However, the numbers and distribution patterns of cells that show variations in GABA immunoreactivity are unknown. Therefore, we observed and quantitatively analyzed this diversity in the GCL of the mouse OB using immunogold, high-voltage electron microscopy, combined with light microscopy. Consequently, our results clearly show variations in the GABA immunoreactivity among GCL interneurons, which suggested heterogeneity in the amount of GABA present in each interneuron and reflected the possibility that different amounts of neuroactive substances may be associated with different functions for the various GABAergic interneuron groups. Variations in GABA immunoreactivity could be a novel criterion for classifying interneuron subpopulations.

Telencephalon Cytoarchitecture of tsinling dwarf skinks (Scincella tsinlingensis).

The telencephalon of adult Scincella tsinlingensis was detected by light and electron microscopy, which will be used as the basis for further neurobiological comparative studies. The telencephalon of S. tsinlingensis was consisted of paired olfactory bulbs, paired cerebral hemispheres, and a telencephalon medium or impar. Main-olfactory bulb can be classified into six layers such as olfactory nerve fibers layer, glomerular layer, external plexiform layer, mitral layer, internal plexiform layer, granular layer and ependyma layer. The dorsal part of telencephalon contained the cortex and dorsal ventricular ridge. The cerebral cortex of S. tsinlingens was relatively thin, while the dorsal cortex was the thinnest, but gradually thickened as it extended to the medial and lateral cortex. The neural cells, glial cells and ependymal cells widely distributed in the cerebral cortex represented similar ultrastructural characteristics to those described in other vertebrates. Golgi staining revealed multipolar cell, bitufted cell and monotufted cell in three cortical layers of medial cortex. The results indicated that the cytoarchitectonic characteristics of telencephalon in S. tsinlingensis resembled those found in other lizards.

Dynamic Changes in Ultrastructure of the Primary Cilium in Migrating Neuroblasts in the Postnatal Brain.

New neurons, referred to as neuroblasts, are continuously generated in the ventricular-subventricular zone of the brain throughout an animal's life. These neuroblasts are characterized by their unique potential for proliferation, formation of chain-like cell aggregates, and long-distance and high-speed migration through the rostral migratory stream (RMS) toward the olfactory bulb (OB), where they decelerate and differentiate into mature interneurons. The dynamic changes of ultrastructural features in postnatal-born neuroblasts during migration are not yet fully understood. Here we report the presence of a primary cilium, and its ultrastructural morphology and spatiotemporal dynamics, in migrating neuroblasts in the postnatal RMS and OB. The primary cilium was observed in migrating neuroblasts in the postnatal RMS and OB in male and female mice and zebrafish, and a male rhesus monkey. Inhibition of intraflagellar transport molecules in migrating neuroblasts impaired their ciliogenesis and rostral migration toward the OB. Serial section transmission electron microscopy revealed that each migrating neuroblast possesses either a pair of centrioles or a basal body with an immature or mature primary cilium. Using immunohistochemistry, live imaging, and serial block-face scanning electron microscopy, we demonstrate that the localization and orientation of the primary cilium are altered depending on the mitotic state, saltatory migration, and deceleration of neuroblasts. Together, our results highlight a close mutual relationship between spatiotemporal regulation of the primary cilium and efficient chain migration of neuroblasts in the postnatal brain.SIGNIFICANCE STATEMENT Immature neurons (neuroblasts) generated in the postnatal brain have a mitotic potential and migrate in chain-like cell aggregates toward the olfactory bulb. Here we report that migrating neuroblasts possess a tiny cellular protrusion called a primary cilium. Immunohistochemical studies with zebrafish, mouse, and monkey brains suggest that the presence of the primary cilium in migrating neuroblasts is evolutionarily conserved. Ciliogenesis in migrating neuroblasts in the rostral migratory stream is suppressed during mitosis and promoted after cell cycle exit. Moreover, live imaging and 3D electron microscopy revealed that ciliary localization and orientation change during saltatory movement of neuroblasts. Our results reveal highly organized dynamics in maturation and positioning of the primary cilium during neuroblast migration that underlie saltatory movement of postnatal-born neuroblasts.

Balancing Extrasynaptic Excitation and Synaptic Inhibition within Olfactory Bulb Glomeruli.

Glutamatergic transmission in the brain typically occurs at well-defined synaptic connections, but increasing evidence indicates that neural excitation can also occur through activation of "extrasynaptic" glutamate receptors. Here, we investigated the underlying mechanisms and functional properties of extrasynaptic signals that are part of a feedforward path of information flow in the olfactory bulb. This pathway involves glutamatergic interneurons, external tufted cells (eTCs), that are excited by olfactory sensory neurons (OSNs) and in turn excite output mitral cells (MCs) extrasynaptically. Using pair-cell and triple-cell recordings in rat bulb slices (of either sex), combined with ultrastructural approaches, we first present evidence that eTC-to-MC signaling results from "spillover" of glutamate released at eTC synapses onto GABAergic periglomerular (PG) cells in glomeruli. Thus, feedforward excitation is an indirect result of and must cooccur with activation of inhibitory circuitry. Next, to examine the dynamics of the competing signals, we assayed the relationship between the number of spikes in eTCs and excitation of MCs or PG cells in pair-cell recordings. This showed that extrasynaptic excitation in MCs is very weak due to single spikes but rises sharply and supralinearly with increasing spikes, differing from sublinear behavior for synaptic excitation of PG cells. Similar dynamics leading to a preference for extrasynaptic excitation were also observed during recordings of extrasynaptic and inhibitory currents in response to OSN input of increasing magnitude. The observed alterations in the balance between extrasynaptic excitation and inhibition in glomeruli with stimulus strength could underlie an intraglomerular mechanism for olfactory contrast enhancement.

Examination of morphological and synaptic features of calbindin-immunoreactive neurons in deep layers of the rat olfactory bulb with correlative laser and volume electron microscopy.

The olfactory bulb (OB) contains various interneuron types that play key roles in processing olfactory information via synaptic contacts. Many previous studies have reported synaptic connections of heterogeneous interneurons in superficial OB layers. In contrast, few studies have examined synaptic connections in deep layers because of the lack of a selective marker for intrinsic neurons located in the deeper layers, including the mitral cell layer, internal plexiform layer (IPL) and granule cell layer. However, neural circuits in the deep layers are likely to have a strong effect on the output of the OB because of the cellular composition of these regions. Here, we analyzed the calbindin-immunoreactive neurons in the IPL, one of the clearly neurochemically defined interneuron types in the deep layers, using multiple immunolabeling and confocal laser scanning microscopy combined with electron microscopic three-dimensional serial-section reconstruction, enabling correlated laser and volume electron microscopy (EM). Despite a resemblance to the morphological features of deep short axon cells, IPL calbindin-immunoreactive (IPL-CB-ir) neurons lacked axons. Furthermore, multiple immunolabeling for plural neurochemicals indicated that IPL-CB-ir neurons differed from any interneuron types reported previously. We identified symmetrical synapses formed by IPL-CB-ir neurons on granule cells (GCs) using correlated laser and volume EM. These synapses might inhibit GCs and thus disinhibit mitral and tufted cells. Our present findings indicate, for the first time, that IPL-CB-ir neurons are involved in regulating the activities of projection neurons, further suggesting their involvement in synaptic circuitry for output from the deeper layers of the OB, which has not previously been clarified.

Repeated gestational exposure to diesel engine exhaust affects the fetal olfactory system and alters olfactory-based behavior in rabbit offspring.

BACKGROUND: Airborne pollution, especially from diesel exhaust (DE), is known to have a negative effect on the central nervous system in exposed human populations. However, the consequences of gestational exposure to DE on the fetal brain remain poorly explored, with various effects depending on the conditions of exposure, as well as little information on early developmental stages. We investigated the short-term effects of indirect DE exposure throughout gestation on the developing brain using a rabbit model. We analyzed fetal olfactory tissues at the end of gestation and tested behaviors relevant to pups' survival at birth. Pregnant dams were exposed by nose-only inhalation to either clean air or DE with a content of particles (DEP) adjusted to 1 mg/m3 by diluting engine exhaust, for 2 h/day, 5 days/week, from gestational day 3 (GD3) to day 27 (GD27). At GD28, fetal olfactory mucosa, olfactory bulbs and whole brains were collected for anatomical and neurochemical measurements. At postnatal day 2 (PND2), pups born from another group of exposed or control female were examined for their odor-guided behavior in response to the presentation of the rabbit mammary pheromone 2-methyl-3-butyn-2-ol (2MB2). RESULTS: At GD28, nano-sized particles were observed in cilia and cytoplasm of the olfactory sensory neurons in the olfactory mucosa and in the cytoplasm of periglomerular cells in the olfactory bulbs of exposed fetuses. Moreover, cellular and axonal hypertrophies were observed throughout olfactory tissues. Concomitantly, fetal serotoninergic and dopaminergic systems were affected in the olfactory bulbs. Moreover, the neuromodulatory homeostasis was disturbed in a sex-dependent manner in olfactory tissues. At birth, the olfactory sensitivity to 2MB2 was reduced in exposed PND2 pups. CONCLUSION: Gestational exposure to DE alters olfactory tissues and affects monoaminergic neurotransmission in fetuses' olfactory bulbs, resulting in an alteration of olfactory-based behaviors at birth. Considering the anatomical and functional continuum between the olfactory system and other brain structures, and due to the importance of monoamine neurotransmission in the plasticity of neural circuits, such alterations could participate to disturbances in higher integrative structures, with possible long-term neurobehavioral consequences.

Deafferentation-induced alterations in mitral cell dendritic morphology in the adult zebrafish olfactory bulb.

The removal of afferent input to the olfactory bulb by both cautery and chemical olfactory organ ablation in adult zebrafish results in a significant decrease in volume of the ipsilateral olfactory bulb. To examine the effects of deafferentation at a cellular level, primary output neurons of the olfactory bulb, the mitral cells, were investigated using retrograde tract tracing with fluorescent dextran using ex vivo brain cultures. Morphological characteristics including the number of major dendritic branches, total length of dendritic branches, area of the dendritic arbor, overall dendritic complexity, and optical density of the arbor were used to determine the effects of deafferentation on mitral cell dendrites. Following 8 weeks of permanent deafferentation there were significant reductions in the total length of dendritic branches, the area of the dendritic arbor, and the density of fine processes in the dendritic tuft. With 8 weeks of chronic, partial deafferentation there were significant reductions in all parameters examined, including a modified Sholl analysis that showed significant decreases in overall dendritic complexity. These results show the plasticity of mitral cell dendritic structures in the adult brain and provide information about the response of these output neurons following the loss of sensory input in this key model system.

Sparsened neuronal activity in an optogenetically activated olfactory glomerulus.

Glomeruli are the functional units of olfactory information processing but little remains known about their individual unit function. This is due to their widespread activation by odor stimuli. We expressed channelrhodopsin-2 in a single olfactory sensory neuron type, and used laser stimulation and simultaneous in vivo calcium imaging to study the responses of a single glomerulus to optogenetic stimulation. Calcium signals in the neuropil of this glomerulus were representative of the sensory input and nearly identical if evoked by intensity-matched odor and laser stimuli. However, significantly fewer glomerular layer interneurons and olfactory bulb output neurons (mitral cells) responded to optogenetic versus odor stimuli, resulting in a small and spatially compact optogenetic glomerular unit response. Temporal features of laser stimuli were represented with high fidelity in the neuropil of the glomerulus and the mitral cells, but not in interneurons. Increases in laser stimulus intensity were encoded by larger signal amplitudes in all compartments of the glomerulus, and by the recruitment of additional interneurons and mitral cells. No spatial expansion of the glomerular unit response was observed in response to stronger input stimuli. Our data are among the first descriptions of input-output transformations in a selectively activated olfactory glomerulus.

Barcoded solid-phase RNA capture for Spatial Transcriptomics profiling in mammalian tissue sections.

Spatial resolution of gene expression enables gene expression events to be pinpointed to a specific location in biological tissue. Spatially resolved gene expression in tissue sections is traditionally analyzed using immunohistochemistry (IHC) or in situ hybridization (ISH). These technologies are invaluable tools for pathologists and molecular biologists; however, their throughput is limited to the analysis of only a few genes at a time. Recent advances in RNA sequencing (RNA-seq) have made it possible to obtain unbiased high-throughput gene expression data in bulk. Spatial Transcriptomics combines the benefits of traditional spatially resolved technologies with the massive throughput of RNA-seq. Here, we present a protocol describing how to apply the Spatial Transcriptomics technology to mammalian tissue. This protocol combines histological staining and spatially resolved RNA-seq data from intact tissue sections. Once suitable tissue-specific conditions have been established, library construction and sequencing can be completed in ~5-6 d. Data processing takes a few hours, with the exact timing dependent on the sequencing depth. Our method requires no special instruments and can be performed in any laboratory with access to a cryostat, microscope and next-generation sequencing.

Immunohistochemical analysis of the development of olfactory organs in two species of turtles Pelodiscus sinensis and Mauremys reevesii.

The nasal cavity of turtles is composed of the upper and lower chambers, lined by the upper and lower chamber epithelia, respectively. In many turtles including the Reeve's turtle Mauremys reevesii, the upper chamber epithelium contains ciliated olfactory receptor neurons (ORNs) and the lower chamber epithelium contains microvillous ORNs. However, in the olfactory organ of the Chinese soft-shelled turtle Pelodiscus sinensis, both the upper and lower chamber epithelia contain ciliated ORNs. In the present study, we immunohistochemically examined the developmental process of olfactory organs in soft-shelled turtle and the Reeve's turtle to clarify the developmental origins of the lower chamber epithelium in these turtles. Obtained data indicate that olfactory organs of these turtles have identical origin and follow similar process of development, suggesting that, in the lower chamber epithelium of the nasal cavity, ciliated ORNs differentiate in soft-shelled turtle whereas microvillous ORNs differentiate in the Reeve's turtle.

Microvascular anatomy of the brain of the adult pipid frog, Xenopus laevis (Daudin): A scanning electron microscopic study of vascular corrosion casts.

To demonstrate the 3D microvascular anatomy of the brain of the model organism Xenopus laevis Daudin scanning electron microscopy of vascular corrosion casts was correlated with light microscopy of stained 7 µm thick serial tissues sections. Results showed that supplying arteries descended from the leptomeningeal surface without remarkable branchings straight to the subventricular zone where they branched and capillarized. Capillaries showed few H- and/or Y-shaped anastomoses during their centrifugal course toward the leptomeningeal surface where they drained into cerebral venules and veins. Apart from the accessory olfactory bulb and the vestibule-cochlear nucleus where capillaries were densely packed, capillaries formed a wide-meshed 3D network throughout the brain parenchyma and thus contrasted to urodelian brains where hairpin-shaped capillaries descend from the leptomeningeal vessels into varying depths of the brain parenchyma. In about two-third of specimens, a closed arterial circle of Willis was found at the base of the brain. If this circle in Xenopus might serve the same two functions as in men is briefly discussed. Choroid plexuses of third and fourth ventricle were found to have a high venous, but a low arterial inflow via one small choroidal artery only. Findings are compared with previous studies on the vascularization of the anuran brain and discrepancies in respect to presence or absence of particular arteries and/or veins in Ranids, Bufonids, and Pipids studied so far are discussed with particular emphasis on the techniques used in the various studies published so far.

Age-dependent decrease in glomeruli and receptor cells containing α1-2 fucose glycan in the mouse main olfactory system but not in the vomeronasal system.

Receptor cells of the olfactory epithelium (OE) and vomeronasal organ (VNO) project axons to glomeruli in the main olfactory bulb (MOB) and accessory olfactory bulb (AOB), respectively and undergo continuous turnover throughout life. Alpha1-2 fucose (α1-2Fuc) glycan mediates neurite outgrowth and synaptic plasticity and plays important roles in the formation of the olfactory system during development. We previously confirmed the localization of α1-2Fuc glycan in the olfactory system of 3- to 4-month-old mice but whether such localization persists throughout life remains unknown. Here, the MOB, AOB, OE and VNO of 1-, 3- and 8-month-old mice were histochemically examined using Ulex europaeus agglutinin-I (UEA-I) that specifically binds to α1-2Fuc glycan. Binding sites for UEA-I in the MOB were similar among all age groups but the ratio of UEA-I-positive glomeruli significantly decreased with aging. The frequency of UEA-I-positive receptor cells in the OE of the two older groups was also significantly lower than that of 1-month-old mice. On the other hand, UEA-I binding in the AOB and VNO did not significantly differ among all three groups. These findings suggest that the primary pathway of the main olfactory system requires the role of α1-2Fuc glycan in young mice rather than old mice, while the vomeronasal pathway equally requires this glycan in both young and old mice.

Architecture of a mammalian glomerular domain revealed by novel volume electroporation using nanoengineered microelectrodes.

Dense microcircuit reconstruction techniques have begun to provide ultrafine insight into the architecture of small-scale networks. However, identifying the totality of cells belonging to such neuronal modules, the "inputs" and "outputs," remains a major challenge. Here, we present the development of nanoengineered electroporation microelectrodes (NEMs) for comprehensive manipulation of a substantial volume of neuronal tissue. Combining finite element modeling and focused ion beam milling, NEMs permit substantially higher stimulation intensities compared to conventional glass capillaries, allowing for larger volumes configurable to the geometry of the target circuit. We apply NEMs to achieve near-complete labeling of the neuronal network associated with a genetically identified olfactory glomerulus. This allows us to detect sparse higher-order features of the wiring architecture that are inaccessible to statistical labeling approaches. Thus, NEM labeling provides crucial complementary information to dense circuit reconstruction techniques. Relying solely on targeting an electrode to the region of interest and passive biophysical properties largely common across cell types, this can easily be employed anywhere in the CNS.