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Three-dimensional visualization of the human membranous labyrinth - The

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DOI: 10.1002/ar.24675

AR WOW - VIDEO ARTICLES

Three-dimensional visualization of the human

membranous labyrinth: The membrana limitans and its
role in vestibular form

Christopher M. Smith1,2,3 | Ian S. Curthoys4 | Payal Mukherjee5 |

Christopher Wong4 | Jeffrey T. Laitman1,2,3,6

1
Department of Anthropology, The
Graduate Center, City University of Abstract
New York, New York City, New York The inner ear contains the end organs for balance (vestibular labyrinth) and
2
Center for Anatomy and Functional hearing (cochlea). The vestibular labyrinth is comprised of the semicircular
Morphology, Icahn School of Medicine at
canals (detecting angular acceleration) and otolith organs (utricle and saccule,
Mount Sinai., New York City, New York
3
New York Consortium in Evolutionary
which detect linear acceleration and head tilt relative to gravity). Lying just
Primatology, New York City, New York inferior to the utricle is the membranous membrana limitans (ML). Acting as
4
Vestibular Research Laboratory, School a keystone to vestibular geometry, the ML provides support for the utricular
of Psychology, University of Sydney,
macula and acts as a structural boundary between the superior (pars superior)
Sydney, New South Wales, Australia
5 and inferior (pars inferior) portions of the vestibular labyrinth. Given its
RPA Institute of Academic Surgery,
Royal Prince Alfred Hospital, Sydney, importance in vestibular form, understanding ML morphology is valuable in
New South Wales, Australia establishing the spatial organization of other vestibular structures, particularly
6
Department of Otolaryngology, Icahn the utricular macula. Knowledge of the 3D structure and variation of the ML,
School of Medicine at Mount Sinai,
New York City, New York however, remain elusive. Our study addresses this knowledge gap by visualiz-
ing, in 3D, the ML and surrounding structures using micro-CT data. By doing
Correspondence
so, we attempt to clarify: (a) the variation of ML shape; (b) the reliability of
Christopher M. Smith, Center for
Anatomy and Functional Morphology, ML attachment sites; and (c) the spatial relationship of the ML to the stapes
Icahn School of Medicine at Mount Sinai, footplate using landmark-based Generalized Procrustes, Principal Component
Annenberg Building Room 12-90, 1468
and covariance analyses. Results indicate a consistent configuration of three
Madison Ave., New York, NY 10029.
Email: christopher.smith1@mssm.edu distinct bony ML attachments including an anterolateral, medial, and posterior
attachment which all covary with bony structure. Our results set the stage for
Funding information
further understanding into vestibular and more specifically, utricular macula
Garnett Passe and Rodney Williams
Memorial Foundation; New York spatial configuration within the human head, offering the potential to aid in
Consortium in Evolutionary Primatology; clinical and evolutionary studies which rely on a 3D understanding of vestibu-
Center of Anatomy and Functional
Morphology at the Icahn School of
lar spatial configuration.
Medicine at Mount Sinai; Graduate
Center, City University of New York KEYWORDS
balance, bony labyrinth, inner ear, membrana limitans, vestibular system

1 | INTRODUCTION
*This article includes AR WOW Videos, which can be viewed at https://
players.brightcove.net/656326989001/default_default/index.html? The vestibular system is an evolutionarily ancient system
videoId=6256917031001. that provides a sense of 3D spatial awareness. In humans,

Anat Rec. 2021;1–14. wileyonlinelibrary.com/journal/ar © 2021 American Association for Anatomy. 1

2 SMITH ET AL.
the end organs responsible for this sense are comprised
of fluid-filled membranous structures housed within a
bony labyrinth inside the petrous portion of the temporal
bone. These include five paired organs: three semicircu-
lar canals (SCCs) with their constituent ducts and two
otolith organs (the utricle and the saccule) housed within
the bony vestibule (entryway). The utricle is a sac-like
membranous structure to which all three semicircular
ducts connect while the saccule is a relatively smaller
membranous sac lying just inferior to the utricle.
Establishing the spatial organization of vestibular
structures is key to understanding the functional proper-
ties of the vestibular system. Great strides have been
made over the last 50 years to establish the spatial organi-
zation of the SCCs, especially regarding their arrange-
ments within the human head (see, e.g., Blanks,
Curthoys, & Markham, 1975; Curthoys, Blanks, &
Markham, 1977; Della Santina, Potyagaylo, Migliaccio,
Minor, & Carey, 2005; Bradshaw et al., 2010). We now
seek to establish the spatial configuration of the utricular
macula (the sensory portion of the utricle) both in rela-
tion to other structures of the membranous labyrinth and
within the human head. In the case of the SCCs, esta-
blishing their configuration with respect to the head is
relatively straightforward since the membranous semicir-
cular ducts lie within well-defined bony canals. Statistical
analysis of direct measures and reconstructions from CT
scans have allowed the location and configuration of the
SCCs to be established (Blanks et al., 1975; Curthoys
et al., 1977). These data have been valuable in treating
vestibular disorders [e.g., Benign paroxysmal positional
vertigo (BPPV)] and diagnosing the functional status of
individual SCCs (Cremer et al., 1998).
Knowledge of otolith organ spatial organization in
the head and spatial configuration of the maculae in rela-
tion to other structures of the membranous labyrinth will
also be of great value as evidenced by new developments
in otolith diagnostic testing. For example, a recent
hypothesis proposes that displaced saccular otoconia may
block the ductus reuniens and so may have widespread
effects on the labyrinth (Hornibrook, 2018; Hornibrook &
Bird, 2017; Phillips & Prinsley, 2009; Phillips &
Prinsley, 2017; Yamane et al., 2010; Yamane et al., 2012).
In addition, the location and configuration of the oto-
lithic maculae in relation to SCC ampullae and ducts are
important in determining the cause of BPPV and related
phenomena.
Understanding otolith organ orientation within the
head is also important for the accuracy of novel tests of
otolith organ function. For instance, varying the location
of a bone-conducted vibration stimulus on the skull affects
the characteristics of ocular vestibular evoked myogenic
potentials (oVEMP) (Govender & Colebatch, 2018) which
may be due to the differential activation of various recep-
tor segments of the utricular macula that have differential
projections to ocular muscles (Szent
agothai, 1962). To
establish that relationship, information about utricular
macula orientation in the human head is required.
In addition to clinical contributions, elucidating the
spatial configuration of the maculae in relation to
the membranous labyrinth and head would allow future
comparative investigations into otolith organ orientations
among mammals. This could aid in determining potential
species-specific orientations relative to cranial structure
and SCCs (e.g., Smith & Laitman, 2021) and lead to inno-
vative research paradigms into the evolution of the ves-
tibular system.
However, the challenge of determining the spatial
configuration of the utricular macula within the human
head and to other structures of the membranous laby-
rinth is much greater than for the SCCs. Several studies
in the early 20th century have made attempts to recon-
struct the positioning of the maculae to one another and
within the human skull (e.g., Corvera, Hallpike, &
Schuster, 1958; de Burlet, 1930; Quix, 1925). Neverthe-
less, reconstructing the 3D spatial configuration of the
utricular macula in any amount of detail requires a more
established understanding of its supporting structures. In
humans and guinea pigs more than half the area of the
utricular macula is not attached to bone but is supported
on a thin membrane composed of several layers of cyto-
plasmic extensions of fibrocytes traversing the membra-
nous labyrinth—the membrana limitans (ML) first
described by de Burlet (1920) as the “Grenzmembran”
(see also Hara & Kimura, 1993; Lagally, 1912; Tanaka,
1975) (Figure 1). The only area of the utricular macula
attached to bone is the rostral region where the myelin-
ated nerve fibers leave the macula to course in
the superior division of the vestibular nerve (Uzun-Cor-
uhlu, Curthoys, & Jones, 2007). The receptor surface of
the utricular macula lies dorsally and the afferents inner-
vating those receptors emerge from the neuroepithelium
on the ventral side of the macula. The ML lies just infe-
rior to those myelinated fibers. Due to the unique
supporting role the ML has in relation to the utricular
macula, the ML serves as a logical starting point to begin
work on establishing the spatial configuration of the
utricular macula relative to the membranous labyrinth
and cranium. While evidence about the ML from tempo-
ral bone sections has been published (e.g., Hara &
Kimura, 1993) it is very difficult to identify the spatial
configuration of the ML and its 3D relationship to other
vestibular and membranous structures from two-
dimensional temporal bone sections.
This study attempts to clarify the morphology and
attachment sites of the ML. Specifically, we identify and
SMITH ET AL.
F I G U R E 1 The membranous tissues of the inner ear. (a) Left
lateral view of human cranium with window in temporal bone
showing position of left bony labyrinth; (b) Enlarged view of the
left labyrinth from A, highlighting the bony labyrinth, otic
labyrinth (green), ML (blue), utricular macula, and saccular
macula. The stapes is shown in position ghosted on top of the bony
labyrinth; (c) Left bony labyrinth and ML shown in medial view.
(d) Left bony labyrinth and ML shown in superior view. Crania
show orientation of the head in respective views. References for the
illustration were created by overlaying 3D surface renders from two
sets of micro-CT data (one cranial scan and one otic capsule scan)
using the orientation of the bony labyrinth from the cranial scan as
reference. For a discussion on modeling canal geometry see
Bradshaw et al. (2010). Figure and illustrations by CMS. © 2021
Christopher M. Smith
assess the consistency of ML anchor points to the bony
wall of the labyrinth in humans and test the hypothesis
that there are distinct ML attachments, consistently
anchoring onto specific regions of the bony wall. Many
dissections have confirmed that guinea pig ML anchor
points are regular and so serve as reference for the identi-
fication of similar points in the human (Uzun-Coruhlu
et al., 2007). This reference serves as a guide to begin
defining the location and orientation of the human
ML. Clarifying ML morphology in this way would thus
be a valuable tool to establish the orientation and spatial
configuration of the human utricular macula by provid-
ing a way to link the macula to the bony structure of the
labyrinth. In addition, establishing a more quantifiable
framework of ML proximity to the stapes footplate would
3
ensure better protection of the delicate tissues of the ves-
tibule during surgical approach (particularly procedures
that include stapes footplate manipulation).
In regard to the terminology used herein, we use the
term vestibular labyrinth to refer to all hard and soft tis-
sues that comprise the inner ear excluding the cochlea
while our use of the term vestibular system refers to the
vestibular labyrinth, projections to the vestibular nuclei
in the brainstem, and vestibular pathways throughout
the thalamus, cerebellum, spinal cord, and cerebral
cortex (for a review see Goldberg et al. 2012). We use the
term bony labyrinth to refer to the endocast of the otic
capsule devoid of soft tissue. Membranous labyrinth here
is used to refer to both the otic labyrinth of endodermal
origin (containing endolymph) and the soft tissues of
mesodermal origin which occupy the space between the
otic labyrinth and bony otic capsule (also known as
the periotic labyrinth; for further discussion see Bast &
Anson, 1949; Streeter, 1918).
2 | METHODS
2.1 | Data acquisition and
reconstruction
Our sample comprises nine human temporal bones from
eight individuals and one guinea pig temporal bone. PM
did the initial surgical preparation of the temporal bones,
ISC did the staining, micro-CT (μCT) scans were carried
out by CW. μCT was chosen as it shows the configuration
and spatial relationship of the ML and utricular macula
to other membranous and bony structures at high resolu-
tion (Mukherjee et al., 2011; Mukherjee, Cheng, &
Curthoys, 2019).
All procedures on human temporal bones were
approved by the NSW Department of Health and proce-
dures were approved by the Royal Prince Alfred Hospital
Ethics Office with protocol number X19-0480. All proce-
dures on guinea pigs were approved by the Animal Ethics
Committee of the University of Sydney (protocol number
L29/4-2010/3/5266) and conformed to the Principles of
Laboratory Animal Care.
Nine cadaveric human temporal bones were drilled to
isolate the otic capsule. One guinea pig temporal bone
from an animal used in a physiological study (Curthoys,
Vulovic, Sokolic, Pogson, & Burgess, 2012) was scanned
and included in this study to serve as comparison for ML
attachment sites in humans (the guinea pig is commonly
used as an animal model in inner ear studies). Age, sex,
and provenance were not available for our sample. All
temporal bones were fixed for 24 hr in Karnovsky's fixa-
tive (3% paraformaldehyde, 0.5% glutaraldehyde in
4 SMITH ET AL.
phosphate buffer) which is known to minimize shrinkage
(Anniko & Lundquist, 1977). They were then soaked in
2% osmium tetroxide, for up to 9 days, and scanned in a
64-bit μCT scanner (MicroXCT-400; Xradia, Pleasanton,
CA) with scan width ranging from 4.90 to 25 μm. Aver-
age scan time was 8 hr. The μCT data in tagged image file
format (.TIFF) files were segmented to focus on the vesti-
bule. The methods, including details about the scanning
parameters, have been described in detail in earlier publi-
cations (Mukherjee et al., 2019; Uzun, Curthoys, &
Jones, 2007; Uzun-Coruhlu et al., 2007).
The stained μCT stack of .TIFF images were imported
into the visualization software 3D Slicer, version 4.10.1
(3D Slicer, 2020; Fedorov et al., 2012; Kikinis, Pieper, &
Vosburgh, 2014). This allowed axial, sagittal, and coronal
plane views of each structure, and enabled 3D recon-
struction. Six structures were segmented for each speci-
men, including the bony labyrinth, ML, utricular macula,
saccular macula, stapes, and a portion of the facial canal.
Segmentation of each bony labyrinth and facial canal
was completed through a combination of manual and
semiautomated thresholding to define the boundary
between the air-filled space of the labyrinth or canal and
surrounding bone. Each macula was segmented manually
by outlining the macula at every three slices, filling in
between slices (using the “fill between slices” tool in 3D
Slicer), and smoothing the resulting 3D surface render.
Segmentation of the ML was carried out as follows:
1. Automatic thresholding was used to isolate the den-
sity range corresponding to the ML with no manual
alterations of the segmentation (Segmentation 1). This
prevented user error from being introduced into the
initial segmentation.
2. A second segmentation was performed manually by
selecting visible boundaries of the ML and its bony
anchors (Segmentation 2). This allowed for finer con-
trol in order to segment the edges of the ML that are
difficult to visualize.
3. Both segmentations were exported as. OBJ files and
imported into the software Zbrush version 4R8
(Pixologic, 2020) where they were superimposed.
4. Segmentation 2 was used as a guide to crop Segmentation 1.
5. Segmentation 1 was remeshed using the “dynamesh”
tool (maintaining polygon count) in Zbrush. This was
done to smooth surface topology and remove artifacts
in the mesh, resulting in the final 3D model.
2.2 | Data analyses
In addition to a qualitative assessment of overall mor-
phology, three quantitative analyses were performed to
assess: (a) variation of ML shape; (b) reliability of ML
attachment sites; and (c) spatial relationships of the
ML to the stapes footplate.
2.2.1 | Analysis 1—Variation of ML shape
The cleaned 3D models of each ML were imported into
the software package Stratovan Checkpoint (Stratovan
Corporation, 2018) where six landmarks were manually
placed on the surface of the 3D model to mark the bou-
nds of each of the three ML attachments to the bony wall
(Table 1). All landmarks were placed by CMS and sub-
jected to a generalized procrustes analysis (GPA) using
the “geomorph” package (v. 3.3.2; Adams, Collyer,
Kaliontzopoulou, & Baken, 2021) in the software R
(v. 3.6.3; R Core Team, 2020). All following statistical ana-
lyses were performed in R unless otherwise noted. The GPA
registers multiple landmark configurations to one another
(via X, Y, and Z coordinates) by superimposing and scaling
all instances to a reference set of landmarks. This process
removes orientation and isometric size differences, thereby
resulting in a collection of shape variation only (for a review,
see O'Higgins, 2000). Intraobserver error was assessed by
repeating all six measurements (10 times) on one specimen
(voxel size 24.7 μm3). Procrustes distances recovered from
this repeatability analysis (0.003–0.01) did not overlap with
any Procrustes distances recovered for the sample. There-
fore, no systematic errors in recognizing individual land-
marks substantially influenced landmark placement.
Procrustes superimposed landmark coordinates were then
subject to a principal components analysis (PCA) in order to
examine the shape differences related to attachment sites
that contribute the most to observed shape variation.
2.2.2 | Analysis 2—Reliability of ML
attachment sites
In order to examine the reliability of ML attachment
sites, we performed two assessments: First, a two-block
TABLE 1 ML landmarks used in this study
Landmark Description
1 Posteriormost point of anterolateral attachment
2 Anteriormost point of anterolateral attachment
3 Anteriormost point of medial attachment
4 Posteriormost point of medial attachment
5 Medialmost point of posterior attachment
6 Lateralmost point of posterior attachment
SMITH ET AL. 5

partial least-squares analysis (2B-PLS) was used to assess superimposition would distribute shape variance amongst
the degree of association between the ML and bone, and landmarks in order to minimize the sum of squared dis-
second, we examined the coefficient of variation (CV) for tances among shapes and, in doing so, may reduce or con-
each interlandmark distance in order to assess relative flate the “perceived variance” of highly variable or more
variation of attachment site length. stable landmarks, respectively (for a review see Goswami &
A 2B-PLS assesses the degree of association between Polly, 2010; see also Rohlf, 1990). Our size-corrected
two blocks of Procrustes shape variables (in this case, the pairwise interlandmark distance data were determined to
ML and bone). Three landmarks were placed on the bony be heteroscedastic (where an increase in the mean value
wall to approximate the clearest points of ML attachment relates to increases in variance). Therefore, we used the
(Table 2). These three landmarks made up “Block 1,” CV to assess degree of variation of interlandmark distances
while “Block 2” consisted of the ML landmarks from relative to each specimen's distance mean.
Analysis 1. All landmarks were placed on a single 3D
model that was cropped to allow visualization of both the
bony wall and ML. Each landmark was then assigned to 2.2.3 | Analysis 3—Spatial relationships of
one of two subsets (the ML or bony wall). Each landmark the ML to the stapes footplate
belongs to only one subset. Since the ML is intimately
connected to the bony wall, all nine landmarks under- The spatial relationships of the ML in relation to the sta-
went a simultaneous Procrustes superimposition. This pes footplate were assessed by measuring the following
“simultaneous fit” approach allows assessment of covari- four distances using the “transpose” tool in the software
ation of the structure as a whole and retains information program Zbrush (see Figure 2 for an illustration of each
about how the subsets are connected to one another. This distance):
method was chosen to allow for comparison between
multiple response and explanatory variables and it pro- 1. Distance between anteriormost point of ML bony
vides a way to treat them symmetrically, rather than one attachment and midpoint of anterior edge of stapes.
variable predicting variation in the other (Rohlf & 2. Shortest distance between the anterolateral ML bony
Corti, 2000; for a review see Goswami & Polly, 2010). attachment and superior border of the stapes.
Both PLS and RV coefficient (RV) were used to measure 3. Distance between posteriormost point of anterolateral
the association between the two blocks. RV measures the ML bony attachment and midpoint of posterior edge
amount of covariation scaled to the total variation pre- of stapes.
sent within both sets of variables (for a review see
Klingenberg, 2009).
In order to assess relative variation in attachment site
length, we examined CV based on a matrix of pairwise
interlandmark distances that were size adjusted by divid-
ing each inter-landmark distance by its respective cen-
troid size. Centroid size is the square root of the sum of
squared distances of all landmarks from a center point
defined by averaging the x, y, and z coordinates of all
landmarks. This approach was chosen instead of using
distances between superimposed Procrustes coordinates
since we needed to retain as much raw positional infor-
mation about each landmark as possible. Procrustes

TABLE 2 Bony labyrinth landmarks used in this study

Landmark Description
7 Posteriormost point of supraovalic fossa
8 Inferiormost point where the posterior bony
F I G U R E 2 Distances measured from the left ML to the left
ampulla meets vestibular aqueduct
stapes footplate. Abbreviations: S, superior; A, anterior; mm,
9 Superiormost point where spherical recess meets
millimeter. The ML is depicted in blue. ML attachments to bone are
vestibule [viewed in vestibular orientation
in yellow. Cranium shows orientation of the head from this
(Delattre & Fenart, 1958)]
viewpoint
6 SMITH ET AL.

4. Distance between lateralmost point of posterior ML

attachment and midpoint of posterior edge of stapes.

All distances were measured by CMS with the stapes

footplate placed parallel to field of view. Intraobserver
error (0.03–0.06 mm) was assessed by repeating all four
measurements (10 times) on one specimen (voxel size
10.7 μm3).

3 | R E SUL T S

3.1 | Human sample

In all specimens, the ML has a consistent pattern of config-

uration, with local variation in the extent of attachment to
the bony wall. As observed in prior studies (e.g., Backous,
Aboujaoude, Minor, & Nager, 1999; de Burlet, 1929a;
Hara & Kimura, 1993; Mukherjee et al., 2019) the ML is
positioned directly inferior to the utricle and its neural sub-
strate. Overall, the ML is elongated anteroposteriorly,
forming an “arch”-like shape when viewed laterally. Poste-
rior to the utricular macula the ML is tightly adhered to the
inferior wall of the utricle. We observe three distinct ML
attachments to the bony wall; (a) an anterolateral attach-
ment; (b) a medial attachment; and (c) a posterior attach-
ment (see Figure 1 for anatomical illustration of human ML
in relation to cranium and labyrinth; see Figure 3 for 3D
visualizations and Video 1 for 3D models).

3.1.1 | Anterolateral attachment

The anterolateral attachment is where the ML attaches

just superior to the posteriormost point of the stapes foot-
plate and stretches anteriorly, tethering itself along the
inferior border of a slight concavity in the bony wall just
superior to the fenestra ovalis. We call this bony feature
the “supraovalic fossa” (Figure 4). The anterolateral
attachment begins at the posterior end of this fossa and
terminates at or just beyond the anterior boundary of the
stapes footplate, often matching the termination of
the supraovalic fossa. This attachment is often discontin-
uous (observed in seven of nine ears sampled). In three
of nine ears, this attachment extends far enough anteri-
orly to contact the anterosuperior edge of the spherical
recess.

F I G U R E 3 3D visualizations of the ML in nine humans (a–i)

3.1.2 | Medial attachment
Superior to the saccule, the ML sends out an extension to
attach along the superior border of the spherical recess.
and one guinea pig (J). c, d, and g–i are mirrored right ears for ease
of comparison. All others are left ears. The ML is depicted in light
blue. ML attachments to bone are in yellow, while dark blue
denotes edges of the ML that do not attach to bone. Cube is 1 mm.
Video of 3D models can be found here: (Video 1)
SMITH ET AL.
F I G U R E 4 (a) Micro-CT image showing the supraovalic fossa superior to the ML; (b) 3D segmentation of the bony labyrinth illustrating
the overall form of the supraovalic fossa and its proximity to the stapes footplate
The extent of attachment to this bony site varies greatly
with some specimens possessing a small tethering to the
bony wall and others with a much broader attachment.
Like the anterolateral attachment, the medial attachment
presents as either continuous (eight out of nine speci-
mens) or discontinuous (one out of nine). This attach-
ment terminates just anterior to where the utricular duct
enters the endolymphatic sinus.
3.1.3 | Posterior attachment
The posterior attachment is where the ML inserts into
the inferiormost edge of where the posterior ampulla
meets the vestibule. The ML often spans the entire width
of this junction and may even extend into the non-
ampullated side of the lateral canal (observed in one
specimen; see Figure 3c and Video 1C). This attachment
site is continuous in all nine specimens. Prior to
attaching here, the ML is tightly adhered to the underside
of the posterior region of the utricle.
While these bony attachments are similar in configu-
ration, they do vary locally in their length and position-
ing. In addition, the ML occasionally projects inferiorly
to lie upon the surface of the lateral saccular wall
(observed in seven of nine ears). One important variant
of ML attachment is the tethering of the ML to the super-
oposterior aspect of the stapes footplate (as seen in
Mukherjee et al., 2019; Backous et al., 1999). Here it is
observed in two specimens, where it attaches to the pos-
terior third of the footplate. Although, it is possible that
this tethering may have been affected by postmortem tis-
sue changes (Mukherjee et al., 2011).
7
3.2 | Guinea pig sample
In the guinea pig all attachment sites were similar to
humans, apart from a fourth attachment site just anterior
to the non-ampullated side of where the lateral canal
joins the vestibule. This site of ML attachment is continu-
ous with the anterolateral and posterior attachments for-
ming an uninterrupted insertion of the ML into the bony
wall spanning from just anterior to the stapes footplate to
the bony lip of the vestibular aqueduct. In addition, com-
pared to humans, the guinea pig ML spans a greater pro-
portion of the vestibule and exhibits a more distinct and
continuous configuration. An invagination of the ML at
the bony lip of the vestibular aqueduct has also been
observed in guinea pigs (Hara & Kimura, 1993). In our
sample, the posterior bony attachment of the ML seems
to extend up to the inferior edge of the bony lip of the
vestibular aqueduct, but distinct invagination of the ML
could not be determined. Superior to this attachment is a
condensation of periotic connective tissue which sepa-
rates the vestibular aqueduct from the vestibule.
3.3 | Analysis 1—Variation of ML shape
The PCA of Procrustes-fitted coordinate values results in
51.32, 29.35, and 12.20% of shape variation distributed
among the first, second, and third PC vectors, respec-
tively. PCs four and above comprise 3.09% or less of the
total shape variation. PC1 and PC2 relate to the position-
ing and width of the medial ML attachment in the trans-
verse plane, respectively (Figure 5). Positive movement
along the PC1 axis positions the medial attachment closer
8 SMITH ET AL.

F I G U R E 6 Plot of PC1 and PC3 showing shape variation

among ML attachment points. PC1 axis corresponds to the position
F I G U R E 5 Plot of PC1 and PC2 showing shape variation
of the medial attachment. The PC3 axis relates to the superoinferior
among ML attachment points. PC1 axis corresponds to the position
positioning of the anterolateral and posterior attachments relative
of the medial attachment, while the PC2 axis corresponds to width
to one another. Letters correspond to specimen labels from
of the medial attachment. 80.67% of the total shape variation can be
Figure 3. Wireframes (depicting shape changes along axes) are
attributed to the size and relative positioning of the medial
shown in superior view (PC1) and anterior view (PC3)
attachment. Letters correspond to specimen labels from Figure 3.
Red dot on stapes denotes anterior side. Wireframes show shape
changes along axes in superior view

to the anterolateral attachment while negative move-

ment places it closer to the posterior attachment. Posi-
tive movement along the PC2 axis widens the medial
attachment and negative movement decreases the
width. PC3 corresponds to the superoinferior position-
ing of the anterolateral and posterior attachments
relative to one another (Figure 6). This component is
driven primarily by specimen ‘C’ where the posterior
ML attachment extends into the non-ampullated side
of the lateral canal.

F I G U R E 7 Two-block partial least-squares (PLS) plot of ML

3.4 | Analysis 2—Reliability of ML
attachment sites
The 2B-PLS analysis indicates a close association between
the bony labyrinth and ML attachment sites (R2 = 0.97,
RV coefficient = 0.81, p = .001), with the ML attach-
ments reliably tracking displacements of the bony sites.
The first PLS axes describe the majority (56.48%) of the
total squared covariance between the shape of ML attach-
ment sites and bony labyrinth (Figure 7). The CV analysis
shows most variation in attachment length is relegated to
the medial attachment and its positioning relative to the
anterolateral attachment. The length of the posterior
attachment showed the third lowest amount of relative
variation and the least variation compared to the other
two attachment sites (Figure 8).
and bony labyrinth. Block 1 axis shows difference in shape of the
three landmarks (labeled 7–9) placed on the bony labyrinth. Grey
signifies negative values; blue signifies positive values. Block 2 axis
shows difference in shape of the six landmarks (labeled 1–6) placed
on ML. Grey signifies negative values, orange signifies positive
values. Wireframes are shown in anterior view for the bony
labyrinth and lateral view for the ML. A significantly strong
association between shape variation of bony labyrinth and ML
landmarks is recovered
3.5 | Analysis 3—Spatial relationships of
the ML to the stapes footplate
In the human sample, we assessed four distances
between the ML and footplate of the stapes. Distance
SMITH ET AL.
F I G U R E 8 Coefficient of variation (CV) among interlandmark
distances. The posterior attachment exhibits the lowest level of
relative variation in attachment site width, slightly less than the
anterolateral attachment. The medial attachment width is the
second most variable interlandmark distance, with the width
between the anterolateral and medial attachments sites varying
the most
TABLE 3 Variation among ML and stapes footplate distances
Distance X̄ SD
1 1.59 0.51
2 0.40 0.28
3 1.34 0.37
4 2.81 0.46
Abbreviations: ML, membrana limitans; X̄ , sample mean; SD, standard
deviation; CV, coefficient of variation. See text for a description of distances.
averages are as follows: (a) Mean (X  ) = 1.59 mm
(±0.51 mm); (b) X̄ = 0.40 mm (±0.28 mm);
(c) X̄ = 1.34 mm (±0.37 mm); (d) X̄ = 2.81 mm
(±0.46 mm). Distance 1 ranges from 0.89 to 2.44 mm.
Distance 2 is shortest, ranging from 0 (where the ML
attaches directly onto the footplate) to 0.68 mm, and
has the highest amount of relative variation
(CV = 70.16). This difference corresponds to the super-
oinferior positioning of the anterolateral ML attach-
ment superior to the footplate. Distance 3 ranges from
0.86 to 1.83 mm. Distances 1 and 3 are comparable in
average distance (1.59 and 1.34 mm, respectively),
while distance 1 exhibits slightly greater variability.
All distances related to the anterolateral attachment
lie superior to the long axis of the footplate. Distance
4 ranges from 2.07 to 3.60 mm and exhibits the
lowest amount of relative variation (CV = 16.35). This
distance consistently lies inferior to the footplate
long axis and is primarily influenced by the position
9
F I G U R E 9 Variation in the direction of each ML attachment
site distance (labeled 1–4) viewed from the plane parallel to the left
stapes footplate (see Table 3 for metrics of each distance). Drawing
is an approximation of average stapes shape [average human stapes
footplate “long length” has been reported as 2.81 ± 0.158 mm (Sim,
Röösli, Chatzimichalis, Eiber, & Huber, 2013)]. Black dots signify
the midpoints of the anterior and posterior edges of the footplate.
Cranium shows orientation of the head from this viewpoint.
CV Distances are relative in scale
31.87
70.16 of the lateral ampulla relative to the posterior
27.91 edge of the footplate. See Table 3 for a summary of
16.35 results and Figure 9 for an illustration of distance
distributions.
4 | DISCUSSION
Our results show a consistent pattern of three distinct
ML attachments to the bony wall of the labyrinth. This
supports our initial hypothesis and shows a predictable
3D arrangement of the ML in humans which covaries
with bony structure. The spatial configuration of the
guinea pig ML differs from the human by attaching to a
greater proportion of the bony wall of the labyrinth in
a more continuous fashion (Figure 3j; Video 1J). This
observation is similar to reports from previous studies
(Hara & Kimura, 1993; Mukherjee et al., 2019). In con-
trast, our human sample shows diminished anchoring of
the ML to the bony wall and more discontinuity in
attachment sites. This differs from early descriptions of
the ML including those of de Burlet's dissections of non-
mammalian tetrapods showing the ML as dividing the
labyrinth into the pars superior and pars inferior
(de Burlet, 1929b). His figures suggested that the ML was
like a drumhead separating the two halves of the
10 SMITH ET AL.

membranous labyrinth. This study and our previous
investigations of fixed guinea pig and human membra-
nous labyrinths have not confirmed that representation
(Mukherjee et al., 2019; Uzun et al., 2007; Uzun-Coruhlu
et al., 2007). The ML is not a uniform membrane like a
drumhead but is instead discontinuous with gaps where
the perilymph from pars superior and pars inferior can
mix. This is especially clear in the data from guinea pigs
where the fixation was by intracardiac perfusion of
Karnovsky's fixative (which causes minimal tissue
shrinkage [Anniko & Lundquist, 1977]) indicating that
the gaps are not due to shrinkage. Dissections of the
guinea pig temporal bone showed that the ML is tautly
attached to the bony wall of the labyrinth and has the
appearance of a trampoline-like structure upon which
the caudal part of the utricular macula rests. It should be
noted that, in the human specimens, postmortem deterio-
ration or failure of the stain to pick up portions of the
ML may influence the visibility of attachment sites,
including their discontinuous nature and/or slippage
onto the stapes footplate. Previous work has, however,
indicated that the scanning and staining methods used in
this study minimize any distortion of the membranous
vestibular structures (Anniko & Lundquist, 1977).
Upon visual inspection of the 3D reconstructions, it is
apparent that the most variable of attachments is the
medial attachment. Our PCA results and CV assessment
both support this assertion indicating the greatest
amounts of ML shape differences can be attributed to the
length and positioning of the medial attachment.
The reason for this may relate to the presence of many
nearby membranous structures in this region including
the saccule, Bast's Valve, utricular duct, and endolym-
phatic sinus. Morphological variation of these structures
may thus influence the length and positioning of the
medial attachment of the ML.
amount of relative variation in width and positioning,
likely due to its intimate connection with the vestibular
aqueduct. The consistency of these two attachments indi-
cates the possibility of establishing predictable soft tissue
geometry based on bony data. Such data may be useful
for future investigation into modeling spatial orientation
of the utricle within the labyrinth and moreover, within
the human head. Establishing a consistent spatial rela-
tionship of the utricle relative to the SCCs would also add
additional data to refine such models.
4.2 | Relationships to intralabyrinthine
structures
Our visualizations of the ML illustrate its spatial relation-
ships to structures of the vestibular labyrinth, particularly
its close proximity to the utricle, Bast's valve (also known
as the utriculoendolymphatic fold), saccule, lateral canal,
posterior ampulla, endolymphatic duct, and the opening
to the vestibular aqueduct (as also reported in Hara &
Kimura, 1993) (Figure 10). In addition, the perilymphatic
space of the pars superior is filled with a trabecular mesh-
work of periotic connective tissue which is closely associ-
ated with the ML. This meshwork varies in density and
may also vary ontogenetically, with reduced trabecular
density in the adult (as discussed by Bast & Anson, 1949;
see also Hara & Kimura, 1993). No meshwork is observed

4.1 | Relationship to bony labyrinth

Our 2B-PLS analysis suggests a close association between

the ML and bony labyrinth. Combining these data with
results from our CV analysis suggests the posterior and
anterolateral attachment sites share similar levels of reli-
ability in terms of bony attachments. This conclusion is
also supported by the observed consistency in these
attachments related to bony landmarks in humans
(i.e., the posterior bound of the anterolateral attachment
matching the posteriormost point of the supraovalic fossa
and medialmost point of the posterior attachment
inserting at the inferiormost junction of the vestibular F I G U R E 1 0 Superomedial view of the left membranous
aqueduct and inferior edge of the bony posterior labyrinth. Otic labyrinth (in green) is seen through the transparent
ampulla). The posterior attachment shows the lowest bony labyrinth
SMITH ET AL.
on the inferior surface of the ML facing the pars inferior.
This observation is consistent with previous literature.
Indeed, the ML provides a structural support to a
majority of the utricular macula and underlying neural
substrate. In all cases observed here, the posteriormost
tip of the anterolateral attachment is tethered along the
same trajectory as posteriormost tip of the utricular mac-
ula, suggesting a tight structural relationship between the
two. As the ML traverses the vestibule posteriorly, it
adheres tightly to the inferior wall of the utricle, breaking
away just before its posterior attachment.
Before reaching its posterior attachment, the ML can be
found just inferior to Bast's valve, forming what Bast and
Anson described as a “septum of periotic connective tissue”
(1949). The role for this connective tissue at Bast and Anson's
time of publication was not known, but 3D visualizations of
the ML now raise the possibility that this septal tissue may
belong to the ML itself. It is this portion of the ML that inserts
into the bony lip of the vestibular aqueduct (Figure 10). When
viewed on an endocast of the bony labyrinth, this insertion
lies at the inferiormost junction of the vestibular aqueduct
and inferior edge of the bony posterior ampulla, forming the
medial bound of the posterior attachment.
As noted in previous studies (Hara & Kimura, 1993;
Mukherjee et al., 2019), the ML often overlies a portion
of the saccule, although the extent to which this overlap
extends is difficult to pinpoint. Some have suggested that
the tissue tethering the saccule to the inferior bony vesti-
bule belongs to the ML. However, our observations favor
a more limited extension of the ML over the saccule due
to observable ML overlaying only the superior portion of
the saccule if at all. The membrane attaching the saccule
to the bony wall inferiorly is instead likely related to the
thickened portion of the inferior saccular wall first
reported by Perlman (1940) (for a review see Bast &
Anson, 1949; Anson & Donaldson, 1967) which may have
a protective function (Igarashi, 1964).
The posterior attachment of the ML attaches consis-
tently to the inferior lip that marks the junction of the
bony posterior ampulla and vestibule. While the length
of this attachment varies (observed to extend into the lat-
eral canal itself in one specimen) it stays anterior to the
non-ampullated side of the lateral semicircular duct.
4.3 | Relationship to utricular macula
The consistency of ML attachments suggests value in utiliz-
ing this structure for estimating orientation and spatial con-
figuration of the utricular macula, particularly the
anterolateral attachment which extends out from under-
neath the utricular macula. The anterolateral attachment
reliably attaches just superior to the fenestra ovalis. This
11
attachment consistently lies along the inferior boundary of
the supraovalic fossa, observed as a subtle bulge in the bony
labyrinth endocast (Figure 4b). This feature, however, is not
observed in the guinea pig included in this study. Anteri-
orly, the anterolateral attachment underlies the inferior bor-
der of the superior division of the vestibular nerve. The
region of the ML adjacent to the anterolateral attachment
provides structural support for the utricular macula and
neural substrate. Consistent insertion of this attachment site
at the base of the superior division of the vestibular nerve
and supraovalic fossa may prove useful in establishing the
lower bound of the plane of utricular macular orientation
using bony data. In addition, the posterior end of this
supraovalic fossa matches the trajectory of the posterior tip
of the utricular macula in all specimens. It is unclear as to
what may be influencing the appearance of this feature,
although, to our knowledge, the presence or absence of the
supraovalic fossa has not been determined in nonhuman
taxa. Further investigation into this feature is currently
underway.
4.4 | Surgical implications
The proximity of the ML to the stapes footplate and its
attachment to the utricular macula poses a significant issue
for surgeons. Surgical procedures on the footplate (such as
a stapedotomy) may well interfere with the ML and so indi-
rectly affect the utricular macula (see also Mukherjee
et al., 2019). Such interference could be responsible for
some reports of disorientation or postural unsteadiness that
some patients experience following stapedotomy or stape-
dectomy operations (Bartel et al., 2020; Mun et al., 2019;
Singh et al., 2018; Vaughan et al., 2020). Importantly, the
anterolateral attachment of the ML lies superior to the
footplate's long axis, coming in closest proximity to the foot-
plate in its posterior two-thirds. Therefore, this site is partic-
ularly vulnerable in procedures that include footplate
manipulation. Indeed, disruption of the anterolateral
attachment may prove the most severe of any attachment
site disturbance as this portion of the ML directly supports
the utricular macula and neural substrate. Caution should
thus be exercised when approaching the footplate as to not
disturb the ML attachments along its superior border and
potentially directly inserted into the posterior portion of the
footplate itself.
5 | CONCLUSION
The ML serves as a keystone to vestibular geometry as
seen in this study of its morphology. It provides a bound-
ary between the pars inferior and pars superior, supports
12
the utricular macula, and is intimately associated with
the membranous vestibular labyrinth. The consistency of
ML attachment sites and their covariance with bony laby-
rinth shape reported here indicate that we can now reli-
ably predict the spatial configuration of the ML based on
the morphological observation of the bony labyrinth. Our
results support the possibility of using these data to
inform reconstruction of utricular macula spatial config-
uration within the human head.
A C K N O WL E D G M E N T S
The authors acknowledge the facilities and the scientific
and technical assistance of Microscopy Australia at the
Australian Centre for Microscopy & Microanalysis at
the University of Sydney. This work was supported by
the Garnett Passe and Rodney Williams Memorial Foun-
dation, the Graduate Center, City University of
New York; the Center of Anatomy and Functional Mor-
phology at the Icahn School of Medicine at Mount Sinai,
and the New York Consortium in Evolutionary Primatol-
ogy. We would also like to thank Prof. William Harcourt-
Smith from the City University of New York for valuable
feedback on our shape analyses.
A U T H O R C ON T R I B U T I O NS
Christopher Smith: Conceptualization; data curation; for-
mal analysis; investigation; methodology; resources; soft-
ware; visualization; writing-original draft; writing-review &
editing. Christopher Wong: Data curation; methodology;
resources; writing-review & editing. Ian Curthoys: Con-
ceptualization; investigation; methodology; project adminis-
tration; resources; supervision; validation; writing-original
draft; writing-review & editing. Jeffrey Laitman: Concep-
tualization; project administration; supervision; validation;
writing-original draft; writing-review & editing. Payal
Mukherjee: Data curation; methodology; resources;
writing-review & editing.
V ID E O LE G E N D F O R LI N K ED V ID E O S
VIDEO 1: 3D visualizations of the membrana limitans
(ML) in nine humans (A-I) and one guinea pig (J). Struc-
tures include the bony labyrinth (transparent), utricular
macula (orange), saccular macula (red), facial canal (yellow
transparent). The ML is depicted in light blue. ML attach-
ments to bone are in yellow, while dark blue denotes edges
of the ML that do not attach to bone. Cube is 1 mm.
ORCID
Christopher M. Smith https://orcid.org/0000-0001-9259-
1079
Ian S. Curthoys https://orcid.org/0000-0002-9416-5038
Jeffrey T. Laitman https://orcid.org/0000-0002-9629-
946X
SMITH ET AL.
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How to cite this article: Smith, C. M., Curthoys,
I. S., Mukherjee, P., Wong, C., & Laitman, J. T.
(2021). Three-dimensional visualization of the
human membranous labyrinth: The membrana
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