Beyond Perineural Drainage: Revisiting the Olfactory Nerve as an Active Immunological Interface During Neuroinflammation.

Bacterial infections of the central nervous system (CNS), though uncommon, are associated with very high rates of morbidity and mortality. Recent research has also highlighted the correlation between pathogens and chronic diseases of the CNS, such as neurodegenerative disorders, particularly Alzheimer’s disease. Whilst some bacteria can cross the blood-brain/blood-cerebrospinal fluid barriers, to date, other pathways by which bacteria enter the CNS remain largely unknown. Identifying alternative paths by which pathogens can enter the CNS is thus important for developing novel strategies preventing CNS infection and potential long-term sequelae. Novel evidence suggests some bacterial species (as well as certain viruses and amoebae) can enter the brain via the cranial nerves innervating the nasal cavity, particularly the olfactory nerve that mediates the sense of smell and connects the nasal cavity with the olfactory bulb in the forebrain. The trigeminal nerve also innervates the nasal cavity and constitutes another invasion path. Only a handful of pathogens are thought to use cranial nerves to reach the brain; certain Chlamydia species (spp.) being amongst these. Chlamydia pneumoniae is to date the bacterium with the strongest established link to Alzheimer’s disease. Previous research by our laboratory has also demonstrated that the bacterium causing the tropical disease melioidosis, Burkholderia pseudomallei, can invade both the olfactory and trigeminal nerves, travel along these nerves, to then infect the CNS (the olfactory bulb and brainstem, respectively). We have also previously shown that in outbred mice, the olfactory nerve is resistant to B. pseudomallei infection. The nasal mucosa contains both innate and adaptive immune components and prevents many infections. If pathogens penetrate the mucosal barrier and reach nerves, glial cells of the nerves can also combat the infection. Whilst only a few macrophages are present inside the olfactory nerve fascicles, olfactory nerve glial cells, termed olfactory ensheathing cells (OECs), are powerful phagocytes with innate immune functions. Thus, in addition to the immune cells and other components of the immune system in the nasal mucosa, cranial nerve glia are thus thought to be key for preventing CNS infection, explaining why such infections are relatively rare. Some pathogens, however, can evade destruction by these cells and invade the nerves, however, it remains largely unknown which pathogens are capable of doing so. Furthermore, injuries to the nasal epithelium are common, and if the mucosal barrier is removed by injury, perhaps it is easier for pathogens to infect the underlying nerves (in particular the olfactory nerve) and then reach the CNS. With the exception of one bacterium (Staphylococcus aureus) for which injury has been shown to allow infection of the olfactory nerve, it also remains unknown whether epithelial injury increases the risk of pathogens invading the CNS via these paths. Thus, we need to determine which pathogens are capable of invading the CNS via nerves connecting the nasal cavity and the brain, and whether epithelial injury increases the risk of infection. Furthermore, determining the cellular mechanisms that protect against microbial invasion of the CNS via nerves, as well as why certain pathogens can evade destruction of the immune system may pave the way for the development of novel therapies preventing and treating CNS infections. The key aims of this thesis were to determine (1) whether prior injury to the nasal epithelium could allow B. pseudomallei to invade the olfactory nerve and bulb in the mouse strain where this nerve is usually resistant to this infection, (2) whether the bacterium Chlamydia muridarum (which infects mice and is commonly used to study Chlamydia spp. infection in rodents) can utilise cranial nerves that innervate the nasal cavity to invade the CNS and, if C. muridarum can invade the CNS, to then determine whether the bacteria remained viable and (3) whether C. muridarum can infect OECs, and how OECs respond to C. muridarum in vitro. This thesis demonstrated that injury to the olfactory epithelium allowed the invasion of the olfactory nerve and bulb by B. pseudomallei in S100β-DsRed Quackenbush mice, in which the olfactory nerve is otherwise typically resistant to infection. This work also showed that C. muridarum can rapidly (within 48 h) reach the CNS (olfactory bulb and cerebral cortex) via the olfactory nerve, as well as infect the trigeminal nerve, in mice. Immunohistochemistry showed the presence of C. muridarum inclusion bodies (membrane-bound components inside which the bacteria replicate intracellularly) and viable C. muridarum bacteria were also isolated from these regions. C. muridarum was shown to readily infect OECs in vitro, which led to the upregulation of a range of cytokines. The outcomes from this project will contribute to an increased understanding of how bacteria can reach the CNS and has revealed that injury to the nasal epithelium may increase the risk of CNS bacterial invasion via the olfactory nerve. The outcomes also include an increased understanding of how olfactory nerve glia become infected by and respond to bacteria. This work may also contribute towards the growing body of knowledge regarding the link between pathogens and certain diseases of the CNS, such as Alzheimer’s disease. Furthermore, with an increased understanding of how glial cells respond to bacteria, new therapies may be developed that stimulate bacterial degradation by the glia. Such therapies may provide valid future alternatives to antibiotics, also combating the growing problem of antibiotic resistance. Thus, this work may contribute to the foundation required to develop therapies to treat diseases that are currently not curable, as well as to better diagnose and identify susceptibilities to certain conditions.

The purpose of the study was to establish whether autobiographical memories differ when a stimulus producing olfactory or/and trigeminal sensations was used as memory cue. The following hypothesis was formulated: memories evoked by odors activating the trigeminal and olfactory nerves would be subjectively assessed as more detailed, more clear, more important and less happy, as compared to memories evoked by odors activating exclusively the olfactory nerve. The hypothesis was based on the assumption that trigeminal odors are perceived as signaling potential threats for the organism. 30 Polish psychology students (M age = 22 yr.; 20 women, 10 men) were tested using six odors: three stimulating the olfactory nerve only and three stimulating both the olfactory and trigeminal nerves. Participants were asked whether a particular odor evoked any memories, and if they answered "yes," they were to respond to four questions related to the qualities of the memory. Ratings of memories evoked by odors that stimulated the olfactory nerve and those that stimulated both the olfactory and trigeminal nerves differed in clarity. Odors stimulating the trigeminal nerve may induce less happy memories. The results are promising as to the role of the trigeminal system in coding and retrieval of survival-related memories.

The infectious disease melioidosis is caused by the bacterium Burkholderia pseudomallei. Melioidosis is characterised by high mortality and morbidity and can involve the central nervous system (CNS). We have previously discovered that B. pseudomallei can infect the CNS via the olfactory and trigeminal nerves in mice. We have shown that the nerve path is dependent on mouse strain, with outbred mice showing resistance to olfactory nerve infection. Damage to the nasal epithelium by environmental factors is common, and we hypothesised that injury to the olfactory epithelium may increase the vulnerability of the olfactory nerve to microbial insult. We therefore investigated this, using outbred mice that were intranasally inoculated with B. pseudomallei, with or without methimazole-induced injury to the olfactory neuroepithelium. Methimazole-mediated injury resulted in increased B. pseudomallei invasion of the olfactory epithelium, and only in pre-injured animals were bacteria found in the olfactory nerve and bulb. In vitro assays demonstrated that B. pseudomallei readily infected glial cells isolated from the olfactory and trigeminal nerves (olfactory ensheathing cells and trigeminal Schwann cells, respectively). Bacteria were degraded by some cells but persisted in other cells, which led to the formation of multinucleated giant cells (MNGCs), with olfactory ensheathing cells less likely to form MNGCs than Schwann cells. Double Cap mutant bacteria, lacking the protein BimA, did not form MNGCs. These data suggest that injuries to the olfactory epithelium expose the primary olfactory nervous system to bacterial invasion, which can then result in CNS infection with potential pathogenic consequences for the glial cells.

WE have found the candidate neurotransmitter γ-aminobutyric acid (GABA) and its biosynthetic enzyme in the olfactory nerve of two fish. In the light of these findings current concepts of GABA as an exclusively inhibitory transmitter may have to be reconsidered. The olfactory nerve is a component of the rhinencephalon, a phylogenetically primitive part of the central nervous system (CNS). In addition to its receptive sensory function, the olfactory nerve conducts impulses. Fish have either short olfactory nerves and long olfactory tracts, as do most vertebrates, or long nerves and short tracts, as found in the gar fish (Lepisosteus osseus) and the pike (Esox estor)1. The olfactory cilia and nerve cell body are in the olfactory mucosa, from where the nerve action potentials are propagated to the olfactory bulb, 3 cm (pike) to 15 cm (gar) away, depending on the size of the animal. The primary synapse occurs within the olfactory bulb with the mitral cells. The olfactory nerve in the gar and the pike contains no motor nor other known sensory fibres2,3. Furthermore, it is neurally homogeneous, sensory, unmyelinated and is of a reasonable size for biochemical study (50–150 mg per nerve per fish).

Glycosaminoglycans in the olfactory epithelium and nerve of chick embryos: an immunocytochemical study

A new perspective on imaging of olfactory dysfunction: Does size matter?

The potential oscillations appearing in the olfactory epithelium, nerve and bulb were studied in the toad and the frog.1. Characteristic potential oscillations were found very frequently in the olfactory epithelium of the toad and the frog. They appeared superimposed on the on-or the off-slow potential at the onset or the cessation of olfactory stimulation.2. The olfactory nerve and bulb show potential oscillations in response to odour stimulation. They are mutually identical in frequency, phase and shape.3. The oscillations in the olfactory epithelium are entirely different in frequency, magnitude, shape and time of appearance from those in the olfactory nerve and bulb. It was concluded that there are two kinds of potential oscillations in the lower olfactory nervous system.4. The potential oscillations in the olfactory bulb of the toad and the frog were compared with those in mammals. The origin of the oscillation was considered.

Chlamydia pneumoniae can infect the brain and has been linked to late-onset dementia. Chlamydia muridarum, which infects mice, is often used to model human chlamydial infections. While it has been suggested to be also important for modelling brain infection, nervous system infection by C. muridarum has not been reported in the literature. C. pneumoniae has been shown to infect the olfactory bulb in mice after intranasal inoculation, and has therefore been suggested to invade the brain via the olfactory nerve; however, nerve infection has not been shown to date. Another path by which certain bacteria can reach the brain is via the trigeminal nerve, but it remains unknown whether Chlamydia species can infect this nerve. Other bacteria that can invade the brain via the olfactory and/or trigeminal nerve can do so rapidly, however, whether Chlamydia spp. can reach the brain earlier than one-week post inoculation remains unknown. In the current study, we showed that C. muridarum can within 48 h invade the brain via the olfactory nerve, in addition to infecting the trigeminal nerve. We also cultured the glial cells of the olfactory and trigeminal nerves and showed that C. muridarum readily infected the cells, constituting a possible cellular mechanism explaining how the bacteria can invade the nerves without being eliminated by glial immune functions. Further, we demonstrated that olfactory and trigeminal glia differed in their responses to C. muridarum, with olfactory glia showing less infection and stronger immune response than trigeminal glia.

The nasal cavity acts as a gateway through which pathogens can invade the human body. Streptococcus pneumoniae can infect the central nervous system directly through the nasal cavity. However, the regulatory mechanisms that govern the direct nose-to-brain invasion of S. pneumoniae remain unclear. The nasal cavity is innervated by both olfactory and trigeminal nerves, which will modulate the defense mechanism of the olfactory epithelium in different ways. Transient receptor potential vanilloid 1 (TRPV1), which is expressed in both nerves, maintains the regeneration ability of the olfactory epithelium. In this study, we investigated the role of TRPV1 against the nose-to-brain invasion of S. pneumoniae. Six-week-old mice were treated with methimazole and/or resiniferatoxin to ablate TRPV1 on the olfactory and trigeminal nerves, respectively. Bacterial density, cytokines/chemokine induction, and histologic changes were investigated after intranasal infection with S. pneumoniae. Olfactory epithelium ablation by methimazole increased intracranial invasion and mortality without bacteremia. Trigeminal TRPV1 ablation suppressed mortality. Intracranial invasion of S. pneumoniae induced IL-6 in the olfactory bulb. Olfactory epithelium ablation was associated with significantly higher TNF-α compared to trigeminal TRPV1 ablation. Intracranial invasion was suppressed by intranasal administration of a TRPV1 stimulant. TRPV1 on the olfactory nerve worked against intracranial invasion, whereas TRPV1 on the trigeminal nerve enhanced lethality via excessive cytokine/chemokine production. The effect of intranasal TRPV1 stimulation was dominant on the olfactory nerve and could suppress central nervous system infection via the nose-to-brain route. The current finding can bring new strategies against infection targeting TRPV1.IMPORTANCEThis study updates the current clinical concept of the pathogenesis of pneumococcal meningitis and bacteremia. The findings provide new insight into how the non-hematogenous nose-to-brain route is controlled via transient receptor potential vanilloid 1 (TRPV1) of the olfactory and trigeminal nerve. A mouse model demonstrates the non-hematogenous invasion of Streptococcus pneumoniae from the nasal cavity directly into the cranium. TRPV1 on the olfactory nerve protected against intracranial invasion, whereas TRPV1 on the trigeminal nerve could induce a lethal course due to excessive inflammatory responses. Intranasal TRPV1 stimulation inhibited intracranial invasion and could control lethal central nervous system infection by S. pneumoniae. These findings suggest a novel preventive strategy targeting TRPV1 against invasive pneumococcal infections.

Composition and characterization of the lipids of garfish ( Lepisosteus osseus) olfactory nerve, a tissue rich in axonal membrane

Primary sensory olfactory axons arise from the olfactory neuroepithelium that lines the nasal cavity and then project via the olfactory nerve into the olfactory bulb. The beta-galactoside binding lectin, galectin-1, and its laminin ligand have been implicated in the growth of these axons along this pathway. In galectin-1 null mutant mice, a subpopulation of primary sensory olfactory axons fails to reach its targets in the olfactory bulb. In the present study we examined the spatiotemporal expression pattern of galectin-1 in normal mice in order to understand its role in the development of the olfactory nerve pathway. At E15.5, when olfactory axons have already contacted the olfactory bulb, galectin-1 was expressed in the cartilage and mesenchyme surrounding the nasal cavity but was absent from the olfactory neuroepithelium, nerve and bulb. Between E16.5 and birth galectin-1 began to be expressed by olfactory nerve ensheathing cells in the lamina propria of the neuroepithelium and nerve fibre layer. Galectin-1 was neither expressed by primary sensory neurons in the olfactory neuroepithelium nor by their axons in the olfactory nerve. Laminin, a galectin-1 ligand, also exhibited a similar expression pattern in the embryonic olfactory nerve pathway. Our results reveal that galectin-1 is dynamically expressed by glial elements within the nerve fibre layer during a discrete period in the developing olfactory nerve pathway. Previous studies have reported galectin-1 acts as a substrate adhesion molecule by cross-linking primary sensory olfactory neurons to laminin. Thus, the coordinate expression of galectin-1 and laminin in the embryonic nerve fibre layer suggests that these molecules support the adhesion and fasciculation of axons en route to their glomerular targets.

Olfactory sensory neuron (OSN) axons coalesce to form the olfactory nerve (ON) and then grow from the olfactory epithelium to the olfactory bulb (OB), enter the olfactory nerve layer (ONL), reorganize extensively, and innervate specific glomeruli. Within the ON and ONL a population of glial cells, the olfactory ensheathing cells (OECs), surround OSN axon fascicles. To better understand the relationship between OECs and axon fascicles in the ONL of the adult mouse, we used confocal microscopy and antibodies to the low affinity nerve growth factor receptor p75 (p75), glial fibrillary acidic protein (GFAP), neuropeptide Y (NPY), and S-100 to identify glia. Antibodies to olfactory marker protein (OMP) and neuronal cell adhesion molecule (NCAM) were used to identify OSN axons. Electron microscopy characterized the ONL ultrastructure. We found that glial processes were not uniformly distributed in the ONL of the mouse. The p75(+) OEC processes were restricted to the ON and the outer ONL sublamina, and oriented parallel to the plane of the OB layers. In the inner ONL NPY(+) OEC-like processes were seen. GFAP(+) processes were restricted to the inner ONL sublamina, the ONL/GL boundary, and the GL, where they delineated loosely aggregated axon fascicles that entered the glomeruli obliquely. S-100(+) processes and somata were distributed throughout the ONL; the outer and inner ONL were equivalent in their S-100 staining. Ultrastructural studies showed that, although OECs could be identified in both the outer and inner ONL, in the latter, their relationship to bundles of OEC axons appeared less orderly than seen in the outer ONL. Our data demonstrate a differential organization of the ONL that could subserve distinct functions; axon extension may occur predominantly in the outermost ONL, whereas glomerular targeting occurs in the inner sublamina of the ONL.

Olfactory receptor neurons of the nasal epithelium send their axons, via the olfactory nerve (ON), to the glomeruli of the olfactory bulb (OB), where the axon terminals form glutamatergic synapses with the apical dendrites of mitral and tufted cells, the output cells of the OB, and with juxtaglomerular (JG) interneurons. Many JG cells are GABAergic. Here we show that, despite the absence of conventional synapses, GABA released from JG cells activates GABA(B) receptors on ON terminals and inhibits glutamate release both tonically and in response to ON stimulation. Field potential recordings and current-source density analysis, as well as intracellular and whole cell recording techniques were used in rat OB slices. Baclofen (2-5 microM), a GABA(B) agonist, completely suppressed ON-evoked synaptic responses of both mitral/tufted cells and JG cells, with no evidence for postsynaptic effects. Baclofen (0.5-1 microM) also reversed paired-pulse depression (PPD) of mitral/tufted cell responses to paired-pulse facilitation (PPF), and reduced depression of JG cell excitatory postsynaptic currents (EPSCs) during repetitive ON stimulation. These results suggest that baclofen reduced the probability of glutamate release from ON terminals. The GABA(B) antagonists CGP35348 or CGP55845A increased mitral/tufted cell responses evoked by single-pulse ON stimulation, suggesting that glutamate release from ON terminals is tonically suppressed via GABA(B) receptors. The same antagonists reduced PPD of ON-evoked mitral/tufted cell responses at interstimulus intervals 50-400 ms. This finding suggests that a single ON impulse evokes sufficient GABA release, presumably from JG cells, to activate GABA(B) receptors on ON terminals. Thus GABA(B) heteroreceptors on ON terminals are activated by ambient levels of extrasynaptic GABA, and by ON input to the OB. The time course of ON-evoked, GABA(B) presynaptic inhibition suggests that neurotransmission to M/T cells and JG cells will be significantly suppressed when ON impulses arrive in glomeruli at 2.5-20 Hz. GABA(B) receptor-mediated presynaptic inhibition of sensory input to the OB may play an important role in shaping the activation pattern of the OB glomeruli during olfactory coding.

Olfactory dysfunction may influence the quality of life tremendously. This study investigated the strength of the human olfactory nerve at the frontal skull base using cadavers. A total of 180 olfactory nerves were examined in 90 human cadaveric heads. The cut edges of the olfactory nerves were pulled until they were pulled out from the skull base. In the first set of 30 cases, each right olfactory nerve was pulled 0° laterally and 0° upward, and each left olfactory nerve was pulled 0° laterally and 15° upward. In the second set of 30 cases, each right olfactory nerve was pulled 0° laterally and 15° upward, and each left olfactory nerve was pulled 15° laterally and 15° upward. In the third set of 30 cases, each right olfactory nerve was pulled 15° laterally and 15° upward, and each left olfactory nerve was pulled 30° laterally and 15° upward. The strength of the olfactory nerve was measured when pulled in each direction. There was no significant difference in the strength of the olfactory nerves when pulling them in the postero-upward direction between 0° and 15° upward. The strengths of the olfactory nerves when pulling them in the postero-lateral direction 0° and 15° laterally were 3.14±1.87 and 4.05±1.70 g (mean ± standard deviation [SD]), respectively; the difference was almost significant. The olfactory nerve could be pulled more laterally than posteriorly because the retraction force is absorbed by the lateral wall of the olfactory fossa.

The Olfactory Nerve and Not the Trigeminal Nerve Is the Major Site of CNS Entry for Mouse Hepatitis Virus, Strain JHM