J. Cell Sd. 78, 283-310 (1985)
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Printed in Great Britain © The Company of Biologists 1985
GENESIS OF CILIA AND MICROVILLI OF RAT NASAL
EPITHELIA DURING PRE-NATAL DEVELOPMENT
I. OLFACTORY EPITHELIUM, QUALITATIVE STUDIES *
BERT P H . M. MENCOf AND ALBERT I. FARBMAN
Department of Neurobiology and Physiology, O.T. Hogan Building,
Northwestern University, Evanston, IL 60201, U.SA.
SUMMARY
Rat foetuses from intra-uterine days E13 through E22 (day before parturition) and adults were
used for a qualitative electron-microscopic investigation of the development of ciliated/microvillous
surfaces of the olfactory epithelium. In the E13 and most of the E14 embryos the epithelial surface
is not yet characteristically olfactory. Apical cell profiles show primary cilia. These can arise at the
epithelial surface or below. From E14 onwards the epithelial surface acquires olfactory characteristics. Dendritic endings of the olfactory receptor cells can be found amidst microvillous profiles of
supporting cells. Either cell type may bear primary cilia. From E16 onwards the receptor cells sprout
multiple olfactory cilia, but cells with primary cilia are found throughout pre-natal development.
These primary cilia are, at least for a while, retained during the formation of the secondary cilia.
Primary cilia always have distinct necklaces at their base. Otherwise, especially with respect to their
tips, their morphology can vary. Originally they have expanded tips (up to E14); later on such wide
tips are no longer encountered (E16 and E17). Primary cilia of receptor cells never have wide tips.
Appreciable numbers of endings with tapering olfactory cilia are discerned around E18 and especially E19.
Throughout pre-natal development posterior/superior parts of the septal olfactory epithelium are
more precocious than anterior/inferior parts, in particular in the region of transition with the
respiratory epithelium. This advance in development includes total densities of dendritic endings
of olfactory receptor cells, densities of multiciliated endings alone and lengths of supporting cell
microvilli. This difference is discussed with respect to the topography of the olfactory epithelial
surface in adult animals.
In addition to the systematic topographic variation, a number of more local, apparently notsystematically distributed, topographic variations present during development are described. Most
of these also occur in adult animals and they include heterogeneity in length of supporting cell
microvilli and the presence of patches of supporting cells with rounded apical protuberances, of
patches displaying dendrites with polyaxonemes rather than individual cilia and of scattered atypical
cells (neither typical olfactory receptor nor olfactory supporting cells). At their surfaces such
atypical cells can resemble inner-ear hair cells. Relative to olfactory receptor and supporting cells
there are only very few atypical cells.
Finally, we describe some differences in the appearance of membrane surfaces of olfactory cilia
and supporting cell microvilli as a function of development.
INTRODUCTION
It is generally assumed that the apical cilia extending from the dendrites of olfactory
receptor cells are responsible for the reception and initial transduction processes of
• This series is dedicated to the memory of Bert Menco's father, Mr Ad. Menco, who died on July
12, 1984.
j" Author for correspondence.
Key words: olfactory epithelium, cilia, primary cilia, microvilli, pre-natal development,
electromicroscopy.
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smells (Holley & Mac Leod, 1977; Mair, Gesteland & Blank, 1982; Menco, 1983).
Electrophysiological studies on rat foetuses have shown that initially (around E17 and
El8) olfactory receptor cells are non-selectively responsive to a wide range of odorous
stimuli. Later on in development (around E19) most of the cells are responsive to a
limited number of stimuli (Gesteland, Yancey & Farbman, 1982). It is reasonable to
assume that a morphological change in cilia of olfactory receptor cells may accompany
the physiological changes in behaviour of these cells. The main objective of this and
the accompanying paper (Menco & Farbman, 1985a) is to study in detail the morphological events taking place during olfactory ciliogenesis and to consider the functional implications of these events (see also Menco & Farbman, 19856). In the present
paper the emphasis is placed on ultrastructural changes occurring during ciliogenesis
of the olfactory epithelium during pre-natal development, whereas in the accompanying one (Menco & Farbman, 1985a) a quantitative account will be presented. Observations on the formation of microvilli of supporting cells surrounding the olfactory
receptor cells are also included.
There are many studies on the development of olfactory epithelium in various
orders of vertebrates (see Breipohl & Fern&ndez, 1977; Breipohl, Mestres & Meller,
1973;Breipohl&Ohyama, 1981; Cuschieri& Bannister, 1975a,6; Kerjaschki, 1977;
Kerjaschki & Horandner, 1976; Klein & Graziadei, 1983; Mulvaney & Heist, 1971;
Noda & Harada, 1981; Pyatkina, 1982; Smart, 1971; Taniguchi, Taniguchi &
Mochizoki, 1982; Waterman & Meller, 1973; Yamada, 1983). However, none of these
were focused on the events occurring during the formation of the olfactory cilia.
An additional objective of this part of the study was to investigate the topography
of the olfactory epithelial surface during development. Anatomical (Allison & Turner
Warwick, 1949; Breipohl, Moulton, Ummels & Matulionis, 1982; Breipohl &
Ohyama, 1981; Kolb, 1979; Mackay-Sim & Patel, 1984; Menco, 1977; Yamamoto,
1982) and electrophysiological (Daval, Leveteau & Mac Leod, 1980; Erickson &
Caprio, 1984; Mackay-Sim & Shaman, 1984; Mackay-Sim, Shaman & Moulton,
1982; Moulton, 1976; Mozell & Hornung, 1984; Mustaparta, 1971; Thommesen,
1982, 1983; Thommesen & D0ving, 1977) investigations demonstrated that the olfactory epithelial surface in adult vertebrates is not homogeneous.
Apart from a morphological study on the mouse (Breipohl & Ohyama, 1981), no
topographical developmental data have previously been published. Therefore, we
decided to repeat, in part (for the rat) and extend the above study. Various regions
of the rat's septal olfactory epithelium were compared from day to day in embryos
from the 14th day of gestation up to the day before birth and in adults. In addition
we investigated more local variations taking place in the olfactory epithelial surface
during development.
MATERIALS AND METHODS
Materials and specimen preparation
Pregnant Sprague-Dawley rats were obtained from Holtzman Co. (Madison, WI, U.S.A.). They
were killed by decapitation from day 14 (E14) through day 22 (E22) of gestation for scanning
Olfactory epithelial development. I
285
electron microscopy (E1 is the day that the dams are sperm-positive; E23 = PI is the day of birth), and
from E13 through E17 for transmission electron microscopy. Foetuses were removed from the uterus,
decapitated, and after a quick rinse with phosphate-buffered saline (PBS), whole noses (upper jaw
from tip of nose until just rostral to the eyes) were placed in 5 % glutaraldehyde/4 % formaldehyde,
i.e. Karnovsky's (1965) fixative in 0-1 M sodium cacodylate buffer (pH 7-4, room temperature). When
specimens from all foetuses of the dam in question were collected in that way, nasal septa containing
olfactory and respiratory epithelial surfaces (Menco & Farbman, unpublished) were dissected out for
further aldehyde fixation. The total fixation time was about 2 h. Six to ten embryos were collected from
each dam, and for each embryonic age group one dam was used, though embryos from different dams
were used for scanning and for transmission electron microscopy. Septa of the dams and of two male
adults served as comparison tissue for scanning electron-microscope observations.
After rinsing in 0-1 M sodium cacodylate buffer (pH7-4) the samples were post-fixed in 1 %
OsC>4 for 1 h (also in cacodylate buffer). Specimens to be used for scanning electron microscopy
were, after a rinse in water or buffer, dehydrated through a graded series of ethanol, whereas samples
to be used for transmission electron-microscopic observation were block-stained with 3 % uranyl
acetate in 50% ethanol for 1 h before dehydration. For transmission electron-microscopy the samples were then infiltrated with Epon through a graded series of propylene oxide/Epon (1:1, 1:2 and
Epon alone, each l h ) . The samples were subsequently embedded in Epon (60 °C, 1 day) and
sectioned in coronal planes with a diamond knife. Thin sections were stained with 3 % uranyl acetate
(in distilled water) and counterstained with lead citrate (Reynolds, 1963). The sections were
examined at 80 kV in a JEOL JEM 100CX scanning-transmission electron microscope equipped
with a rotating stage and a eucentric goniometer.
For scanning electron microscopy the specimens, after dehydration, were critical-point dried
from CO2 and sputter-coated with gold/palladium (in Polaron E3000 and ESI00 devices, respectively) and either examined in the scanning mode of the JEM 100CX electron microscope at 40 kV,
using the ASID attachment, or in a JEOL JEM 35CF scanning electron microscope at 35 kV.
Topography
From each embryonic age group one animal served as a sample for a systematic topographic study
of the development of the septal olfactory epithelial surface from region to region, using scanning
electron microscopy only. The same was done for one adult. The nasal septa to be mapped were
photographed at low magnifications (20-50 times) and the outlines of the samples were drawn.
Twenty to thirty areas were photographed at two or sometimes three magnifications: the lower ones
ranged from X300 to X2000, the medium ones from X5000 to X9000 and the high (optional) ones
from X 15 000 to X 60 000. The photographed areas were numbered and these numbers were mapped
in the outlined cartoons of the samples. When all photographs of all age groups were taken they were
laid out according to the mapping. Since in all cases the mapping included the respiratory
epithelium, and most often also the transitional region between the olfactory and the respiratory
epithelium, it was easy to determine which sides of the septa were posterior/superior (i.e. closest
to the cribriform plate) and which anterior/inferior (i.e. closest to the respiratory epithelium).
Various features were indicated in newly drawn outline maps and these maps were compared for the
various age groups from region to region. These maps, together with the numbered photographs,
were used for compiling the topographic part of the present study.
RESULTS
E13 and El4 embryos; general appearance
The putative olfactory area is found towards the most dorsal region of the nasal
cavity. At E13 this region is characterized mainly by the presence of elongated cells.
Nuclei with mitotic figures are amply present in the vicinity of the epithelial surface.
Primary cilia (for the distinction between primary and secondary cilia see the
Discussion) are rather frequently found near the centre of the apical surfaces of the
cells (Fig. 1).
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In E14 embryos two surface appearances can be discerned, hereafter called El4.1
and E14.2. E14.1 animals included six out of the eight foetuses from the same dam
used for scanning electron microscopy. As in E13 embryos, the surfaces have not yet
acquired a recognizable olfactory epithelial morphology. Areas with knob-shaped and
flat apical cell profiles (Fig. 2) and areas with mainly flat apical cell profiles (Fig. 3)
alternate. The area of Fig. 3 occurred slightly more anterior and inferior than that of
Fig. 2. Most of the otherwise fairly bare surface profiles bear primary cilia.
In contrast to E14.1, the dorsal nasal cavity of E14.2 embryos displays surface
features reminiscent of those of the olfactory epithelium (Fig. 4). Individual (Figs 4,9)
and clusters (Figs 4, 7) of knob-shaped structures, with an appearance resembling that
of olfactory dendritic endings, are found amidst olfactory supporting cells with
microvilli. Both cell types bear primary cilia.
E14.1 and E14.2 can also be distinguished in lateral aspect. The undifferentiated
epithelium of E14.1 is characterized by the presence of numerous slender processes,
often occurring in groups. Nuclei are found at various levels along these processes,
though more frequently in basal than in distal regions (Fig. 5). The differentiated
epithelium of E14.2 shows fewer slender processes and begins to exhibit the layering
of nuclei typical of the olfactory epithelium, i.e. the supporting cell nuclei are distal
to those of the receptor cells (Fig. 6).
In addition to their primary cilia the receptor cells have numerous centrioles. The
Fig. 1. Transmission electron micrograph of the surface of the putative olfactory
epithelium of an E13 rat embryo. Primary cilia (arrows) are expressed at the surfaces of
some cells. Slender, rather electron-opaque cell processes alternate with larger, rounded
cells displaying mitotic figures. X6000.
Fig. 2. Scanning electron micrograph of the posterior/superior surface of the putative
olfactory epithelium of the septum of an E14 (E14.1) rat embryo. The appearance of the
epithelial surface is not yet characteristic of olfactory epithelium, in that there is no clear
distinction in surface profiles apart from that some are more globular and others are flat.
Areas like these were not seen in E13. Most cells bear primary cilia (arrows). X6000.
Fig. 3. Scanning electron micrograph of the putative olfactory epithelium of an E14.1
embryo. The area shown here, which is obtained from a more anterior/inferior region than
that in Fig. 2, demonstrates mainly rather flat cell surface profiles. Areas like these were
seen in E13. Most cells bear primary cilia. The cilium marked with an arrow is shown at
a higher magnification in Fig. 10. X6000.
Fig. 4. Olfactory epithelial surface of an E14 (E14.2) embryo. Typical knob-shaped
endings of dendrites of olfactory receptor cells alternate with microvillous surface profiles
of olfactory supporting cells. Both bear primary cilia (small arrows: those of receptor cells;
large arrows: those of supporting cells). Surface profiles of some supporting cells are
virtually completely covered with short microvilli; others have hardly any. X6000.
Fig. 5. Lateral aspect of undifferentiated olfactory epithelium (E14.1). Numerous slender processes project onto the epithelial surface, resulting in an appearance like that shown
in Fig. 2. Nuclei are mainly found near the base, but also more distally near the epithelial
surface. X1400.
Fig. 6. Lateral aspect of olfactory epithelium of an E14.2 embryo. The slender processes
of E14.1 (Fig. 5) are no longer present and are replaced by ovoid supporting cell nuclei,
which are beginning to be organized in a layer above that of the receptor cell nuclei.
Dendrites (arrows) surround the supporting cells. X1300.
Olfactory epithelial development. I
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distal parts of the dendrites have some mitochondria and free ribosomes (Figs 7, 9,
13, 14; compare with Figs 42-45), though fewer ribosomes than the surrounding
supporting cells. Supporting cell apices show a remarkable heterogeneity. Some are
virtually bare whereas others are covered with microvilli; both appearances may occur
side-by-side (Fig. 4).
El 3 and El 4 embryos; primary cilia
Primary cilia can be found at the epithelial surfaces (Figs 2-4, 7, 8, 10, 11, 14; see
also Figs 44, 45) and below Figs 9, 14; see also Fig. 43). Within the cell apices one
finds usually one, but sometimes two, diplosomes (Fig. 8). Hence, although the cells
usually give rise to only one primary cilium, two can be generated. Primary cilia are
frequently encircled by a membrane-lined cytoplasmic annulus (Fig. 9), indicating
that they 'escaped' the cell, regardless of whether they face the extra- or intra-epithelial
environment (e.g. see Fig. 9). They clearly display necklace regions at their base (Fig.
10), and tend to taper above that region (Figs 10, 11, 13, 14). Axonemes of primary
cilia are devoid of dynein arms and other microtubule-attached structures and their
central core is usually rather electron-opaque (Fig. 12 for E14.1). Primary cilia often
show reduced numbers of microtubules immediately above the necklace region, irrespective of whether they are found at the epithelial surf ace or sub-surf ace (Figs 13, 14).
Primary cilia of all cells at E13 and E14.1, and of supporting cell profiles of E14.2,
Fig. 7. A cluster of two dendritic endings of olfactory receptor cells in an E14.2 embryo.
One of the endings demonstrates the presence of a primary cilium terminating in an
electron-opaque, in this case not-expanded, tip. The supporting cell apex nearby displays
a basal body of a primary cilium (arrow). X45000.
Fig. 8. Two diplosomes in a cell of the putative olfactory epithelium of an El3 embryo.
One of the diplosomes gave rise to a primary cilium. Otherwise the cell has numerous
ribosomes and some endocytotic vesicles. X23 000.
Fig. 9. Olfactory epithelial surface of an El4.2 embryo. A supporting cell shows a subsurface epithelial primary cilium surrounded at its basal body region by a cytoplasmic
ribosome-free halo. The base of the cilium is enclosed within a membrane-lined cytoplasmic annulus. The cilium terminates, in contrast to the primary cilium of the olfactory
receptor cell shown in Fig. 7, in an expanded electron-opaque tip. The presence of a
primary cilium in another supporting cell is indicated by a basal body (arrow). The
dendritic ending of the olfactory receptor cell contains some mitochondria, centrioles and
ribosomes. The latter structures are more abundant in the supporting cells, which also
display some short microvilli. X30000.
Fig. 10. A primary cilium of the putative olfactory epithelial surface of an E14.1 embryo.
At its base the cilium clearly displays some necklace strands (arrow), above which the
cilium tapers to terminate in a club-shaped tip. X107 000.
Fig. 11. Primary cilium of the putative olfactory epithelium of an El3 embryo in a
longitudinal section. In the electron-opaque expanded tip of the cilium next to the ciliary
membrane, a series of evenly spaced granules can be discerned (arrow). The cilium tapers
immediately after its necklace region and the microtubules run all the way into the ciliary
tip. X44000.
Fig. 12. Cross-section through a primary cilium of an E14.1 embryo at or near its necklace
region. The cross-section displays no axonemal features apart from nine sets of doublet
microtubules. The outer leaflet of the ciliary membrane is more electron-opaque than the
inner leaflet. X100 000.
Olfactory epithelial development. I
Figs 7-12
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terminate in rather wide, club-shaped, electron-opaque tips (Figs 9-11, 15). The tips
of the primary cilia of the receptor cells are less wide (Fig. 7). In contrast to the
microtubules of olfactory cilia, which terminate as singlets (Seifert, 1970; Menco,
1977), the tips of primary cilia contain singlets and/or doublets (Fig. 15).
El5 through E22 and adult; topography
In E15 embryos the surface of the olfactory epithelium resembles that of E14.2 in
that most dendritic endings bear just a single cilium or none (Figs 16—18). From El6
onwards appreciable numbers of receptor cells become multiciliated (Figs 19, 21).
Receptor cells with numerous, fairly long, cilia are only occasionally present at E16
and E17, but in greater numbers at E18 (Fig. 22). The cilia continue to increase in
length and, at E19, rather long tapers are discerned (Figs 23, 24). At E20 the entire
course of most individual cilia can no longer be followed (Fig. 25 for an E21 embryo).
Nevertheless, areas of endings with mainly primary cilia are still seen throughout
prenatal development (Fig. 26 for an E22 embryo). None of the embryos showed the
extensive carpets of tapering olfactory cilia typical of adults (Figs 27, 41, 49).
Concomitant with the above pattern of cilium outgrowth, microvilli of supporting
cells tend to increase in length from around E18 onwards (Figs 16—25). However, at
least up to E19 (Menco & Farbman, 1985a), supporting cells also bear primary cilia
(Figs 16, 17, 20, 21, 44, 46).
Apart from the overall developmental pattern of the olfactory epithelial surface
outlined above, there is a systematic topographical variation throughout development.
Fig. 13. Olfactory dendritic ending of an E14.2 embryo. Tilting (SO °) of the section was
required to enhance the profile of the primary cilium in cross-section. The cilium is clearly
continuous with the dendritic ending (arrow). The reduced numbers of microtubules
suggest that the cilium appears to taper immediately after its necklace region. The ending
displays a fair number of centrioles. X 60 000.
Fig. 14. Developing dendritic ending (sub-surface) of an E14.2 embryo containing
numerous centrioles and, in cross-section, an enclosed primary cilium with a reduced set
of microtubules. X 30 000.
Fig. 15. Stereo-pair (tilt: SO°to 62°) of the tip of aprimary cilium (E14.2 embryo). One
set of doublet (arrow) microtubules and one singlet microtubule run into this tip, which
has an electron-opaque matrix. X70000.
Fig. 16. Olfactory epithelial surface of an E15 embryo in the posterior/superior aspect of
the nasal septum. The density of receptor cell endings is fairly high, and most of these
endings bear single primary cilia. The surface appearance of the surrounding supporting
cells is heterogeneous. Most of these also bear primary cilia (arrow). X6000.
Fig. 17. Olfactory epithelial surface of the same embryo as shown in Fig. 16, but from a
more anterior/inferior part of the nasal septum. Though supporting cell apices are as
heterogeneous as those in the previous figure, the density of receptor cell dendritic endings
is considerably lower. X6000.
Fig. 18. Olfactory epithelial surface of the posterior/superior part of the nasal septum of
an El5 embryo. A cell type with large microvilli and a cluster of receptor cell endings and
supporting cell microvilli can be seen. Supporting cells also have primary cilia (arrow).
X14 000.
Fig. 19. Epithelial surface of an E16 embryo. Endings cluster and begin to become
multiciliated. X6000.
Olfactory epithelial development. I
Figs 13-19
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Figs 20-27
Olfactory epithelial development. I
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In general, receptor and supporting cells in posterior/superior regions of the nasal
septum are further advanced in development than those located more anteriorly/
inferiorly. At least up to E19 receptor cell dendritic endings tend to occur in higher
densities in the former (Figs 16, 20) than in the latter (Figs 17, 21) regions. Around
E17 more multiciliated endings can be discerned posteriorly/superiorly (compare
Figs 20 and 21). Finally, at least up toE19, supporting cell microvilli tend to be longer
in those regions than in more anterior and inferior regions (Figs 20, 21, 23, 24).
The transitional area with the respiratory epithelium is found at the most anterior/inferior part of the olfactory epithelium. Throughout development the transition is either abrupt (Figs 28-31) or gradual (Fig. 32). In contrast to the respiratory
part of the transitional area (Menco & Farbman, unpublished), the development of
the olfactory part of the transitional area tends to lag behind that of the olfactory
epithelial surface elsewhere: cilia and microvilli are shorter (Figs 30, 33). Even in the
adult one finds many endings that did not properly develop ciliary tapers (stereo-pair
of Fig. 31). Receptor cells also tend to 'cross' the transitional area; despite the transition, olfactory receptor cell dendritic endings are, from E1S on and at least up to
E18, occasionally encountered within respiratory areas (Fig. 34).
In addition to the systematic topographical developmental variation described
above, various topographical features occur in an apparently random manner
throughout development. From E14 and at least up to E20, over the epithelial surface
Fig. 20. Olfactory epithelial surface of an E17 embryo in a rather posterior/superior
region of the nasal septum. The density of dendritic endings is fairly high and many
endings are multiciliated. Some endings are further developed than surrounding ones in
that they have more cilia. Supporting cell surfaces tend to be fully covered with microvilli,
but remain heterogeneous in appearance. X3000.
Fig. 21. Olfactory epithelial surface of the same E17 embryo as in Fig. 20, but in a more
anterior/inferior region. The density of dendritic receptor cell endings is lower and the
endings tend to bear fewer cilia than in the area depicted in Fig. 20. Surrounding supporting cells are more rounded and are often bare instead of covered with microvilli as those
seen in Fig. 20. Arrows indicate supporting cell microvilli. X3000.
Fig. 22. Olfactory epithelial surface of an E18 embryo. The cilia begin to taper in appreciable numbers. Supporting cells with long and with shorter microvilli are present. X6400.
Fig. 23. A posterior/superior region of the septal olfactory epithelium of an E19 embryo.
The epithelial surface is mainly covered by a heterogeneous blanket of supporting cell
microvilli through which some olfactory cilia emerge. Several of the ciliary processes
belong to axonemal aggregates, rather than to single cilia (arrows). X3000.
Fig. 24. A more anterior/inferior area of the septal olfactory epithelial surface of the same
E19 embryo asthatintheprevious micrograph. Supporting cell microvilli are less long and
less heterogeneous than in Fig. 23. Olfactory receptor cell dendritic endings with
numerous, rather long, cilia can be discerned next to ones with short cilia (arrow). X 3000.
Fig. 25. Olfactory epithelial surface of an E21 embryo. Receptor cell cilia and supporting
cell microvilli intermingle. X5000.
Fig. 26. Olfactory epithelial surface of the nasal septum of an E22 embryo. In areas, one
can still find receptor cell dendritic endings with primary cilia (arrows). X5000.
Fig. 27. Olfactory epithelial surface of the nasal septum of an adult rat covered with
tapering parts of olfactory cilia. A cell with numerous rigid-appearing microvilli (or cilia?)
can be discerned in between the layer of tapers of olfactory cilia. X10000.
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Figs 28-33
Olfactory epithelial development. I
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and in most developmental age groups, receptor cell dendritic endings frequently
cluster (Figs 4, 7, 18-21, 28, 35, 42). Numbers of cilia per ending are highly
variable throughout development, but, especially earlier on, some endings with
many cilia are found amidst a majority of endings with considerably fewer (Fig.
28). From E18 on and up to the adult stage we rather frequently saw endings having
polyaxonemes distally, i.e. axonemal features of several cilia are contained within
a common membrane; necklaces of individual cilia can often be clearly distinguished
in such polyaxonemes. Polyaxonemes tend to occur in randomly distributed patches
(Figs 36, 37).
Supporting cells, too, display randomly distributed topographical variations. From
cell to cell and area to area the distribution and lengths of the supporting cell microvilli
differ considerably throughout development (Figs 4, 16, 17, 20-24, 38) and also in
the adult (Fig. 41). It may well be that in some areas the supporting cells comprise
a heterogeneous population of various types of cells (Figs 33, 38). At least from E18
onwards rather large scattered areas display supporting cells with apical globular
protuberances (Figs 29, 39). In other areas (from E20 onwards) various types of
inclusions are present amidst the olfactory cilia and supporting cell microvilli (Figs
40, 41). Fig. 41 (adult) shows very well how heterogeneous a small patch of epithelial
surface may appear.
Fig. 28. Transitional region between the olfactory (left) and the respiratory (right)
epithelium in the septum of an E16 embryo. The transition is rather abrupt. Surface
profiles of the supporting cells are more rounded than those of the respiratory cells. Both
cell types bear primary cilia (arrows), though respiratory cells in particular are otherwise
fairly bare. Most receptor cells also bear single cilia, but some have rather large numbers
of cilia. X2000.
Fig. 29. Transitional region between olfactory (left) and respiratory (right) epithelium in
the nasal septum of an E19 embryo. As in the previous micrograph the transition is rather
abrupt. The respiratory epithelium is characterized by many more ciliated cells than in
E18 (compare with Fig. 32). An area with supporting cells with globular apical
protuberances is present (arrow; see also Fig. 39). X1200.
Fig. 30. Adult olfactory epithelial surface near that of the respiratory epithelium (far
right). The olfactory cilia can be followed over fairly long distances and do not completely
cover the epithelial surface in this area. They show numerous vesicular expansions.
X8000.
Fig. 31. Stereo-pair of the region of transition between the olfactory and respiratory
epithelium of the nasal septum of an adult rat. The respiratory epithelium displays rather
distinct ciliated bands, whereas the olfactory epithelium is more patchy, and present in a
different plane (more elevated, as can be seen in stereo). Most receptor cell dendritic
endings have only short cilia in this area. X 1000.
Fig. 32. Transitional region between olfactory and respiratory epithelium in an E18
embryo. The transition is rather diffuse; amidst a heterogeneous population of
microvillous cells, scattered ciliated olfactory receptor cells (small arrows) and ciliated
respiratory cells (large arrow) can be discerned. X2000.
Fig. 33. An olfactory receptor cell dendritic ending of an E20 embryo in the area of
transition to the respiratory epithelium. The olfactory cilia are rather short; the ending is
surrounded by two types of microvillous cells, the microvilli of which differ in appearance
from those of the ciliated respiratory cell nearby. X9000.
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Figs 34-41
Olfactory epithelial development. I
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E15 through E22 and adult: intracellular features, ciliary and microvillous
ultrastructure
At E16 (Fig. 42) and to a lesser degree at E17 (Figs 42-45) the endings still
demonstrate the presence of ribosomes. In contrast to the situation in E14.2 (Fig. 7)
mitochondria are virtually absent. Endocytotic, most probably coated, vesicles
(Menco, 1984) occur more often in E17 than E16.
Up to E17, primary cilia of sensory (Fig. 43) and supporting cells (as Fig. 9) can
still be present below as well as at the epithelial surface. They still tend to taper
immediately after their necklace region (Figs 16—18, 44); their axonemal microtubules sometimes twist (Fig. 44). In proximal regions these axonemes resemble
those of E13 and E14 primary cilia (Fig. 12). The large electron-opaque tips, typical
of primary cilia at earlier stages (Figs 9, 10, IS), are no longer encountered in E16 and
E17 embryos. The tips of primary cilia of olfactory receptor and supporting cells
resemble those of olfactory cilia of adult animals (Lidow & Menco, 1984; Menco,
1977; Menco, 1984). With the apparent loss of microtubules beyond the necklace
region, primary cilia may be enclosed by nearby (supporting) cells (Fig. 45).
Even when the receptor cells have only primary cilia they may bear short microvilli
in addition to those cilia (Fig. 46 for E15 and Fig. 47 for E17; necklaces of recently
formed olfactory cilia can be seen especially clearly in the latter stereo-micrograph).
From E18 onwards, and in some instances already at E16 and E17, we observed that
the surfaces of supporting cell microvilli are rather corrugated, whereas those of the
olfactory cilia are smoother (Fig. 48). We found the same distinction between surfaces
Fig. 34. A well-developed dendritic ending of an olfactory receptor cell of an E18 embryo
in the putative respiratory epithelial area. X3000.
Fig. 35. Stereo-pair of a cluster of multiciliated receptor cell endings (E17) amidst supporting cell apices with short microvilli. The cilia are short and vary more in length among
different than on individual endings. X6500.
Fig. 36. An area with several polyaxonemes (E21), i.e. the ciliary membranes are fused
above necklace regions (arrows). Distally, at least some of the cilia become separate again.
X8200.
Fig. 37. An area of a possible wound (adult) with numerous polyaxonemes. Beyond the
displayed region the area is surrounded by normal-appearing tapering parts of olfactory
cilia. Many of the aggregate endings display ciliary necklaces (arrows) but lack properly
developed cilia. X14 000.
Fig. 38. A region of supporting cells with rather long microvilli (E19) (left) borders on
one of supporting cells with considerably shorter microvilli (right). Olfactory receptor cell
dendritic endings with primary cilia can be discerned among multiciliated ones. X4000.
Fig. 39. Septal olfactory area of E18 embryo demonstrating supporting cell profiles with
globular apical protuberances. X4000.
Fig. 40. A rather heterogeneous patch of olfactory epithelium in an E20 embryo. The
endings display cilia with granular (small arrow) and cilia with smoother membrane
surfaces. Amidst the receptor cell cilia and supporting cell microvilli a 'brain'-shaped
mucous inclusion (large arrow) can be discerned. X6000.
Fig. 41. Numerous large globules alternate with ciliated and microvillous profiles in a
heterogeneous patch of septal olfactory epithelium of an adult rat. X2000.
B. Ph. M. Menco and A. I. Farbman
Figs 42-45
Olfactory epithelial development. I
299
of both structures in the olfactory epithelium of adult frogs (unpublished). From E20
onwards we observed that the surfaces of olfactory cilia themselves may also have a
heterogeneous appearance (Fig. 49).
E15 through E22; atypical cells
More or less directly from the moment that the typical appearance of the olfactory
epithelium is apparent, i.e. E15, atypical cells are occasionally seen at its surface (Fig.
18). The cell type of Fig. 50 is rather frequently encountered in E17 and sometimes
in E16; that of Fig. 51 was only seen in E18. Both types of cells also occur in regions
of putative respiratory epithelium and display one cilium (perhaps a kinocilium) and
numerous slender microvilli. That shown in Fig. 18 only shows slender microvilli. In
other instances the cell type in Fig. 51 was seen to have popped out of the epithelial
surface, thus exposing several circular arrays of microvilli and a single cilium on an
otherwise featureless membrane (not presented). Since all three cell types shown here
are characterized by slender microvilli (Figs 18, 50, 51), they may represent various
developmental stages of the same type of cell. The final (adult) appearance of that cell
could be the one seen in the midst of the olfactory cilia in Fig. 27.
DISCUSSION
The El 4 transformation
During its development the olfactory epithelial surface undergoes a number of
transformations. A major one occurs around E14 when, within a single day (most
probably a considerably shorter period) the whole epithelial surface begins to acquire
ciliated and microvillous features characteristic of the olfactory epithelial surface.
From then on dendritic endings of olfactory receptor cells supplement or replace their
Fig. 42. Transmission electron micrograph of a cluster of dendritic endings of olfactory
receptor cells (El6) with numerous centrioles inside..These have electron-opaque dots in
their centres. The left ending has a fair number of ribosomes, whereas the one on the right
amply displays microtubules. X17 000.
Fig. 43. A cluster of olfactory receptor cell dendritic endings (E17), which have not yet
reached the epithelial surface. In addition to centrioles, the endings have sub-surface
primary cilia (small arrows). A primary cilium of a nearby supporting cell is seen in crosssection (large arrow). X17000.
Fig. 44. Apposing surface profiles in an E17 olfactory epithelium. Many dendrites have
not yet reached the epithelial surface. In all cases the endings have numerous centrioles.
In contrast to apical profiles of the supporting cells, those of the receptor cells have
virtually no ribosomes, whereas microtubules are abundant. Supporting cell (small arrow)
and receptor cell (large arrow) primary cilia demonstrate twisting of axonemal
microtubules. The further developed ending with two cilia has several endocytotic vesicles. X15 000.
Fig. 45. Primary cilium of an E17 embryo olfactory receptor cell enclosed by the distal
part of a supporting cell (arrow). Apart from its basal body and necklace region the cilium
is devoid of all axonemal substructures. The ending has numerous additional centrioles
and some ribosomes and microtubules. Mitochondria are absent. Nearby supporting cells
do have mitochondria and also more ribosomes than the endings. X15 000.
300
B. Ph. M. Menco and A. I. Farbman
Figs 46-51
Olfactory epithelial development. I
301
primary cilia with a set of secondary cilia, which steadily increase in number and
length. The E14 transformation apparently.reflects a change in pattern formation
throughout the epithelial lining of the nasal cavity; it is accompanied by a transformation in the putative respiratory epithelial areas (Menco & Farbman, unpublished).
However, even before the epithelial surface has become olfactory, it already shows a
spatial differentiation (Figs 2, 3).
As first shown by Sauer (1935), and reviewed by Jacobson (1978), nuclei of cells
of neuroepithelia, and also of cells of other pseudostratified epithelia (Sauer, 1936),
migrate from close to the base qf the epithelium to the luminal side, where they divide
(e.g. see Fig. 1). When the cells are not dividing they display solitary cilia (shown in
the diagrams of Sauer (1935) and Jacobson (1978) and electron-microscopy, see, e.g.
Rash, Shay & Biesele (1969)). The presence of single cilia has been demonstrated on
many types of eukaryotic cells during development (listed by Scherft & Daems (1967)
and more recently and extensively by Wheatley (1982)).
Surface profiles with single cilia in E13 and E14.1 probably represent cells that are
not in mitosis. Flat surface profiles most likely belong to dividing cells and/or cells
that will eventually have a wider diameter than receptor cells, i.e. putative olfactory
supporting cells (Figs 1-3). In the latter case such flat profiles usually have solitary
cilia. From the above it can be inferred that the single cilia seen in E13 and E14.1 are
probably primary; their presence, in combination with the general appearance of the
epithelial surface, indicates that the cells have not left the cell cycle.
It is interesting that at an equivalent stage of development in the mouse, i.e. around
E12 (Cuschieri & Bannister, 19756; Noda & Harada, 1981), the number of apical
mitotic figures decreases dramatically (Smart, 1971). This supports our supposition
that the change seen in the 14th day of pre-natal development of the rat, i.e. that
Fig. 46. Dendritic ending of an E16 olfactory receptor cell with a primary cilium and two
short microvilli (small arrows; compare with Fig. 47). Microvilli of surrounding supporting cells are short. One of these cells demonstrates-a primary cilium (large arrow).
X15 000.
Fig. 47. Stereo-pair of a dendritic ending of an E17 olfactory receptor cell with several
cilia, the necklaces of which can clearly be discerned; the cilia narrow immediately above
the necklace region. The ending also displays a short microvillus (arrow). The surface
appearance of the membrane of the ending and of those of the surrounding supporting cell
microvilli are rather similar (compare with Fig. 48). X26000.
Fig. 48. In contrast to the membrane surface of the dendritic endings of the olfactory
receptor cells and their cilia (El9), those of the surrounding supporting cell microvilli are
rather coarse. X18 000.
Fig. 49. Surface of olfactory epithelium with tapers of olfactory cilia (adult). These are
expanded in several regions and either have a smooth or a coarser surface appearance.
X18 000.
Fig. 50. Atypical cells with numerous long microvilli and single cilia (arrows) and otherwise bare apical profiles amidst microvillous cell profiles (E17). A dendritic ending of a
receptor cell can also be discerned. X6000.
Fig. 51. Atypical cell (E18) with a horseshoe-shaped array of rather long microvilli and
a single cilium, surrounded by an olfactory receptor cell and microvillous cells. X9000.
302
B. Ph. M. Menco and A. I. Farbman
occurring between E14.1 and E14.2, denotes a period at which many cells leave the
cell cycle and enter G0-phase.
In contrast to our observations on the rat, mouse (Noda & Harada, 1981) and
hamster (Waterman & Meller, 1973) nasal epithelial surfaces display numerous
microvilli when the epithelium is not yet characteristically olfactory (i.e. no sensory
endings at the surface).
A case for primary cilia
Most features described in this study, i.e. the presence of a basal pouch (Fig. 9),
reduced numbers of micro tubules above necklace regions (Figs 13, 14), twisting of
these microtubules (Fig. 44) and the peculiar shape of the tips (Figs 9, 11, 15), have
been described for primary cilia of many other metazoan cell types (Albrecht-Buehler
& Bushnell, 1980; Fonte, Searls & Hilfer, 1971; Rash et al. 1969; Sorokin, 1968;
Tachi, 1984; Tucker & Pardee, 1982; Wheatley, 1982). Primary cilia are expressions
of centrioles involved in division rather than of later formed basal bodies, which are
merely responsible for the formation of subsequent (or secondary (Wheatley, 1982))
cilia. A characteristic of primary cilia is that their presence is linked to that of
diplosomes (Fig. 8; see references cited above). It is thought (Masuda & Sato, 1984;
Tucker & Pardee, 1982; Tucker, Pardee & Fujiwara, 1979; Tachi, 1984) that primary
cilia form some time in G\ and resorb some time in 5 or Gz. According to these
authors, primary cilia are markers for mitotically quiescent cells, but their formation
is associated with a time in G\ important for growth control.
Primary cilia have also been seen in cells that have left the cell cycle for Go (Tucker
et al. 1979). This may explain why they are present on young olfactory receptor and
supporting cells (Figs 7, 9, 13, 14, 43-46; Furuta, Tsukise, Okano & Sugawa, 1974).
On the receptor cells they are still found just before (Fig. 26) and at birth (Furuta et
al. 1975). Primary cilia are found on basal cells, too (Klein & Graziadei, 1983;
authors' unpublished observations). Primary cilia of receptor cells tend to be wider
over longer distances than those of supporting cells. Moreover, primary cilia of olfactory receptor cells never show the wide expanded tips seen early on in those of
supporting cells (Figs 7, 9, 15). Primary cilia of both cell types lack central
microtubules and microtubule-attached structures, however. Also, the morphology
of the primary cilia in the supporting cell apices changes with development; the
expanded electron-opaque tips seen in E14.2 (Figs 9, 11, 15) were no longer found
in E16 and E17.embryos. In the nasal respiratory epithelium the morphology of
primary cilia was similarly observed to undergo changes during development (Menco
& Farbman, unpublished).
On the following grounds we believe that olfactory receptor cells bear primary cilia
before secondary ones. (1) In addition to their presence at epithelial surfaces, we
encountered solitary cilia fairly deep below the epithelial surface. These cilia are often
at least partially enclosed by the cell from which they sprout (Figs 9, 14, 43; Rash et
al. 1969; Sorokin, 1968; Tucker & Pardee, 1982; Wheatley, 1982; Whiteley &
Young, 1982); they can also be enclosed by cells nearby (Fig. 45). Even when there
were many centrioles we never saw more than one sub-surface cilium. Early genesis
Olfactory epithelial development. I
303
of secondary cilia is also accompanied by apparently enclosed compartments. Several
of these tend to be near the epithelial surface rather than deeper within the cell
(Kalnins & Porter, 1972; Sorokin, 1968). Olfactory knobs which did not yet reach the
surface can also already bear several secondary cilia (Kerjaschki & Horandner, 1976).
(2) The results of the quantitative analysis presented in the accompanying paper show
that the distribution of olfactory cilia per receptor cell dendritic ending, as a function
of pre-natal developmental age, is highly concentrated around one cilium (Menco &
Farbman, 1985a). (3) Although it is hard to detect which centrioles and basal bodies
belong to diplosomes and which will eventually give rise to non-primary cilia, when
other centrioles than those of the diplosomes are present (Figs 7, 9, 13, 42-45), we
occasionally saw that the primary cilium was at a distance from the centrioles that
eventually give rise to secondary cilia (Fig. 14). (4) Primary cilia are found on many
metazoan cells during development (Wheatley, 1982) including sensory and supporting cells of vomero nasal olfactory epithelia where cilia are usually lacking (Bhatnagar,
Matulionis & Breipohl, 1982; Kratzing, 1971). There is no reason to assume that
vertebrate olfactory receptor cells are exceptional in that respect. Unfortunately,
secondary olfactory cilia of most mammals, including the rat, also lack microtubuleattached structures, and the distance over which this is the case is within range of that
of the lengths of primary cilia (Lidow & Menco, 1984; Menco, 1984; Menco &
Farbman, 1985<a). However, the above four points strongly support the suggestion
that olfactory receptor cells first generate primary cilia; others are subsequently
added. Hence, the first olfactory cilium is apparently a (possibly modified) primary
cilium, whereas additionally formed olfactory cilia are modified secondary ones.
It is thought that basal bodies of secondary cilia are replicated near the nucleus and
that the diplosomes are involved in this replication process, although the exact
mechanism is unknown. Secondary basal bodies migrate to the cell apices to give rise
to secondary cilia (Heist & Mulvaney, 1968; Mulvaney & Heist, 1971; Sorokin,
1968). Despite this, and in contrast to the situation in respiratory ciliated epithelia
(Kober, 1975), since primary cilia and the centrioles of presumptive secondary cilia
may coexist (Figs 7, 9, 13, 14, 43-45), they seem to be retained, at least for a while,
during the formation of secondary cilia.
Primary cilia have, like most other types of cilia (listed by Menco, 1980c), a
necklace-like structure at their base (Fig. 10, Whiteley & Young, 1985). This feature
can be resolved clearly with the scanning electron microscope and resembles the
equivalent membrane structures of secondary cilia (Fig. 47). Since primary cilia tend
to taper immediately above their necklace region, these necklaces, in addition to other
possible functions (Gilula & Satir, 1972; Menco, 1980c), at least provide a region of
mechanical support for the cilium, that is, a region of microtubule-membrane interaction providing a rigid tube through which the rest of the ciliary axoneme can grow.
This supposition is supported further by the fact that we encountered polyaxonemes
throughout development in which, apart from the necklace regions, the circumferences of individual cilia could not be discerned (Figs 36, 37).
As shown for adult vertebrates (Cuschieri & Bannister, 19756; Glees & Spoerri,
1978; Graziadei & Bannister, 1967; Menco, 1980a), dendritic endings of embryos
304
B. Ph. M. Menco and A. I. Farbman
may have short microvilli in addition to primary (Fig. 46) and secondary (Fig. 47) cilia.
Systematic developmental topography
In the accompanying paper we shall deal with the overall temporal events occurring
at the developing surface of the olfactory epithelium. Here we discuss spatial differentiations as far as these temporal events are concerned. Our results confirm and
extend those of Breipohl & Ohyama (1981) who showed, for mouse and chick, that
the dendritic endings of the olfactory receptor cells and the microvillous apices of the
olfactory supporting cells are more precocious in the posterior than in the anterior
regions of the nasal septum. We showed that this lead in development concerns
densities of receptor cell dendritic endings, numbers of olfactory cilia per ending, and
presence and lengths of olfactory supporting cell microvilli; this lead sets in as soon
as the olfactory epithelium can be recognized as such and possibly even earlier,
assuming that the globular profiles in Fig. 2 represent putative receptor cells (Figs 2,
3). This lead is retained in adults of most vertebrate species investigated (Menco,
1983) and is at least partly responsible for differences in quantitative response to
smells as measured by the electro-olfaerogram (Breipohl et al. 1982; Daval et al.
1980; Mackay-Sim & Shaman, 1984).
The persistence of the anatomical gradient in adult vertebrates implies that it is not
a developmental feature, but rather a permanent one, which is established at the onset
of the formation of the recognizable olfactory epithelial surface. Studies in adult
vertebrates using tracers suggest that different parts of the olfactory epithelium are
projected consistently towards certain regions of the olfactory bulb (DuboisDauphin, Tribollet & Dreifuss, 1980, 1981; Jastreboff et al. 1984; Kauer, 1981;
Mackay-Sim & Nathan, 1984). In the rat, the spatial gradient in the olfactory
epithelium (occurring from E14 on) is established before the receptor cells synapse
in the olfactory bulb (from E18 on; Farbman, 1985). However, the gradient may be
expressed because posteriorly located receptor cells are closer to the presumptive
olfactory bulb and have shorter distances to grow: the axons reach the bulb two to
three days before they synapse (Farbman, 1985; Farbman & Squinto, 1985; Hinds,
1972; Hinds & Hinds, 1976). Furthermore, the bulb exerts retrograde trophic effects
on the development of the receptor cells (Chuah & Farbman, 1984; Chuah, Farbman
& Menco, 1985). With these points in mind, the gradient could be used for a functional mapping study, by investigating whether the furthest developed areas are projected
first with regard to regions in the olfactory bulb.
The present developmental study provides no evidence for the formation of different types of receptor cells in different parts of the olfactory epithelium and confirms
previous studies in adults (Menco, 1977, 1983). Therefore, any qualitative variation
in topographical coding (Mackay-Sim et al. 1982; Mackay-Sim & Patel, 1984;
Mackay-Sim & Shaman, 1984; Thommesen & D0ving, 1977) needs to be sought at
a molecular rather than a morphological level. Even when various types of receptor
cells co-exist in the same vicinity, as is the case in most fishes (ciliated and microvillous
cells), there is no conclusive evidence that both cell types code for different types of
smells (Erickson & Caprio, 1984).
Olfactory epithelial development. I
305
Non-systematic developmental topography
The non-systematic variation occurring during development of the rat's olfactory
epithelium, especially from E18 onwards, is also similar to that of adults. This variation concerns in particular the appearance of supporting cell surfaces. As in the
embryos (e.g. see Figs 16-18, 23, 38), lengths of supporting cell microvilli vary from
area to area (Menco, 1984; Naessen, 1971; Yamada, 1983) and globular protuberances at the cell apices (Figs 29, 39) are also present in adults (Meinel & Erhardt,
1978; Menco, 1977). The variation in length of supporting cell microvilli was also
found in 1-week-old explants of E15 rat olfactory epithelium (Chuah et al. 1985).
Inclusions like those seen in Figs 40 and 41 can also be an expression of supporting
cell activity. There are several indications that supporting cells are rather polymorphic and react with a change in morphological appearance to environmental (Ekblom,
Flock, Hansson & Ottoson, 1984; Okano & Takagi, 1974), hormonal (Balboni &
Vanelli, 1982; Da Pos & Arimondi, 1983; Saini & Breipohl, 1976) and aging (Da Pos
& Arimondi, 1983; Vanelli, 1978) conditions. Moreover, there is considerable
evidence that the supporting cells comprise a heterogeneous population (Erhardt &
Meinel, 1979; Rafols & Getchell, 1983) (see also Figs 33, 38). The present study
provides evidence that supporting cell polymorphism occurs throughout development.
Olfactory polyaxonemes (Figs 36,37), also can be induced by, for example, noxious
compounds (Ekblom et al. 1984) and may also occur as a consequence of degeneration
as shown for non-sensory cilia by Boisvieux-Ulrich, Sandoz & Chailley (1980). However, in most instances the distribution of intramembranous particles, as seen by
freeze-etch techniques, is similar for membranes of polyaxonemes and individual cilia
(Menco, 19806,c, 1984). Hence, it may be that dendrites that terminate in
polyaxonemes are normally functional. However, we believe that a case like that
shown in Fig. 37 (adult) is due to degeneration, since the area was clearly surrounded
by ciliary tapers and situated as an isolated patch (wound) within an area that appears
normal. It remains enigmatic why polyaxonemes occur so frequently during development.
The variation described here in the appearance of the membranes of olfactory cilia
and of the microvilli of surrounding supporting cells is compatible with freeze-etch
observations (Menco, 19806, 1984).
Atypical cells
The occurrence of atypical cells (neither receptor nor supporting cells) in the
olfactory epithelium is not uncommon (Andres, 1975; Bertmar, 1972; Jourdan, 1975,
1984; Menco, 1980a; Miragall, Breipohl, Naguro & Voss-Wermbter, 1984; Moran,
Rowley & Jafek, 1982). Here we have shown that such cells also occur during pre-natal
development (Figs 18, 50, 51). It should be stressed that atypical cells comprise only
a tiny fraction of the total cell population. Therefore, their possible function in the
olfactory reception process should not be overemphasized (Jourdan, 1984; Moran et
al. 1982). Some of these cells occur also in the respiratory epithelium, namely the
306
B. Ph. M. Menco and A. I. Farbman
brush cell (Jeffery & Reid, 1975; Menco, 1977). This is also the case for the atypical
cells found in this study (Figs 27, 50, 51; Menco & Farbman, unpublished) which do
not resemble brush cells but, rather, look like inner-ear hair cells, especially the cell
type shown in Fig. 51 (Tilney & Tilney, 1984).
We thank Mr Gene Minner for help with the EM facility. This work was supported by NIH grants
5PO1 NS-18490 (Program Project) and 1RO1 NS-21555.
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{Received 26 March 1985-Accepted 15 April 1985)