Development of the human olfactory system

Handbook of Clinical Neurology, Vol. 164 (3rd series) Smell and Taste R.L. Doty, Editor https://doi.org/10.1016/B978-0-444-63855-7.00003-4 Copyright © 2019 Elsevier B.V. All rights reserved

Chapter 3

Development of the human olfactory system HARVEY B. SARNAT1,2,3* AND LAURA FLORES-SARNAT1,2 Department of Paediatrics, University of Calgary Cumming School of Medicine and Alberta Children’s Hospital Research Institute, Calgary, AB, Canada

1

2

Department of Clinical Neurosciences, University of Calgary Cumming School of Medicine and Alberta Children’s Hospital Research Institute, Calgary, AB, Canada

3

Department of Pathology and Laboratory Medicine (Neuropathology), University of Calgary Cumming School of Medicine and Alberta Children’s Hospital Research Institute, Calgary, AB, Canada

Abstract This chapter focuses on the development of the human olfactory system. In this system, function does not require full neuroanatomical maturity. Thus, discrimination of odorous molecules, including a number within the mother’s diet, occurs in amniotic fluid after 28–30 weeks of gestation, at which time the olfactory bulbs are identifiable by MRI. Hypoplasia/aplasia of the bulbs is documented in the third trimester and postnatally. Interestingly, olfactory axons project from the nasal epithelium to the telencephalon before formation of the olfactory bulbs and lack a peripheral ganglion, but the synaptic glomeruli of the future olfactory bulb serves this function. Histologic lamination of the olfactory bulb is present by 14 weeks, but maturation remains incomplete at term for neuronal differentiation, synaptogenesis, myelination, and persistence of the normal transitory fetal ventricular recess. Myelination occurs postnatally. Although olfaction is the only sensory system without direct thalamic projections, the olfactory bulb and anterior olfactory nucleus are, in effect, thalamic surrogates. For example, many dendro-dendritic synapses occur within the bulb between GABAergic granular neurons and periglomerular neurons. Moreover, bulbar synaptic glomeruli are analogous to peripheral ganglia of other sensory cranial nerves. The olfactory tract contains much gray as well as white matter. The olfactory epithelium and bulb both incorporate progenitor cells at all ages. Diverse malformations of the olfactory bulb can be detected by clinical examination, imaging, and neuropathology; indeed, olfactory reflexes of the neonate can be reliably tested. We recommend that such testing be routine in the neonatal neurologic examination, especially in children with brain malformations, endocrinopathies, chromosomopathies, genetic/metabolic disorders, and perinatal hypoxic/ ischemic encephalopathy.

INTRODUCTION The ability to perceive odorous molecules in water or air and tracking them is the earliest special sense to appear in the evolution of animal life and is also the first special sense in human embryonic and fetal development. Even protozoans and simple organisms, such as medusae (jellyfish) and polyps (hydra; sea anemone), which have

no central nervous system but only a diffuse nerve net, react differently to water-soluble molecules depending on whether they perceive them as toxic and threatening or attractive as food. The human fetus perceives odorous molecules dissolved in amniotic fluid that flows through developing nasal passages with specific reactions. Anatomical structures enabling this function appear early in ontogenesis. The olfactory bulb exhibits

*Correspondence to: Dr. Harvey B. Sarnat, Alberta Children’s Hospital Research Institute (Owerko Centre), #395, 3820-24 Avenue NW, Calgary, AB, Canada T3B 2X9. Tel: +1-403-441-8409, Fax: +1-403-955-7609, E-mail: [email protected]; [email protected]

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essentially the same laminar structure in all vertebrates with minor differences, such as whether the same zone of olfactory epithelium projects to several or only one synaptic glomerulus (Ariëns Kappers et al., 1936; Sarnat and Netsky, 1981; Komiyama and Luo, 2006; Kuciel et al., 2016). The olfactory bulb was the first structure of the human brain to be studied by Golgi impregnations, described in 1875 by Camille Golgi himself. Olfaction is unique in many ways relative to the other special sensory systems of the brain. Some neurobiologists do not consider the olfactory nerve to be a true cranial nerve because the axons from neurons in the olfactory epithelium pass individually through multiple fenestrations of the bony cribriform plate rather than forming a compact nerve fascicle. These axons remain unmyelinated throughout life. They do not synapse in a peripheral ganglion because the synaptic glomeruli with the ventral surface of the olfactory bulb function as a ganglion. Olfaction is the only sensory system that does not directly project to the thalamus. Instead, it incorporates its own intrinsic thalamic equivalent, which is derived segmentally from embryonic prosomere 2, similar to the thalamus (see following text). Criteria that we suggest to define a sensory cranial nerve are: (1) specialized peripheral receptor; (2) primary synapse in a ganglion or ganglionic equivalent; (3) projection of secondary neurons to thalamus or thalamic equivalents; and (4) projection of tertiary and quaternary neurons to specialized regions of the neocortex for conscious recognition of the stimuli. Myelination is not a requirement; autonomic afferents are unmyelinated. Cranial nerve I thus fulfills the criteria of a true cranial nerve, despite its unique features unlike the optic nerve, CN II, which really is a central fasciculus that forms within the diencephalic diverticulum of the optic stalk and optic cup and is enclosed within a dural sheath. Despite its unique development, neuroanatomy, and function, olfactory reflexes are rarely tested in the neonatal neurologic examination—olfactory bulbs are not constantly reported by radiologists in neonatal MRI, and their histopathology is inconstantly described in post-mortem autopsy reports, even in infants with major malformations in other parts of the brain or with genetic and metabolic diseases. Testing olfactory reflexes in the neonate examines regions of the brain not shown by neurologic examination of any other part, requires no elaborate technology, and should be incorporated into the routine physical examination of the newborn.

system was fully mature by 14 weeks gestation. This assessment was based upon the histologic morphology of the olfactory bulb (Humphrey, 1940). Modern histochemical and immunocytochemical techniques not available in her day demonstrate that the olfactory bulb and tract remain far from mature even in the full-term neonate (Sarnat and Yu, 2016). Nevertheless, it is difficult to ascertain olfactory perception in the first half of gestation, but it cannot be appreciated at the cerebral cortical level because synaptogenesis does not begin in the cortical plate until 22 weeks gestation (Sarnat, 2013, 2015). Fetuses and preterm neonates regularly respond to olfactory stimuli after 28 weeks gestation, as can be seen in utero in the increased heart rate recorded by fetal heart monitoring or in the increased movement of the head, trunk, or extremities identified by real-time ultrasound (Mennella et al., 1995; Schaal et al., 1998, 2000). Continuous flow of amniotic fluid through fetal nasal passages enables both human and rat fetuses to detect odors of strong foods ingested by the mother, such as garlic, onion, curry, and spices, the odorous molecules of which pass the placental circulation to enter the amniotic fluid (Hepper, 1995; Mennella et al., 1995; Schaal et al., 1998, 2000). Responses are detected as changes in fetal respiratory patterns, increased fetal movements, and even augmented facial grimacing and lingual movements detected by real-time ultrasound. The rate of fetal swallowing increases when amniotic fluid is sweetened and decreases when it is made to taste bitter (Hepper, 1995). Preterm and term neonates perceive differences in odor or flavor of maternal milk from substitute formulae (Schaal et al., 2004; Delaunay-El Allam et al., 2006; Abadie and Couly, 2013). However, no difference is found in olfactory responses between term neonates who are small-for-gestational-age or appropriate in size (Ayers et al., 2012; Rotstein et al., 2015), though some investigators do report differences in facial grimacing and even speculate that exposure of preterm and smallfor-gestational-age neonates to sweet tastes and odors may contribute to obesity later in life (Laureano et al., 2016). The possibility that fetal and early postnatal exposure conditions the infant to later food preferences or aversions remains a fascinating but almost untestable hypothesis. Olfactory perception in the fetus might also serve to condition the fetal brain to sensory discrimination, not only for olfaction but also for other sensory stimuli, but this is speculative.

CLINICAL DEVELOPMENT OF HUMAN OLFACTION

Olfactory reflexes in the neonate and early infancy

Olfactory perception in the human fetus In 1940 a world expert in developmental neuroanatomy, Tryphena Humphrey, wrote that the human fetal olfactory

After 28–30 weeks of gestation, infants exhibit reproducible olfactory reflexes that reliably test the integrity of the olfactory system (Sarnat, 1978; Sarnat et al., 2017).

OLFACTORY DEVELOPMENT The test substance must be aromatic rather than an irritant. In reality, there are no substances that are purely aromatic or purely irritative (Cameron and Doty, 2013), but the ratio of stimuli for the olfactory nerve vs. nasal mucosal pain endings of the trigeminal nerve is reversed between peppermint and ammonia. The test substance can be weak concentrations of vanillin, rose oil (phenyl ethanol), peppermint, eugenol, cinnamon, coffee, and cloves, to name a few (Doty et al., 1978; Frasnelli et al., 2011). Testing should not be done during deep or quiet sleep, as responses may be suppressed physiologically in normal infants. Similarly, the best timing is before rather than after a routine feeding. Sedation induced by many medications, particularly antiepileptic drugs, also may suppress neonatal olfactory reflexes. Enriched neonatal exposure to odors increases the number of neurons in the adult olfactory bulb and enhances olfactory memory in rodents and humans (Hauser et al., 1985; Woo et al., 1987; Davis and Porter, 1991; Gheusi et al., 2000; Rochefort et al., 2002). Neonatal olfactory recognition includes the odor of the mother’s axilla and other odorous clues that distinguish her from other women (Macfarlane, 1975; Cernoch and Porter, 1985). Rodents deprived early of olfactory stimulation exhibit deficient inhibition of synaptic function in the olfactory bulb (Wilson et al., 1990). Odor intensity can also lead to greater discrimination after maturity (Lipsitt et al., 1963; Engen et al., 1965; Marlier et al., 2007; Mainland et al., 2014). Olfactory memories often are strong and lead to recall of earlier associated events when the same odor is perceived even years later.

Eliciting the neonatal olfactory reflex This test is performed by introducing the test substance beneath the nostrils without touching the skin (Sarnat, 1978; Sarnat et al., 2017). A standard test substance, such as weak oil of peppermint, can be easily obtained by lightly soaking half a tuft of cotton placed within a small laboratory tube used for blood collection. The advantage of this substance is that its odor lasts for years or even decades, unlike substances such as coffee or cloves, which are satisfactory when fresh but lose their odor upon drying and must be periodically replenished. Each nostril can be tested separately, but usually this is not necessary. Equivocal responses should be retested after a pause. Neonatal responses to peppermint are of two varieties. The majority of times the response is initiation of sucking or lip smacking; an alternative response is some type of arousal/withdrawal, such as retraction of the head, wrinkling of the forehead, facial grimacing, wiping the arm across the nose, or even simply alerting by opening the eyes more widely (Sarnat, 1978; Sarnat et al., 2017).

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Responses can be semi-quantitated in gradients with olfactory sensitivity increasing in the first postnatal days (Lipsitt et al., 1963). Olfactory reflexes can also be used as a sign of alert consciousness because they are depressed in obtunded or encephalopathic infants (Sarnat et al., 2017). In neonates with certain cerebral malformations, endocrinopathies, chromosomopathies and other genetic diseases, hypoxic/ischemic or metabolic encephalopathies, olfactory reflexes may be severely diminished or absent if the olfactory bulbs are hypoplastic or aplastic. Olfactory responses thus should be tested at least once during the neurologic examination of the neonate, especially if the neurologic function is not normal. Lack of response should be reassessed by serial tests at different times and on different days to validate the finding, but olfactory unresponsiveness may be transitory as hypoxic or metabolic cerebral depression recovers. Nasal passages should not be obstructed by thick mucus or copious rhinorrhea at the time of testing. Nasogastric feeding tubes may partially obstruct nasal passages or even abrade the delicate mucosal olfactory epithelium. Intubated infants may not respond, even if alert, because the flow of air through the nasal passages may be insufficient to carry odorous molecules. Negative responses must be interpreted cautiously if physical obstruction of upper airway flow is present.

Olfactory reflexes in late infancy and childhood In infants the responses are similar to those of neonates. In children old enough to express themselves verbally, one can ask what they smelled. Appropriate precise responses to peppermint are “toothpaste” or “candy cane.” Some children even answer with surprising precision by identifying “mint” or “peppermint.” Standardized tests are now available to quantitatively assess the olfactory function of children over the age of four years (Cameron and Doty, 2013).

NEUROIMAGING OF THE OLFACTORY BULBS In post-natal MR imaging, normal olfactory bulbs are nearly always identifiable on 3 mm coronal spin-echo T2-weighted images (Yousry et al., 2000). The recognition is even easier in infants than in adults because of more prominent bifrontal subarachnoid fluid spaces in infants. With three-dimensional high resolution T2-weighted images, the olfactory bulbs can be identified in both axial and coronal planes, the latter at the level of the posterior orbits (Yousry et al., 2000). The olfactory tract is distinguished from the bulb by a change in caliber,

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but a clear neuroanatomical landmark is lacking (Azoulay et al., 2006; Barkovich and Raybaud, 2012; Blitz et al., 2014). A normal olfactory bulb is usually accompanied by an olfactory sulcus, previously called the olfactory groove, on the ventral surface of the gyrus rectus in which the olfactory bulb and tract lie, also readily identifiable in both axial and coronal T2-weighted images (Fig. 3.1A and B). Absence of the olfactory bulbs can be bilateral or unilateral, both conditions diagnosed with confidence by MRI (Sarnat et al., 2017). Absence of the olfactory bulb is nearly always accompanied by absence of the olfactory sulcus. In pre-natal MRI, the olfactory bulbs and sulci can be recognized after 30 weeks gestation (Azoulay et al., 2006; Barkovich and Raybaud, 2012), but the frequency of identification has not been systematically studied to date in a large series of fetal brain MRIs. Failure to visualize olfactory bulbs in fetal MRI is not conclusive

evidence of their absence. Congenital arhinia (absence of the external nose, nasal cavities, olfactory epithelia, and olfactory bulbs) is a rare malformation diagnosed prenatally by fetal MRI (Li et al., 2015). Total absence of the olfactory bulb, either as a paired structure or as a fused midline structure ventral to the rostral prosencephalon, is a characteristic finding in alobar and semilobar holoprosencephaly, but olfactory bulbs may be present, though hypoplastic, in the milder lobar and middle interhemispheric variants. Hypoplastic olfactory bulbs also occur in septo-optic-pituitary dysplasia (Sarnat et al., 2017). All may be demonstrated by MRI at any age of life as well as neuropathologically at autopsy. True agenesis can be distinguished from artifactual tearing away of the olfactory bulbs and tracts in postmortem examination by absence of the olfactory sulcus at the surface of the gyrus rectus, evident in the third trimester fetus, neonate, and older ages.

Fig. 3.1. (A) Coronal T2-weighted MRI (1.5 Tesla) of a 4-month-old boy, at the level of the prefrontal cortex and orbits to demonstrate well formed olfactory bulbs (arrows) in a normal brain. Arrowheads indicate the olfactory sulci (olfactory grooves) on the gyrus rectus, which fail to form in arhinencephaly. (B) Higher magnification of (A). Arrows indicate the cribriform plate through which the axons of the olfactory nerve pass from the nasal epithelium. Arrowhead points to interhemispheric fissure. e ¼ ethmoid sinuses. (C) Coronal T2 MRI at level of orbits in a 9-year-old boy with isolated bilateral agenesis of the olfactory bulbs. Compare with (A and B). Note the well formed interhemispheric fissure denoting that this arhinencephaly is not associated with alobar or semilobar holoprosencephaly. The septum pellucidum and corpus callosum also were well formed (not seen at this rostral plane of section). The patient was anosmic. (D) Coronal T2 MRI in a 5-year-old girl in whom the left olfactory bulb (lower arrow) and olfactory sulcus (upper arrow) are well formed but the right structures are absent. The interhemispheric fissure is well developed, hence this unilateral isolated arhinencephaly is not associated with holoprosencephaly; no other cerebral malformations were detected in other brain sections (not shown). Reproduced from Sarnat, H.B., Flores-Sarnat, L., Wei, X.-C., 2017. Olfactory development. Part 1. Functional, from fetal perception to adult wine-tasting. J Child Neurol 32, 566–578.

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MORPHOGENESIS AND NEURONAL MATURATION OF THE HUMAN OLFACTORY SYSTEM A basic developmental principle that full neuroanatomical maturation is not necessary for function to appear is nowhere better illustrated than in the olfactory lobe. It has been accepted over the past century that the olfactory epithelium, bulbs, and tracts matured early in fetal life because their histologic architecture of apparently mature lamination (Humphrey, 1940). The mitral cell layer is initiated at 9.5 weeks gestation and well formed by 11 weeks; it is more prominent at 18.5 weeks than in the adult (Humphrey, 1940). More recent immunologic studies of the olfactory bulb and tract at fetal and neonatal autopsy points reveal, however, that the olfactory system is far from mature even in the term neonate, in the context of synaptogenesis, myelination, and expression of maturational proteins in all neurons (Sarnat and Yu, 2016; Sarnat and Flores-Sarnat, 2017). These findings have importance for the interpretation of pathologic development of the olfactory system, whether isolated or as a component of more extensive malformation of the brain or in specific genetic and metabolic diseases. The maturation of granular neurons from undifferentiated neuroepithelial cells and their migration is regulated by tee-shirt homeodomain zinc finger family number 1 (TSHZ1), without which the olfactory bulbs fail to form normally in mice (Ragancokova et al., 2014). The macroscopic (gross) appearance of the olfactory bulb is prominent by the early second trimester of gestation, and it is relatively large in relation to the rest of the immature telencephalic hemisphere (Fig. 3.2).

Transitory olfactory ventricular recess The normal fetal olfactory bulb includes a recess in the frontal horn of the lateral ventricle, which becomes lined by a pseudostratified columnar ependymal epithelium from about 16 weeks gestation but does not thin to a simple cuboidal epithelium as in other parts of the ventricular system. This olfactory recess involutes postnatally, leaving only scattered ependymal clusters and rosettes, similar to the fate of the spinal central canal (Sarnat and Yu, 2016). In some older children and adults, persistence of an olfactory recess is identified by MRI (Smitka et al., 2009). In fetal hydrocephalus, the olfactory recess enlarges in proportion to the general ventriculomegaly of the lateral ventricles, but its ependymal lining does not become discontinuous (Sarnat and Yu, 2016). Table 3.1 summarizes the laminae of the olfactory bulb that are histologically evident from the late first trimester of fetal life, even if neuronal protein expression and synapse formation are not mature. Layer 1 is really

Fig. 3.2. Ventral surface of the normal uncut brain at autopsy of a 17-month fetus to demonstrate the position and relatively large size of the olfactory bulb (b) and olfactory tract (tr). Reproduced from Sarnat, H.B., Flores-Sarnat, L., 2017. Olfactory development. Part 2. Developmental neuroanatomy and neuropathology. J Child Neurol 32, 579–592.

the distal part of the olfactory nerve and layer 2 is really the equivalent of an olfactory nerve ganglion. The olfactory tract is not a simple white matter fasciculus. It contains as much gray as white matter, including an extension of the core of granular cells of the olfactory bulb at one end and nodules of the anterior olfactory nucleus at the other (Sarnat and Yu, 2016). Resident progenitor “stem” cells capable of neuronal differentiation are present in both the olfactory bulb and tract. In the adult brain, the olfactory bulb remains one of two reservoirs of such cells, the other being the polymorphic zone on the inner surface of the dentate gyrus of the hippocampus. The olfactory epithelium also possesses many progenitor cells to provide for rapid turnover of its neurons throughout life which might be lost with even trivial upper respiratory infections that may transiently denude the nasal mucosa.

Neuroembryology of the olfactory epithelium The olfactory epithelium is a part of the nasal mucosa that lines the roof of the nasal cavities, the medial part of the nasal septum, the superior and partly extending to the middle, but not the inferior, turbinate bones (Nakashima et al., 1984; Cowart et al., 2011). Epithelial plugs obstruct the external nares in the first trimester of fetal life but regress between 16 and 24 weeks gestation, as demonstrated over a century ago (Schaffer, 1910).

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Table 3.1 Lamination of the human olfactory bulb, histologically evident from 14 weeks gestation to adult Layer

Components

Comments

1

Axons of primary neurons in olfactory epithelium Synaptic glomeruli; periglomerular interneurons

Acellular; unmyelinated, calretinin-reactive axons; specialized glial ensheathing cells Glomeruli form at 14 weeks on the ventral surface of the bulb facing the cribriform plate, with precise synaptic ratios between olfactory axons and mitral cell dendrites for amplification; periglomerular cells co-express GABA and dopamine; equivalent of peripheral ganglion Tufted neurons at rostral end, otherwise cell-sparse; dendro-dendritic synapses: mitral to granule excitatory; granule to mitral inhibitory Large mitral cells form at 9 weeks.; axons extend into olfactory tract Neurites; cell- and synapse-sparse GABAergic axonless granular neurons in core of olfactory bulb; form multiple concentric laminae alternating with sheets of dendro-dendritic synapses; main reservoir of resident progenitor cells; ventricular recess in fetus/neonate within granular core, but eccentric; major part of olfactory thalamus

2

3

External plexiform layer

4 5 6

Mitral neuronal cell layer Internal plexiform layer Granular layer

Adapted from Sarnat, H.B., Yu, W., 2016. Maturation and dysgenesis of the human olfactory bulb. Brain Pathol 26, 301–318.

Volatile molecules penetrate an aqueous mucus layer covering the olfactory epithelium to reach receptor sites on the cilia of primary olfactory dendrites (Cowart et al., 2011). Olfactory mucosal neurons project to the olfactory placode of the telencephalon shortly after cleavage of the prosencephalon at 4–5 weeks gestation, before the olfactory bulb is even formed (Fig. 3.3) (Ashwell and Waite, 2004). The number of bipolar primary olfactory neurons increases greatly with the formation of the turbinate bones that begin at about 8 weeks gestation, which enables a greatly enlarged surface area of olfactory epithelium. An active process of apoptosis occurs simultaneous with the generation of new neurons in the olfactory epithelium (Magrassi and Graziadei, 1995). These resident stem cells are so capable of neuronal regeneration that they have been used as transplants to other damaged parts of the human brain (Chen et al., 2014). Invagination of the olfactory zone neuroepithelium occurs at 7.5 weeks gestation and forms the incipient olfactory bulbs, carrying with it a part of the ventricular cavity to form the fetal olfactory ventricular recess (Bayer and Altman, 2008). Specific olfactory marker proteins are identified in the human olfactory epithelium from 28 weeks gestation (Chuah and Zheng, 1987; Johnson et al., 1997). In rodents, the zonal organization of the olfactory epithelium is preserved in its somatotopic distribution in the olfactory bulb (Mori et al., 1999). Many progenitor cells are present throughout life in the olfactory epithelium. Novel DNA microarrays and other genetic studies also recognize human olfactory receptor gene families and classes of neural precursors in the olfactory epithelium

(Zhang et al., 2007; Tucker et al., 2010). The olfactory epithelium with its primary olfactory receptor neurons also does not approach mature scanning ultrastructural morphology in humans until near-term (Kimura et al., 2009); transmission electron microscopy of the mature olfactory epithelium is well described (Pyatkina, 1982). The olfactory bulb was named by Weitbrecht (1751). The neuroanatomy and axonal connections of the olfactory system were defined in the late 19th century by Golgi (1875) and Ramón y Cajal (1901, 1909–1911), the latter author describing fetal morphogenesis as well as the mature neuroanatomy. Other late 19th and early 20th century developmental neuroanatomists who contributed to the description of the olfactory system morphogenesis were von K€olliker (1882), van Gehuchten and Martin (1895), Probst (1901), Rossi (1907), Pearson (1941), Crosby and Humphrey (1939), and Humphrey (1940). Fascinating details of historical aspects of the naming of structures and understanding of integration of the olfactory system into neural networks have been previously discussed (Sarnat and Yu, 2016; Sarnat and Flores-Sarnat, 2017).

Anatomical architecture and synaptic organization of the developing olfactory bulb and tract Axonal terminals of primary olfactory neurons entering the olfactory bulb form synaptic glomeruli (glomerulus ¼ Greek; ball of threads) with dendrites of the layered mitral and tufted cells. Olfactory bulb glomeruli are generally arranged as a single layer, but in places may be two or three glomeruli thick. These glomeruli make

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Fig. 3.3. Diagram of initial olfactory epithelium and projections of olfactory nerve and accessory olfactory (vomeronasal) nerve with its ganglion toward the telencephalic olfactory fields, the future olfactory bulbs, in a 42-day human fetus. Reproduced from Ashwell, K.W.S., Waite, P.M.E., 2004. Development of the peripheral nervous system. In: Paxinos, G., Mai, J.K., (Eds.), The human nervous system, second ed. Amsterdam: Elsevier/Academic Press, 95–110.

up a layer of the olfactory bulb near its ventral surface, facing the cribriform bony plate through which primary olfactory nerve axons pass. The b-secretase enzyme BACE-1, deficient in Alzheimer disease, is essential for axonal guidance of olfactory sensory neurons and for the formation of synaptic glomeruli during development (Rajapaksha et al., 2011). Netrin is another important molecule that guides olfactory nerve axons to olfactory bulb synaptic glomeruli (Lakhina et al., 2012). Each olfactory epithelial axon is ensheathed by special cells derived from neural crest that are not oligodendrocytes but are more closely related to nonmyelin-forming Schwann cells (Mackay-Sim et al., 2008). Small periglomerular interneurons form sheet-like processes that envelop other small periglomerular neurons (Reese and Brightman, 1970). Spine-laden dendrites of periglomerular cells ramify within two or occasionally more glomeruli. Their axons extend across as many as six glomeruli as they contact local interneurons. These periglomerular neurons are heterogeneous in morphology, neurochemistry, and physiology; about 10% lack synapses with olfactory neurons (Kratskin and Belluzi, 2003). They synthesize gamma-aminobutyric acid (GABA) and dopamine as co-localized transmitters (Kosaka et al., 1985; Gall et al., 1987; Ohm et al., 1990, 1991). Periglomerular neurons contain the calciumbinding proteins calbindin, parvalbumin, and calretinin (calbindin-2). The olfactory bulb is unique in possessing the highest ratio of dendro-dendritic synapses of any part

of the brain. It also expresses the tight-junction protein claudin-3 (Eppler et al., 2017). The synaptic organization of the olfactory bulb includes structural and functional columns (Kauer and Cinelli, 1993; Willhite et al., 2006) similar to the barrels or radial units of the neocortex (Rakic, 2000; Feldmeyer et al., 2013). Lateral synaptic connectivity within the olfactory bulb is much sparser (Kim et al., 2011). Olfactory bulb synaptic glomeruli are thus compartmentalized with primary afferent axodendritic and dendro-dendritic synaptic sub-compartments (Kasowski et al., 1999; Kim and Greer, 2000). This functional organization of synaptic circuitry is initiated with the onset of synaptogenesis. An important difference between neuroblasts that form the laminae of the olfactory bulb and those that form the laminated cerebral cortex is that the former do not arise from the subventricular region of the olfactory recess with primary radial migration but rather migrate into the olfactory bulb from the frontal lateral ventricular wall and then only secondarily migrate radially within the olfactory bulb (Kishi, 1987; Rousselot et al., 1994; Lois et al., 1996). The principal efferent synaptic network connections of the olfactory bulb, directly or through the anterior olfactory nucleus, are with the amygdala, piriform cortex, and entorhinal cortex (a portion of the parahippocampal gyrus) with additional projections to the hippocampus, septal nuclei, and to the contralateral olfactory bulb through the anterior commissure. Commissural

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projections from the anterior olfactory nucleus pass to the contralateral olfactory bulb through the anterior commissure, a structure that forms at 7 weeks gestation with the passage of the first interhemispheric pioneer axons, about 3 weeks earlier than those of the future corpus callosum. Primary olfactory memory is mediated by the hippocampus (Hawkes and Doty, 2009). Olfactory memories of certain experiences form highly durable engrams (Mouly and Sullivan, 2010). Furthermore, multiple olfactory cortical areas transmit signals of the corticotrophin-releasing hormone to the hypothalamic neurons, which control stress responses to the odors of predators in many animals (Teixeira et al., 2010). Mitral cells, the principal efferent neurons of the olfactory bulb, can synchronize their discharges through a shared set of granular neurons when separated by arbitrary distances; a specific distribution of dendro-dendritic synaptic clusters is critical for optimal synchronization of mitral cell spikes in response to their odor inputs (Hinds and Hinds, 1976).

Sequence of synaptogenesis in the olfactory system The sequential appearance of synapses in the developing olfactory bulb can be demonstrated in tissue sections by immunocytochemical reactivities of synaptic vesicular proteins such as synaptophysin. Using this method, synaptic glomeruli exhibit synaptophysin expression before 16 weeks gestation, but it is not uniform and some glomeruli begin to show strong reactivity adjacent to others with no reactivity; all are reactive by term. Calretinin, by contrast, is not yet expressed in most of the synaptic glomeruli at mid-gestation but is strong at all ages in the primary olfactory nerve axons forming layer 1 (Sarnat and Yu, 2016). Calretinin within the synaptic glomeruli is localized in the axons of primary olfactory neurons. This calretinin expression is not true of the mitral cells and their dendrites, which are not GABAergic neurons and do not express calcium-binding proteins. The glomeruli do not mediate synaptic transmission during the first half of gestation. Some neurons remain dormant for long periods before developing synaptic connections, even though pre- and postsynaptic membranes are in proximity. An example is the relation between primary olfactory receptor neurons and mitral cells in the synaptic glomeruli of the olfactory bulb (Johnson et al., 1997). This feature may explain the more delayed expression of calretinin than of synaptophysin in the olfactory glomeruli, even though calretinin-immunoreactive primary olfactory nerve axons in layer 1 are already in proximity (Sarnat and Yu, 2016). About 80% of synaptic contacts between neurons are organized as reciprocal pairs:

mitral-to-granule cell synapses are excitatory in contrast to granule cell-to-mitral neuronal synapses, which are inhibitory (Kosaka et al., 1985). The olfactory bulb thus exhibits a mathematical precision and predictability in its intrinsic synaptic relations, reminiscent of the cerebellar cortex, yet having a synaptic plasticity contrasting with the synaptic stability of the cerebellum. In this regard, the olfactory bulb may be comparable more to the hippocampus, which also exhibits precise neuronal ratios but remains synaptically plastic throughout life. More than 20 known neurotransmitters or modulators are identified in the olfactory bulb (Mouly and Sullivan, 2010), enabling complex coding and processing of odorous information (Mori et al., 1999). Serotonin, acting through 5HT2A receptors on mitral cells, increases synaptic activity in olfactory bulb glomeruli, as measured electrophysiologically (Hinds and Hinds, 1976). Serotonin is a major neurotransmitter in the raphe nuclei of the brainstem and certain other regions of the brain, but at the olfactory synaptic glomeruli it functions to augment activity of other chemical transmitters (Brill et al., 2016). Electrophysiology in the murine olfactory bulb demonstrates that early olfactory deprivation diminishes synaptic organization (Wilson et al., 1990). The maturation of neuronal proteins and synaptogenesis in the olfactory bulb are illustrated in Fig. 3.4. Progressive concentric multiple lamination of the core granular layer of the olfactory bulb is a criterion for neuroanatomical maturation, well demonstrated in tissue sections by synaptophysin that shows synapse formation and neuronal nuclear antigen (NeuN) reflective of late neuronal maturation. Layers of granular neurons alternate with layers of their dendro-dendritic synapses (Fig. 3.4A–C). This laminar maturation begins in the most peripheral part of the granular core; the deepest part of the core is the last to become laminated, i.e., in the early postnatal period. Calretinin, by contrast, is expressed earlier in the granular layer when its neuroepithelial cells are still immature (Ulfig, 2002; Sarnat and Yu, 2016; Fig. 3.4D and E). Lamination in the granular layer, and also the mitral cell layer, is at least partly regulated in fetal mice by the expression of the zincfinger gene Fex in olfactory neurons (Hirata et al., 2006). At term, about 30%–40% of granular neurons remain nonreactive for NeuN, mainly deep in the core. NeuN is a late marker of neuronal maturation expressed only when the neuron is nearly mature and functional (Sarnat, 2013, 2015). Synaptic circuitry in the murine olfactory bulb remains plastic not only in the immature neonatal condition but as long as the body is still growing (Pomeroy et al., 1990). Postnatally, the length of mitral cell dendritic branches increases by a factor of 11; the number

OLFACTORY DEVELOPMENT

Fig. 3.4. See figure legend on next page.

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of glomerular and extraglomerular synapses increases by factors of 90 and 170, respectively (Hawkes and Doty, 2009). In addition, olfactory receptors in the mucosa have many de novo genetic mutations. Axonal target specificity and “sorting” at the neocortical level occurs in the medial entorhinal cortex, as shown by reconstructions from tridimensional electron microscopy in rats, so that excitatory neurons sequentially target inhibitory neurons before excitatory neurons (Schmidt et al., 2017).

(Puche and Shipley, 2001; Au et al., 2002). Astrocytic recording of the rodent olfactory bulb synaptic glomeruli is achieved with electrical stimulation (De Saint and Westbrook, 2005). Odorant receptors in the mucosa also regulate guidance of axons to specific sets of synaptic glomeruli in the olfactory bulb (Imai and Sakano, 2007). Levels of adhesion molecules promote connections of sensory axons with proper synaptic glomerular targets.

Myelination of the olfactory bulb and tract

THE INTRINSIC OLFACTORY THALAMUS

Primary olfactory nerve axons remain unmyelinated throughout life, related to continuous turnover and regeneration from progenitor cells in the olfactory epithelium. Myelin forms around axons of mitral and tufted cells of the olfactory bulb, including their projection through the olfactory tract. In the full-term neonate no myelination has as yet been identified by luxol fast blue myelin stain or even by the more sensitive myelin basic protein immunoreactivity (Sarnat and Yu, 2016). Myelination proceeds postnatally during the first few months of age. Secondary postsynaptic projections of the anterior olfactory nucleus and tertiary olfactory fibers in the amygdala, hypothalamus, and neocortex myelinate even later, their cycles extending beyond infancy.

Role of glia and adhesion molecules in the olfactory system Glial neuronal interactions may play an important role in glomerular formation and contribute to the delay in myelination (Bailey and Shipley, 1993; Doucette, 1993; Bailey et al., 1999). Astrocytes are of different types, with localization in different layers of the olfactory bulb

The olfactory system is unique as the only sensory system of the CNS lacking projections to the thalamus. Indeed, the olfactory bulb incorporates its own thalamic equivalent (Sarnat and Yu, 2016). This intrinsic thalamic equivalent that is present within the olfactory bulb and tract consists of three components: (1) the core of intrinsic axon-less granular interneurons and (2) periglomerular interneurons, both of which form dendro-dendritic synapses, and (3) the anterior olfactory nucleus. The olfactory bulb, as well as the dorsal thalamus that receives somatosensory, visual, and auditory afferents, are both derived from the same neural tube segment, prosomere 2, in early embryogenesis in the new revised scheme of neuromeric segmentation (Puelles and Rubenstein, 2003; Puelles et al., 2013; Carstens and Sarnat, 2018). The inner core of the olfactory bulb with its concentric alternating layers of granular neurons and synaptic sheets extends into the olfactory tract, but these small granular GABAergic interneurons have no extrinsic connections; they form dendro-dendritic synapses with mitral and

Fig 3.4 Immunoreactivities of the olfactory bulb during fetal development. (A, B) Synaptophysin is strong in the synaptic glomeruli (g) since 15 weeks in this term neonate. The mitral cell layer also shows synaptic vesicle reactivity around mitral cells (mitr). The granular cell core of the olfactory bulb exhibits multiple concentric layers of dendro-dendritic synapses between granule cells, but more intense in the outer layers than in the central core where synapse formation is still incomplete. (C) Neuronal nuclear antigen (NeuN) in a 19-week fetus, a later marker of neuronal maturation, shows that the outer part of the molecular zone has mature neurons in concentric layers (alternating with the layers of synapses shown in A and B), but the granular neurons of the deeper part of the core of the granular zone are still immature and do not exhibit reactive nuclei or lamination. (D, E) Calretinin reactivity in this 19-week fetus shows strong expression in the outermost zone, layer 1, consisting of axons of primary olfactory neurons that reside in the nasal epithelium. Most olfactory glomeruli are still nonreactive, by contrast with more mature synaptophysin reactivity (A). Granular cells of the core, including the immature neuroepithelial (future granular) cells around the eccentric olfactory ventricular recess (v), are strongly reactive for calretinin. (F) Vimentin in this 19-week fetus shows strong reactivity of bipolar progenitor “stem” cells. The reactivity in the outer layer of primary olfactory axons is not in the axons themselves as with calretinin (D) but rather in the unique sheathing cells of these axons, modified Schwann cells. Capillary endothelial cells also are reactive for vimentin. (G) Proliferating cell nuclear antigen (Ki67; MIB-1) in a 21-week fetus. The deep core of the granular zone contains proliferative progenitor cells with marked (dark) nuclei (p) among immature granular neurons with unmarked ( pale) nuclei (g). The olfactory ventricular recess (v) contains a few desquamated neuroepithelial cells of both types in its ependymal-lined (ep) lumen. (H) Microtubule-associated protein-2 (MAP2) is useful for demonstrating mitral neurons that normally are nonreactive for both NeuN and calretinin at all ages. This mitral cell (*) is in the normal olfactory bulb of a 23-week fetus. Reproduced with permission from Sarnat HB, Yu W (2016). Maturation and dysgenesis of the human olfactory bulb. Brain Pathol 26: 301–318.

OLFACTORY DEVELOPMENT tufted neurons as well as between themselves within the core granular zone. A ratio of about 50–100 granule cells for each mitral cell is preserved. Periglomerular interneurons are another type of intrinsic cells with no afferent or efferent connections outside the olfactory bulb. The anterior olfactory nucleus was first named (in amphibians and reptiles) by Herrick (1910). In humans it is a series of neuronal aggregates, not a single compact cluster; it extends proximally into the olfactory tract (Herrick, 1910; Crosby and Humphrey, 1939; Hawkes and Doty, 2009; Sarnat and Yu, 2016). This is a source of axons that project to the amygdala, entorhinal cortex, hippocampus, and septal nuclei, and thus serves as the olfactory system equivalent of thalamo-cortical projections. Its lamination may be analogous to the layered lateral geniculate body of the thalamus that subserves the visual system but is still part of the thalamus. The anterior olfactory nucleus contains a heterogeneous population of neurons. The majority are glutamatergic, but there is a minority of GABAergic interneurons that are either reactive to calretinin or to parvalbumin, though the same neurons do not co-express both calciumbinding proteins (García-Ojeda et al., 1998; Barbado et al., 2002; Sarnat and Yu, 2016). The olfactory tract (stalk, peduncle) is much more than a simple white matter fascicle and indeed contains as much gray as white matter (Sarnat and Yu, 2016). In addition to axonal projection fibers and a few anterior commissural afferents from the contralateral olfactory bulb, it also includes longitudinal bundles of progenitor cell processes, a caudal extension of granular neurons from the olfactory bulb, and a rostral extension of pyramidal neurons of the anterior olfactory nucleus, singly and in clusters (Hawkes and Doty, 2009; Sarnat and Yu, 2016). Despite a projection from the entorhinal cortex to the mesio-dorsal thalamic nucleus for relay to the posterior orbital agranular insular cortex, the direct monosynaptic projection of the olfactory cortex to the same orbital/insular area involves many more neurons; this thalamic projection, therefore, is not essential (Schmidt et al., 2017).

TRANSITORY FETAL ACCESSORY OLFACTORY BULB AND VOMERONASAL SYSTEM (NERVUS TERMINALIS; CRANIAL NERVE 0) A small secondary olfactory system also exists in vertebrates. The histologic lamination of the accessory bulb is similar to that of the primary olfactory bulb, but usually not as well developed (Humphrey, 1940; Mackay-Sim et al., 2008; Rajapaksha et al., 2011). In the human fetus it is a transitory structure that atrophies by mid-gestation. Though the accessory olfactory bulb is usually absent in

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adult humans, a vestigial bulb and tubular vomeronasal organ at the base of the nasal septum are sometimes still identified (Nakashima et al., 1985; Zbar et al., 2000; Slotnick et al., 2010). Its afferent nerve twigs constitute the nervus terminalis or cranial nerve 0, and its efferents terminate mainly in the septum and amygdala. In the early fetus, it is found medial to the main olfactory nerve fibers and often forms a peripheral ganglion, unlike the main olfactory nerve that incorporates the ganglion into the bulb itself as the synaptic glomeruli (Fig. 3.3). The accessory olfactory bulb was well illustrated in Golgi silver impregnations in the human fetus by Ramón y Cajal (1901, 1909–1911)). As with the principal olfactory bulb, the accessory bulb (of the mouse, where it persists at maturity unlike in the human) also has large numbers of dendro-dendritic synapses between intrinsic granular neurons and between granular neurons and mitral cells (Kaba and Keverne, 1992). Synaptic glomeruli are lacking or located within the ganglion. One reason for the difference is that the accessory olfactory bulb is located at the surface of the principal olfactory bulb, not above the bony cribriform plate through which axons from the olfactory nasal epithelium pass (Ramón y Cajal, 1909–1911), which was confirmed in late first trimester fetuses in our laboratory (Sarnat H.B., unpublished). In mature rodents the accessory olfactory system is involved in the mediation of sexual attraction, but its function in the human fetus is unknown.

RELATION BETWEEN OLFACTION AND TASTE It is sometimes difficult to classify the perception of odorous molecules as olfaction or taste, particularly in phylogenetically simple animals and in the human fetus in late gestation (Sarnat and Flores-Sarnat, 2016a,b). In all vertebrates, olfaction and taste are widely separated in different parts of the neuroaxis, and their central axonal pathways and synaptic connections are dissimilar. Unlike olfaction, taste centers lack the intrinsic thalamic equivalent and synaptic glomeruli of the olfactory bulb but possess the characteristic peripheral nervous system ganglia of most sensory cranial nerves. Taste is mediated by the facial (CN VII), glossopharyngeal (CN IX), and vagal (CN X) nerves whose centers are in the medulla oblongata. The principal gustatory nucleus lies at the rostral end of the nucleus solitarius at the pontomedullary junction. Though taste buds on the tongue and pharynx have a rapid turnover, unlike the olfactory receptor cells, they are not true neurons that form synapses within the central nervous system (Kasowski et al., 1999). Despite their wide neuroanatomical separation, olfaction and taste are functionally closely related because both discriminate specific molecules in water or air. Both taste

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and olfactory responses are elicited in humans by electrical stimulation of the insula (Mazzola et al., 2017).

NEUROLOGIC AND NEUROPATHOLOGIC MANIFESTATIONS OF ABNORMAL OLFACTORY BULBS The identification of a genetic mutation denotes the etiology of most cerebral malformations, but insight into mechanisms of pathogenesis is best provided by a knowledge of normal developmental neuroanatomical processes and maturation of both individual neurons and of organization of neural tissues. The olfactory bulb is not exceptional in this regard and can become dysmorphic as with other parts of the brain, though it has been less well studied neuropathologically (Sarnat and Yu, 2016). Apart from total aplasia of the olfactory bulb and tract (e.g., alobar holoprosencephaly), the olfactory bulbs may be present but hypoplastic with a reduction of total neurons and synaptic glomeruli and imprecise histologic lamination (e.g., septo-optic-pituitary dysplasia). Fusion of the olfactory bulbs of the two sides also can occur (Fig. 3.5; Sarnat and Yu, 2016). In hemimegalencephaly (HME), the olfactory bulb of the enlarged cerebral hemisphere may be greatly hyperplastic and dysplastic, including an abnormal deep longitudinal groove on the ventral surface (Robain et al., 1988; Sarnat and Yu, 2016). The normal olfactory bulb forms neither fissures nor sulci. Megalocytic dysplastic neurons are seen in the olfactory bulb in tuberous sclerosis complex (TSC) as well as in hemimegalencephaly, both being disorders of the mTOR signaling pathway. Hamartomata similar to cortical tubers may form in the olfactory bulb in TSC (de León et al., 1988; Feliciano et al., 2012). There are no neuropathologic differences between isolated HME and HME associated with epidermal nevus syndrome and other neurocutaneous syndromes, including the olfactory bulb (Nakashima et al., 1984; Sarnat et al., 2012; Flores-Sarnat, 2016). In a case of epileptogenic frontal lobe focal cortical dysplasia type II, the adjacent olfactory bulb was unilaterally greatly enlarged (Minami et al., 2014). In neurocutaneous melanocytosis, another neurocristopathy, the olfactory bulb may be heavily infiltrated by both amelanotic and pigmented melanocytes at an early age (Flores-Sarnat, 2013; Sarnat and Flores-Sarnat, 2017). Supernumerary olfactory bulbs are demonstrated by MRI in some non-TSC and non-HME malformations (Levy et al., 2012) but have not yet been confirmed neuropathologically. Kallmann syndrome is agenesis of the olfactory bulbs with anosmia and hypogonadotropic hypogonadism due to a mutation in the gene PROKR2 that encodes the

Fig. 3.5. Olfactory bulb dysgenesis in a 20 weeks F fetus. Gross: absent R olfactory bulb; hypoplastic L bulb; R anophthalmia, absent R optic nerve; malformed R ear and side of mouth; R cerebral hemisphere smaller than L; brain weight 32.7 g (control 50 g); fused gyri R > L occipital lobes; cardiac VSD. (A) Bilobar or fused, poorly formed and hypoplastic olfactory bulbs with recognizable histologic architecture. Mitral neurons are not well aligned, many being displaced and disoriented. In the area of fusion of the two olfactory bulbs, continuous immature granular cells form a single sheet without concentric layering. H&E. Synaptophysin disclosed a paucity of synaptic glomeruli and less activity than expected for gestational age in the mitral cell layer and neuronal nuclear antigen (NeuN) showed delayed granular cell maturation (not illustrated). Reproduced with permission from Sarnat, H.B., Yu, W., 2016. Maturation and dysgenesis of the human olfactory bulb. Brain Pathol 26, 301–318.

protein prokins. Deficient gonadotropin-releasing hormone (GnRH) is demonstrated in Kallmann syndrome and in olfactory bulb agenesis from a variety of causes. By contrast with the hypothesis of failed embryonic migration of GnRH cells from the nasal epithelium to the forebrain, the large number of these neuroendocrine cells normally found in the fetal hypothalamus, particularly the preoptic nuclei, are sparse or absent from the hypothalamus in the arrhinencephaly of CHARGE syndrome, as well as in holoprosencephaly and other cerebral dysplasias with agenesis of the olfactory bulbs (Chalouhi et al., 2005). The CHD7 gene mentioned earlier is also of major importance. A murine genetic model of CHARGE syndrome also exhibits olfactory bulb agenesis (Layman et al., 2009). Homozygous mutations of Chd7 in mice are linked to neural stem cell proliferation in the olfactory bulb (Bergman et al., 2011), evidence that Kallmann syndrome is a mild form of CHARGE. A genetic relation exists between 22q11.2 deletion Kallmann and CHARGE syndromes (CorstenJanssen et al., 2013). Both are associated with hypoplasia/dysplasia of the olfactory bulbs and, often, with an

OLFACTORY DEVELOPMENT impaired sense of smell (Chalouhi et al., 2005; Pinto et al., 2005; Blustain et al., 2008; Layman et al., 2009; Bergman et al., 2011, 2012), reproducible in a murine model (Bergman et al., 2010). In one cohort of Kallmann syndrome that included 201 female and 85 male adults with gender-matched controls, 31.5% were anosmic and 33.6% were hyposmic, and 34.9% had normal olfactory perception (Lewkowitz-Shpuntoff et al., 2012). Arrhinencephaly in Kallmann syndrome also is associated with anomalies of the ethmoid bone that can be demonstrated by computed tomography (Maione et al., 2013). Other genetic syndromes in which brain with dysplasia of the olfactory bulb or epithelium may occur include TUBA1A mutations (Myers et al., 2015), Prader–Willi syndrome (Khor et al., 2016), Bardet–Biedl syndrome (Suspitsin and Imyanitov, 2016), and various congenital heart malformations (Panigrahy et al., 2016). In Waardenburg syndrome due to SOX10 mutations, 7 of 8 patients were anosmic, and arrhinencephaly was demonstrated by imaging (Elmach-Bergès et al., 2013). Aneuploidies often involve hypoplasia or dysplasia of the olfactory bulbs with variable clinical hyposmia (Park et al., 1995; Kovaleva and Mutton, 2005; Balwan et al., 2008). Olfactory perception may actually be enhanced or made hypersensitive postnatally in the fetal alcohol spectrum (FAS) disorder; this effect also is expressed in behavioral and neural responses to alcohol and is ameliorated by naltrexone administration (Youngentob et al., 2012).

Olfactory auras Whereas the origin of olfactory auras in medial temporal lobe epilepsy is usually attributed to the amygdala, insula, or entorhinal cortex, and these sites are potentially epileptogenic, evidence from adults that the olfactory bulb itself may be the primary source of olfactory auras was presented in 1954 by Penfield and Jasper (1954) and this hypothesis has recently received additional support (Sarnat and Flores-Sarnat, 2016b). Whether immaturity of the olfactory bulb of the human neonate precludes generating the paroxysmal activity that is postulated to occur in the adult is unknown. Personal studies cited here were carried out and approved by the Conjoint Research Health Ethics Committee of the University of Calgary and Alberta Health Services.

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FURTHER READING Brunjes PC, Illig KR, Meyer EAA (2005). A field guide to the anterior olfactory nucleus/cortex. Brain Res Rev 50: 305–335. Nakahashi M, Sato N, Yagishita A et al. (2009). Clinical and imaging characteristics of localized megalencephaly: a retrospective comparison of diffuse hemimegalencephaly and multilobar cortical dysplasia. Neuroradiology 51: 821–830. Sarnat HB, Flores-Sarnat L, Trevenen CL (2010). Synaptophysin immunoreactivity in the human hippocampus and neocortex from 6 to 41 weeks of gestation. J Neuropathol Exp Neurol 69: 234–245.