University of Zurich
Zurich Open Repository and Archive
T h e entorh inal c ortex of M egac h iroptera: a c om parative study of
W ah lberg' s epauletted fruit bat and th e straw - c oloured fruit bat
G atom e , CW ; S lom ianka , L ; M w angi, D K ; Lipp, H - P; A m rein, I
G atom e , CW ; S lom ianka , L; M w angi, D K ; Lipp, H - P; A m rein, I ( 2 0 10 ) . T h e entorh inal c ortex of M egac h iroptera:
a c om parative study of W ah lberg' s epauletted fruit bat and th e straw - c oloured fruit bat. B rain S truc ture and
Func tion, 2 14( 4) : 37 5- 39 3.
Postprint available at:
h ttp: / / w w w . z ora. uz h . c h
Posted at th e Zuric h O pen Repository and A rc h ive, U niversity of Zuric h .
h ttp: / / w w w . z ora. uz h . c h
O riginally publish ed at:
B rain S truc ture and Func tion 2 0 10 , 2 14( 4) : 37 5- 39 3.
The entorhinal cortex of the Megachiroptera: A comparative study of
Wahlberg’s epauletted fruit bat and the Straw-coloured fruit bat
Catherine Gatome1, Lutz Slomianka1, Dieter K. Mwangi2, Hans-Peter Lipp1,
1Institute of Anatomy, University of Zurich, Winterthurerstrasse 190, Zurich, Switzerland
2Department of Veterinary Anatomy and Physiology, University of Nairobi, PO Box 30197 –
00100, Nairobi, Kenya
Number of Tables: 4 Number of Figures: 9
Running title: Entorhinal cortex of the Megachiroptera
Keywords: Epomophorus wahlbergi, Eidolon helvum, parvalbumin, SMI-32, Timm-staining,
*Correspondence to: Irmgard Amrein, Institute of Anatomy, University of Zurich,
Winterthurerstrasse 190, 8057 Zurich. E-mail: email@example.com, Telephone: +41
(0)44 6355340, Fax: +41 (0)44 6355702.
Supporting Grants: Rita Levi Montalcini Fellowship for African Women in Neuroscience,
International Brain Research Organisation, National Centre for Competence in Research
(NCCR) Neural Plasticity and Repair, and Swiss National Science Foundation.
Entorhinal cortex EC
Medial entorhinal cortex MEA
Lateral entorhinal cortex LEA
Caudal-limiting entorhinal field ECL
Caudal entorhinal field EC
Intermediate entorhinal field EI
Lateral entorhinal field EL
Rostral entorhinal field ER
This study describes the organisation of the entorhinal cortex of the Megachiroptera, Straw-
coloured fruit bat and Wahlberg’s epauletted fruit bat. Using Nissl and Timm stains,
parvalbumin and SMI-32 immunohistochemistry, we identified 5 fields within the medial
(MEA) and lateral (LEA) entorhinal areas. MEA fields ECL and EC are characterised by a
poor differentiation between layers II and III, a distinct layer IV and broad, stratified layers V
and VI. LEA fields EI, ER and EL are distinguished by cell clusters in layer II, a clear
differentiation between layers II and III, a wide columnar layer III, and a broad sublayer Va.
Clustering in LEA layer II was more typical of the Straw-coloured fruit bat. Timm-staining
was most intense in layers Ib and II across all fields, and layer III of field ER. Parvalbumin-
like staining varied along a medio-lateral gradient with highest immunoreactivity in layers II
and III of MEA and more lateral fields of LEA. Sparse SMI-32-like immunoreactivity was
seen only in Wahlberg’s epauletted fruit bat. Of the neurons in MEA layer II, ovoid stellate
cells account for ~38%, polygonal stellate cells for ~8%, pyramidal cells for ~18%, oblique
pyramidal cells for ~6%, and other neurons of variable morphology for ~29%. Differences
between bats and other species in cellular make-up and cytoarchitecture of layer II may relate
to their 3-dimensional habitat. Cytoarchitecture of layer V in conjunction with high
encephalisation and structural changes in the hippocampus suggest similarities in efferent
hippocampal-entorhinal-cortical interactions between fruit bats and primates.
Although detailed descriptions of the entorhinal cortex (EC) are available for most of the
common species used in neuroscience research, they comprise only a small segment of the
mammalian radiation. To our knowledge, no contemporary descriptions are available for one
of the most species-rich mammalian orders, the Chiroptera. The few available studies date
back to the beginning of the 20th century. Rose (1912) described this region as bearing a
strong semblance to the EC of other mammals. Brodmann (1925) identified a medial and
lateral subdivision in a megachiropteran species, Pteropus edwardsi. Later, Rose (1926) in
his study of a microchiropteran species, Vesperugo pipistrellus, identified an additional
subdivision, which he named area entorhinalis intermedia. In recognizing only the medial and
lateral entorhinal areas, early works generally do not conform to the parcellation of the EC
developed more recently in other species. Buhl and Dann (1991) traced entorhinal afferents to
the hippocampus in two megachiropteran species, but lacked information on the entorhinal
fields and therefore could not draw conclusions on topographical organisation of the
entorhinal-hippocampal connections. Here we provide a description of the EC in
Megachiroptera (fruit bats), which is comparable with the parcellation that has been derived
from recent cytoarchitectural and connectional studies in other species. The inclusion of two
closely related species, Wahlberg’s epauletted fruit bat (Epomophorus wahlbergi) and the
Straw-coloured fruit bat (Eidolon helvum), was prompted by perceptible differences between
the species in the EC.
While the debate on the phylogenetics of the Chiroptera lingers on, the argument for a
common ancestor of the Megachiroptera and primates (Maseko and Manger 2007; Pettigrew
et al. 1989, 2008), and hence a diphyletic origin of the Mega- and Microchiroptera, has been
weakened by mounting evidence from recent molecular phylogenetic analyses (Jones and
Teeling 2006; Teeling et al. 2000). These favour a monophyletic origin of the two chiropteran
suborders (Simmons et al. 2008). That notwithstanding, the Megachiroptera do share a
number of brain characteristics with primates (Johnson et al. 1994; Lapointe et al. 1999). Of
the analysed characters, most are related to the visual system. The Megachiroptera have a
distinctly primate-like retinotectal pathway; retinal input into the midbrain is decussated so
that only the contralateral hemifield is represented. The location of the lateral geniculate
nucleus magnocellular layers adjacent to the optic tract and the presence of a middle temporal
visual area are characteristics only described in the primates, Megachiroptera and Dermoptera
(Pettigrew et al. 1989). More recently, features of the cholinergic, catecholaminergic and
serotonergic systems were found to exhibit many similarities to those in primates (Maseko et
al. 2007). A general evolutionary trend of the megachiropteran brain has been the
enlargement of the telencephalon, most notably the neocortex - a trend which is also seen in
primates (Baron et al. 1996b). Several cytoarchitectural characteristics of the hippocampus
also resemble that of primates (Buhl and Dann 1991; Rosene and Van Hoesen 1987).
Considering the homologies in brain structure, the Megachiroptera may present a small
mammal model for investigating primate-like neural phenomena.
In the rat and mouse, grid cells have been described in layers II and III of the dorsocaudal
region of the medial EC (Fyhn et al. 2008; Fyhn et al. 2004). Metric information is integrated
in the entorhinal cortex by the grid and head-direction cells and fed to the hippocampal place
cells (Jeffery 2007; Moser et al. 2008; O'Keefe and Burgess 2005). Place cells have recently
been described in moving echolocating bats (Ulanovsky and Moss 2007), and it is probable
that they are also modulated by grid cells. Bats may offer the possibility to study the spatial
firing properties of the grid cells in 3-dimensions. The specific neuronal phenotype of grid
cells has not been identified. Physiological characterisation of the possible phenotypes has
focused on the classical stellate and pyramidal cell (Alonso and Klink 1993). Based on
morphology, our study estimates the relative contribution of neuronal phenotypes to the cell
population of layer II.
MATERIALS AND METHODS
Wahlberg’s epauletted fruit bats were captured in Nairobi, Kenya, and Straw-coloured fruit
bats were captured in Kampala, Uganda. Permits and licences were granted by the National
Museums of Kenya and the Uganda National Council of Science and Technology (No.
024/07/1). The animals were aged as adults based on the following criteria: closure of the
femoral and humeral epiphyseal plate, body weight above 60 grams (Wahlberg’s epauletted
fruit bat) or 200 grams (Straw-coloured fruit bat), forearm length over 70 mm (Wahlberg’s
epauletted fruit bat) or 110 mm (Straw-coloured fruit bat) and sexual maturity (evidence of
lactation or pregnancy). The actual ages of the animals are not known, although individuals
of this family have lived in captivity for over 20 years (Kingdon 1984).
Four adult female animals of each species were used in this study (Straw-coloured fruit bat:
mean body weight, 267 grams, mean brain weight, 4.0 grams; Wahlberg’s epauletted fruit
bat: mean body weight, 82 grams, mean brain weight, 1.9 grams).
Tissue preparation: Animals were deeply anaesthetized with sodium pentobarbital
(Nembutal®, 60 mg/ml; 50 mg/kg) and transcardially perfused first with heparinised 0.9%
saline, followed by 0.6% sodium sulphide solution and then cold 4% paraformaldehyde in 0.1
M phosphate buffer with 15% picric acid. Brains were removed and post-fixed overnight at
4°C in fixative and then transferred to 30% sucrose for 24 hours. The right hemisphere was
processed for immunohistochemistry, while the left hemisphere was embedded in
glycolmethylacrylate (see below).
Immunohistochemistry: Staining batches always contained sections of both species.
Antibody concentrations that gave the best signal to background ratio were determined by
Forty micron sections in the coronal plane were cut into twelve series using a cryotome.
Sections from three series were rinsed in tris-triton buffer (pH 7.4), pre-treated in 0.6%
hydrogen peroxide, rinsed and pre-incubated in normal goat serum for one hour. Two series
were incubated for two nights with SMI-32 monoclonal antibody (1:5000, SMI-32R
Sternberger Monoclonals Inc., Maryland) and one series with monoclonal anti-parvalbumin
antibody (1:30000, P-3171, Sigma, Missouri). This was followed by rinsing with tris buffer
(pH 7.4), incubation at room temperature for two hours with biotinylated goat anti-mouse
immunoglobulin (1:1000, Vectastain® Elite ABC Kit, Vector Laboratories Inc, California)
and incubation for 35 minutes with avidin-biotin-peroxidase complex (Vectastain® Elite
ABC Kit). Immunoreaction was detected using diaminobenzidine staining.
Sections from one SMI 32-immunostained series were counterstained with Giemsa solution
The monoclonal anti-parvalbumin (mouse IgG1 isotype) used is derived from the PARV-19
hybridoma cells. Purified frog muscle was used as the immunogen. The antibody reacts with
parvalbumin of human, bovine, goat, pig, rabbit, dog, cat, rat, frog, and fish (Sigma,
Missouri). The monoclonal anti-SMI-32 is derived from mouse ascites fluid. Homogenised
hypothalami of rats were used as the immunogen. The antibody reacts with a non-
phosphorylated epitope in neurofilament H (180 and 200kDa) of most mammalian species
(Sternberger Monoclonals Inc., Maryland).
The immunoreactive structures corresponded in appearance and general distribution to those
observed in other mammalian species. However, as we cannot be certain of the epitope(s)
recognised by the antibodies in the bat, we refer to the observed immunoreactivity as
parvalbumin-like and SMI-32-like.
To control for non-specific staining related to the secondary antibodies and avidin-biotin-
peroxidase labeling, controls from each species, in which the primary antibody was omitted,
was included in the experiment.
Nissl and Timm staining: Brains were dehydrated in ethanol (70% x 5 hours, 96% x 5 hours,
100% x 48 hours), infiltrated in four changes (24 hours, 3 days, 1 week, 1 week) of
glycolmethylacrylate solution (Technovit 7100, Kulzer GmbH, Wehrheim, Germany) and
embedded. Twenty micron horizontal sections were cut and every sixth section was mounted
and dried for 1 hour at 60°C. Sections were stained with Giemsa solution (Merck, Darmstadt,
Germany) diluted 1:10 in 0.07 M KH2PO4 buffer at room temperature for 1 hour (Iñiguez et
al. 1985). They were then differentiated for 10 seconds in 1% acetic acid followed by 10
seconds in 96% ethanol. Sections were dehydrated in two changes of absolute ethanol,
cleared and coverslipped.
A second series was developed in Timm’s solution prepared from a mixture of 120 ml gum
arabic solution (50% weight/volume in distilled water), 20 ml citrate buffer solution (pH 5),
60 ml of 0.5% hydroquinone solution and 1 ml of 17% silver nitrate solution. After 3 hours
incubation at 37°C, the slides were rinsed in tap water, fixed for 1 minute with 1% sodium
thiosulfate and then dehydrated, cleared and coverslipped.
MEA layer II cell numbers and size
Layer II of medial entorhinal cortex was delineated on 20 µm glycolmethylacrylate-
embedded Nissl-stained horizontal sections. Using the StereoInvestigator® software
(MicroBrightfield Inc, Colchester, Vermont), total cell numbers were estimated using the
Optical Fractionator (West et al. 1991) with a 40x oil-immersion objective (N.A. 1.3) in
every 6th (Wahlberg’s epauletted fruit bat) or 12th (Straw-coloured fruit bat) section with a
counting frame of 40 µm x 40 µm, a disector height of 10 µm and x,y-steps of 100 µm.
Section thickness was measured at every 6th sampling site. Cell numbers were estimated
using number-weighed section thickness (Dorph-Petersen et al. 2001).
The areas of cell profiles were estimated using the Nucleator method (Tandrup 1993) for cells
counted in the disector probes. Cell counts and size measurements were made in 4 animals of
either species. Coefficients of error for number estimates were calculated according to
Gundersen et al. (1999) using the conservative m=0 approach (Slomianka and West 2005).
Statistical analysis were done using SPSS Statistics version 17.0.0 (SPSS Inc., Chicago,
Illinois). Cell sizes were compared by an ANOVA using cell type and species as fixed
factors. Post-hoc comparisons of cell sizes were made using LSD and Bonferroni tests, with a
significance level of P < 0.05.
Images of horizontal and coronal sections were captured at 5x to 40x magnification using a
MBF CX9000 camera (MicroBrightField Inc., Colchester, Vermont). Adjustments were
made to the brightness and colour of the images to restore their appearance in the microscope.
No local changes were made unless noted in the figure legends. For 3-dimensional modelling,
EC fields were traced in Nissl-stained horizontal sections and assigned colours. Images were
aligned using AutoAligner 6.0.0 (Bitplane AG, Zurich, Switzerland) and a 3-dimensional
representation was produced using Imaris 6.2.0 (Bitplane AG, Zurich, Switzerland).
For the delineation of the EC, we initially consulted a Megachiroptera brain atlas (Baron et
al. 1996a). Our description of the laminar structure follows Amaral et al. (1987), who used a
composite of the descriptions provided by Ramón y Cajal (1988) and Lorente de Nó (Lorente
de Nó 1933). We have adopted Brodmann’s cytoarchitectonic division of the EC into lateral
entorhinal area (LEA; area 28a) and medial entorhinal area (MEA; area 28b) based on a
distinction between layer II and layer III, which is clear in LEA but not in MEA (Brodmann
1909). The apparent size and position of the entorhinal fields, as they appeared in the 3-D
reconstructions, matched best with the 2-D map provided by Amaral et al. (1987), and their
nomenclature was adopted. We could however not consistently distinguish the ELc and ELr
fields and treat them here collectively as EL. In the bats, the EL field lies in between fields EI
and ER. Also, the EO field, which appears as a small area rostral to ER in primates, could not
be consistently distinguished from the ER field. MEA is comprised of caudal-limiting (ECL)
and caudal (EC) entorhinal fields. LEA is constituted by two lateral fields, rostral (ER), lateral
(EL), and a transitional area, the intermediate (EI) entorhinal field.
One possible alternative to the primate nomenclature would have been that developed in rats
(Insausti et al. 1997) and mice (van Groen 2001). However, most subfields identified based
on histoarchitectural criteria in bats are very difficult to align topographically with those
identified in rats and mice, which cast doubts on a possible homology of these fields. For
example, the ME field is prominently ventral in location, in contrast to the EC field in the bats
which extends proportionally from dorsal to ventral. The lateral fields, DIE and DLE, extend
rostro-caudally but in bats, as in primates, the corresponding fields, EL and ER, extend
mediolaterally, obliquely to the rhinal sulcus. Although functional specialisations of the EC
necessarily will result in differences in the detailed structure of subfields between species or
larger phylogenetic groups, which have justified species-specific nomenclatures (e.g., guinea-
pig: (Uva et al. 2004); dog: (Wóznicka et al. 2006)), we do not believe that this is a
productive approach in the long-term for a cortical region that most likely shares basal
functional aspects in all mammals.
Cellular morphological phenotypes were distinguished based on the earlier descriptions of
Ramón y Cajal (1988), Germroth et al. (1991), Klink and Alonso (1997a), and Schwartz and
Location and delineation of the entorhinal cortex
The location of the entorhinal cortex in the species investigated here corresponds to
Brodmann’s (1925) description of the area 28 in Pteropus, a related fruit bat species (Fig. 1a,
b). Three–dimensional representations of the EC in Wahlberg’s epauletted fruit bat and the
Straw-coloured fruit bat show the location and relationships of the fields (Fig. 1e-h). This
corresponds to a caudo-ventral and lateral location of the EC in the piriform lobe (Fig. 1c, d).
The rostral and lateral boundary of the EC is formed by the perirhinal and prepiriform cortex.
Caudally and medially, it borders the para- and presubiculum. The ventro-medial boundary is
delimited by part of the hippocampo-amygdaloid transition.
Medial entorhinal area (MEA)
Laminar Structure: Generally, layer I varies little. Layer II is not well delineated from layer
III. A cell-sparse layer IV (lamina dissecans) is visible throughout the MEA, and layer V and
VI are wide and densely populated (Fig. 2a, b).
Caudal-limiting entorhinal field (ECL): This is the most caudal field of MEA, bordering the
para- and presubiculum caudally and medially (Fig. 2a, b). It extends medio-laterally and
briefly apposes the postrhinal cortex laterally (Fig. 1g, h).
Cytoarchitecture: In both species, layer I is broad and has small light-staining neurons that
are sparsely distributed. Layer II is well delineated from layer I and is densely populated in
both species (Fig. 2c, d). Layer III is patchily populated by neurons of similar size. It is
separated from layer V by a distinct, cell-sparse layer IV. Layer V is wide and has three
sublayers. Sublayer Va is well-developed and loosely populated by large intensely staining
pyramidal neurons, and appears stratified in the Straw-coloured fruit bat (Fig. 2a, c). Sublayer
Vb has a dense population of small moderately staining cells (Fig. 2c, d). Sublayer Vc is
inconsistently visible as a narrow, cell-sparse zone separating layer V from layer VI. The
three sublayers are more obvious in the Straw-coloured fruit bat (Fig. 2a, c). Layer VI is wide
and densely populated by small intensely staining neurons. Both layers V and VI are
organized in radial columns. In both species, layer VI is not well demarcated from the white
Chemoarchitecture: Timm-staining of layer I of ECL is pale superficially and dark innermost,
dividing layer I into two parts, Ia and Ib (Fig. 3a, b). This pattern is recurrent in all medial
and lateral entorhinal fields. Staining decreases from a dark layer II adjacent to layer I to a
moderately stained deep layer III. This decrease is gradual in the Straw-coloured fruit bat,
whereas is appears more sudden, about half-way in layer III, in Wahlberg’s epauletted fruit
bat. Layer III is separated from layer V by a narrow light staining band corresponding to
layer IV. Sublayer Va stains moderately in contrast to the lightly stained layers Vb and VI.
There are no further apparent differences between the two species.
A sharp decrease in parvalbumin-like immunoreactivity marks the border of the caudal-
limiting field towards the parasubiculum (Fig. 4c, d). Layer I has several processes oriented
perpendicular to the cortical surface, some of which appear smooth or beaded (Fig. 5a). Layer
II has few polygonal cell bodies with radial processes directed into layers I and III (Fig. 5a,
b). The majority of cell bodies are located either proximally or distally in layer II. An
intensely staining, dense fibre plexus extends in layers II and III (Fig. 5a). Layer III contains
several cell bodies that are variable in size and shape (Fig. 5a, c-e). These include medium-
sized to large polygonal cell bodies with vertically directed processes into layer II and IV,
and spherical cell bodies with horizontal and ascending processes (Fig. 5c-e). Medium-sized
to large polygonal cell bodies with radial processes that can be traced within the layer and
into layer II are also observed (Fig. 5e). Most of these cell bodies are located in the lower half
of layer III. More medial and caudal, immunoreactive cell bodies in layer III send processes
into the parasubiculum (Fig. 5f). Staining sharply drops at the border with layer IV (Fig. 5c).
Layer IV has few small cell bodies with radiating processes and spindle-shaped cell bodies
with short ascending and descending processes (Fig. 5g). Processes from cell bodies in the
layers III and V cross through layer IV (Fig. 5c, h). Layer V has little reactivity. Large and
medium-sized polygonal and conical cell bodies with processes directed into layers III, VI,
and white matter are observed (Fig. 5i, j). A sparse fibre plexus is observed in layers V and
VI (Fig. 5j, h, k). Cells are rare in layer VI (Fig. 5k). There are no apparent species
In Wahlberg’s epauletted fruit bat, a SMI-32-like immunoreactive fibre plexus and few cell
bodies are present in layers II and III (Fig. 4f). SMI-32-like immunoreactivity is not observed
in the EC of the Straw-coloured fruit bat (Fig. 4e), while characteristic SMI 32-like staining
of neocortical neurons is present in this species (Fig. 5n).
Caudal entorhinal field (EC): This is the central field of the MEA, located lateral to field
ECL and adjacent to the LEA (Fig. 2a, b). Dorso-laterally it apposes the perirhinal cortex and
parasubiculum ventro-medially (Fig. 1g, h).
Cytoarchitecture: Layer I is broad and well delineated from the layer II. Layer II is wide,
moderately cell-dense, and some layer II cells scatter into the deep part of layer I. It is less
cell-dense compared to layer II in ECL. Similar to ECL, cells are homogeneously distributed
within the layer (Fig. 2a, b, e, f). In the Straw-coloured fruit bat (Fig. 2e), and Wahlberg’s
epauletted fruit bat (Fig. 2f) layer II is populated by varied neuronal phenotypes. Cell-sparse
zones between layer II and III are infrequent. Layer III pyramidal neurons are homogenously
sized and organised in columns. Layer IV is prominent although narrower in comparison to
ECL. Layer V is wide and sublayers Va and Vb are visible; Va is loosely populated and has
the stratified appearance observed in field ECL in the Straw-coloured fruit bat (Fig. 2e), and
Vb is closely apposed to layer VI. Layers V and VI of EC narrow in comparison to ECL, but
are also organized in a radial columnar pattern. In both species, layer VI is not sharply
demarcated from the white matter.
Chemoarchitecture: The Timm-staining of EC shows similarity in pattern and intensity to
field ECL (Fig. 3a, b). Parvalbumin-like immunoreactivity decreases in comparison to ECL, but
the staining pattern is the same (Fig. 4c, d). Layer I has immunoreactive processes, and the
fibre plexus in layers II and III stains intensely. Cell bodies are observed in layers II to VI.
SMI-32-like staining is weak in Wahlberg’s epauletted fruit bat (Fig. 4f), and absent in the
Straw-coloured fruit bat (Fig. 4e).
Lateral entorhinal area (LEA)
The LEA approximates the perirhinal cortex rostrally and dorsally, the pre-piriform cortex
ventrolaterally, and the hippocampo-amygdaloid transition area ventromedially.
Laminar structure: Within the three fields of the LEA, variations in cellular density and
arrangement across the layers are observed (Figs. 6a, b, 7a, b). Neurons in layer II group
together in patches and clusters. A narrow cell-free zone is visible between layer II and III,
especially in more lateral and rostral fields. Layer IV is generally narrow and visible mainly
in field EI, adjacent to the MEA. Layers V and VI are narrower and organised in rows.
Sublayer Va is wide in most of this subdivision. Layers Vb and VI in the LEA are narrow and
closely apposed in comparison to those in the MEA.
Intermediate entorhinal field (EI): This field is the most caudal and medially located of the
LEA (Fig. 6a, b). It is dorso-medial to rhinal sulcus and it first apposes the perirhinal cortex
dorso-medially and subsequently field EL (Fig. 1g, h). Characteristics of both the MEA and
LEA are observed in the laminar organization of EI; the superficial layers share
characteristics with the LEA, while the deep layers show characteristics more consistent with
MEA. This field could therefore be termed a transition area. It extends to the ventral pole of
the EC in the Straw-coloured fruit bat (Fig. 1c, e), apposing the parasubiculum ventro-
medially and the hippocampal-amygdaloid transition area ventro-rostrally. It is only found in
the dorsal one-half of the EC in Wahlberg’s epauletted fruit bat (Fig. 1d, f).
Cytoarchitecture: Layer I is broad and has lightly stained sparsely distributed small neurons.
Layer II is wide (Fig. 6a, b). Neurons disperse from layer II into layer I and some of them
appear as ectopic neurons in layer I (Fig. 6c-f). Small dark staining cells are noted in
Wahlberg’s epauletted fruit bat (Fig. 6f). Occasionally narrow cell-sparse zones are found
within layer II and between layers II and III. Neurons in layer III are more evenly distributed
than in field EC, and superficially (sublayer IIIa) neurons are larger than those deeper in the
layer (Fig. 6c, d). Layer IV is prominent in both species. Sublayer Va in EI is comparable in
width to EC. The large pyramids are stratified within the layer in the Straw-coloured fruit bat
(Fig. 6a, c), but appear closely associated with sublayer Vb in Wahlberg’s epauletted fruit bat
(Fig. 6b, d). Deeper, layer V is confluent with layer VI and these layers are narrower
compared to those in EC, but retain the radial columnar organisation (Fig. 6c, d). Layer VI
gradually merges with the white matter in the Straw-coloured fruit bat (Fig. 6a, c).
Chemoarchitecture: In both species Timm-staining pattern and intensity in EI, is similar to
that of the ECL and EC fields (Figs. 3a, b, 8a, b). A pale layer IV is occasionally observed
(Fig. 8b). A moderately stained layer V is present (Fig. 8a, b). Parvalbumin-like staining is
faint, comprising a loose fibre plexus, few cell bodies and processes in layers II to V (Fig. 9c,
d). SMI-32-like staining is weak in Wahlberg’s epauletted fruit bat (Fig. 9f) and absent in the
Straw-coloured fruit bat (Fig. 9e).
Lateral-rostral entorhinal field (EL): This field is rostral to EI. It apposes the perirhinal
cortex dorso-laterally, and is caudal to ER ventrally (Fig. 7a, b). Ventro-medially it apposes
the presubiculum and subsequently the hippocampal-amygdaloid transition area.
Cytoarchitecture: Layer I is broad and ectopic layer II neurons are common (Fig. 7c, d).
Layer II in the Straw-coloured fruit bat has dense cell clusters that are separated by areas of
low cell density (Figs. 6a, 7a). Although there are cell dense areas within layer II of
Wahlberg’s epauletted fruit bat, variations in cell density are much less pronounced than in
the Straw-coloured fruit bat (Figs. 6b, 7b). A narrow cell-sparse zone is infrequently visible
between layers II and III. In both species, layer III is wide and moderately populated. In the
Straw-coloured fruit bat, neurons are more dispersed than in field EI (Fig. 7a). In both
species, superficially (sublayer IIIa), neurons are densely distributed and appear larger
compared to those deeper in the layer (Fig. 7c, d). Layer IV is sometimes present. Layer Va is
wider and more populated than in EI in both species (Figs. 6a, b, 7a, b). Sublayer Vb and
layer VI are closely apposed, but, in contrast to EI, they do not appear laminated (Fig. 7c, d).
Layers Vb and VI are narrower in comparison to those in EI, which is more marked in the
Straw-coloured fruit bat (Fig. 7a).
Chemoarchitecture: A strong Timm reaction is noted in the EL field in the Straw-coloured
fruit bat (Fig. 8a), and less so in Wahlberg’s epauletted fruit bat (Fig. 8b). A pale sublayer Ia
contrasts with a darkly stained sublayer Ib. Layers II and III stain moderately in Wahlberg’s
epauletted fruit bat and darkly in the Straw-coloured fruit bat. A moderately stained layer Va
is observed. Layers Vb and VI are lightly stained. In both species, parvalbumin-like
immunoreactive processes and cell bodies stain with moderate intensity (Figs. 4c, d, 9c, d). In
Wahlberg’s epauletted fruit bat, SMI 32-like weakly stained cell bodies and processes in
layer III are noted (Fig. 4f). No staining is observed in the Straw-coloured fruit bat (Figs. 4e,
Rostral entorhinal field (ER): Rostrally, this lateral field is located close to a very shallow
rhinal sulcus, dorso-laterally adjoining the perirhinal (Figs. 1h, 6a, b) and prepiriform cortex
ventrolaterally (Fig. 1g, 7a, b).
Cytoarchitecture: In both species, layer I narrows and layer II is characterised by a decrease
in cell density in comparison to other fields (Figs. 6a, b, 7a, b). Layer II has two sublayers
separated by cell-sparse zones. This is more distinct in the Straw-coloured fruit bat (Figs. 6a,
7a), where superficially sublayer IIa neurons forms narrow and discontinuous bands (Fig. 7e,
g). Sublayer IIb is wider than IIa. Cell clusters and areas of lower cell density alternate in a
manner similar to layer II in EL (Fig. 7a, e). Frequent narrow cell-sparse zones extend
through layer II into layer III (Fig. 7e). In Wahlberg’s epauletted fruit bat, IIa neurons are
few, scattered and tend to be located close to IIb (Figs. 6b, 7b). Sublayer IIb is more cell-
dense (Fig. 7f, h) than in the Straw-coloured fruit bat, and cell clusters are not observed (Fig.
7b, f). Layer II narrows and appears to merge with layer III at the junction of the EC with the
perirhinal (Fig. 6a, b) and pre-piriform cortex (Fig. 7a, b). Elsewhere, layer II is separated
from layer III by cell–sparse zones. Layer III is wide and patchily organised superficially,
less noticeably in Wahlberg’s epauletted fruit bat (Fig. 7e, f). Deeper, layer III has a
population of smaller pyramidal neurons. Layer IV is infrequently observed in Wahlberg’s
epauletted fruit bat and cannot be distinguished in the Straw-coloured fruit bat. Sublayer Va
is wider and more populated in comparison to field EL (Fig. 7a, b). The large pyramidal
neurons are stratified within the layer in the Straw-coloured fruit bat (Fig. 7a, e), but this
organisation is less obvious in Wahlberg’s epauletted fruit bat (Fig. 7b, f). Layers Vb and VI
have the same width as those in EL in Wahlberg’s epauletted fruit bat (Fig. 7b), but these
layers thin along the rostral-lateral extent in the Straw-coloured fruit bat (Fig. 7a). A
columnar organisation of layers Vb and VI is observed in the Straw-coloured fruit bat (Fig.
Chemoarchitecture: Field ER shows a pale layer Ia, dark Ib, II and III, moderately staining Va
and pale Vb and VI (Fig. 8a, b). The Timm-staining pattern and intensity is similar to that of
field EL in the Straw-coloured fruit bat (Fig. 8a). Darkly stained protuberances are observed
in sublayer Ia, giving a wavy appearance (Fig. 8a, b). Parvalbumin-like immunoreactivity in
ER is strong (Fig. 9c, d), with a pattern that is similar to that of ECL (Fig. 4c, d). The fibre
plexus is intensely stained, as are the cell bodies and processes in layers II to V. Layer VI is
weakly reactive. Parvalbumin-like reactivity decreases at the border between ER and
perirhinal (Fig. 9c, d) and pre-piriform cortex (Fig. 4c, d). In Wahlberg’s epauletted fruit bat,
SMI 32-like staining is observed between layers II and VI (Figs. 4f, 9f). Fine and sometimes
thick, vertically oriented processes run between the layers and cross the white matter. Layer
II has few cell bodies. A band of conical and polygonal cell bodies is seen in the deep part of
layer III (Figs. 4f, 5l-m). Some of these cell bodies are small, stain lightly and have apical
and basal processes that can be traced for a short distance within the layer. Other cell bodies
are larger, stain intensely, and their radiating processes can be traced in adjacent layers (Fig.
5l, m). Layer V has rare stained large cell bodies. The intensity of staining increases rostrally
in this field.
Cell numbers and phenotypes in MEA layer II
Morphological phenotypes: Ovoid stellate cells are spherical and oriented vertically (Fig. 2g).
Large, trapezoid stellate cells that are either transversely or vertically oriented are referred to
as polygonal stellate (Fig. 2h). Pyramidal cells are medium-sized to large, conical bodies with
a large apical process oriented perpendicular to the pial surface (Fig. 2i). Oblique pyramidal
cells resemble the pyramidal cells but have an apical process that is directed obliquely to the
pial surface (Fig. 2j). ‘Other’ cells include generally small cells of tripolar (Fig. 2k), round
(Fig. 2l), bipolar (Fig. 2m), or fusiform (Fig. 2n) appearance and medium-sized multipolar
neurons. Cells of this class are located mostly superficially in layer II.
Sectional areas of neuronal phenotypes and the contribution of these phenotypes to the layer
II cell population are listed in table 1. Significant main effects were found for species, cell
type and species × cell type interactions (p < 0.001 for all comparisons), with Wahlberg’s
epauletted fruit bat having a larger mean cell area for the polygonal stellate and pyramidal
cell types, while ‘other cells’ are smaller in this species. In the comparison of cell sizes,
stringent Bonferroni and lenient LSD post-hoc testing produced identical outcomes in terms
of significant differences between phenotypes. Ovoid stellate cells are different in size from
all other cell types (pLSD < 0.001 for all comparisons); polygonal stellate, oblique pyramidal
and pyramidal cells are similar in size (polygonal stellate vs. pyramidal : pLSD = 0.46,
polygonal stellate vs. oblique pyramidal : pLSD = 0.37, pyramidal vs. oblique pyramidal : pLSD
= 0.09) but different from other cell types (pLSD < 0.001 for all other comparisons).
Layer II in the Straw-coloured fruit bat has an estimated mean total cell count (Table 2) of
62,022 (SD 8469, CE = 0.08), and in Wahlberg’s epauletted fruit bat 30,675 (SD 2470, CE =
We have provided a detailed description of the laminar and areal organization of the
megachiropteran entorhinal cortex using markers that have helped to define these regions in
other species as well. In the following, the differences between the two bat species, rodents
and primates, summarized in Table 3, are discussed from an anatomical and functional
Medial entorhinal area (MEA): Layer II of the most caudal and medial field is broad,
continuous and well-delineated from layer I above in the bats, primates and rodents. The
islands described in the adjacent field, EC (primates), (Amaral et al. 1987) are not well-
developed in the bats and rodents (Insausti et al. 1997). Layer III is similarly organised in the
bats, primates and rodents, starting off patchy caudally, but progressively adopting a
columnar arrangement in EC and in the less caudal MEA field (ME) in rodents. Differences in
cellular density in IIIa and IIIb are described in ME, but this is not observed in the bats nor
described in the primates. Layer IV is well-developed in the bats and rodents, but poorly
visible or absent in the primates. Layer V in the bats, primates and rodents is well developed,
with sublayers Va and Vb. The pyramidal cells of Va appear as 1 - 2 bands in Wahlberg’s
epauletted fruit bat, and appear closely apposed to Vb, which is also observed in rodents.
Sublayer Va in the Straw-coloured fruit bat is as well developed as in primates, showing 3-4
bands of pyramids. Sublayer Vc which is prominent in the medial fields in primates, is
infrequently visible in the bats and not described in rodents (Amaral et al. 1987; Blaizot et al.
2004; Insausti et al. 1997; van Groen 2001).
Lateral entorhinal area (LEA): Of the lateral fields, EI shows similarity between the two bat
species. The intermediate field is referred to as VIE in the rodents (Insausti et al. 1997).
Similar characteristics found in rodents, primates and bats include layer II clusters and a
patchy layer III. Layer II islands are a feature in the primates that is absent in both bats and
rodents. The bilaminar layer III, presence of layer IV, and a well-defined Va are
characteristics shared by the bats and primates in EI, but not rodents (Amaral et al. 1987;
Insausti et al. 1997; van Groen 2001).
Clustering in layer II is observed in EL field in the Straw-coloured fruit bat, primates, and the
corresponding DLE in rodents (Amaral et al. 1987; Insausti et al. 1997; van Groen 2001). If
clustering is a distinctive feature of this subdivision, then Wahlberg’s epauletted fruit bat has
a poorly differentiated layer II. Cell-sparse zones infrequently separate layers II and III in the
rodents and bats, but only in a caudal part of the LEA in primates. Layer III has a bilaminar
character, observed in rodents and bats, but not in primates. While layer III is narrow in the
rodents, it is wide in the bats and primates. Layer IV is infrequently visible in the bats and
rodents and absent in the primates. Layer Va is prominent in the bats and primates (Amaral et
al. 1987). Layer V in rodents is narrow and comprised mostly of sublayer Vb, and few
pyramids in Va (Insausti et al. 1997; van Groen et al. 2003). Layer VI has a less stratified
appearance in bats, primates, and rodents.
The layer II clusters in ER field are separated by cell-sparse zones into indistinct islands in the
Straw-coloured fruit bat, but they do not develop into the wide cell islands distinctive of
primate LEA layer II (Amaral et al. 1987; Blaizot et al. 2004). The corresponding field in
rodents, DIE (Insausti et al. 1997; van Groen 2001) appears similar to the Straw-coloured
fruit bat. Cell-sparse zones separating layers II and III in the ER field described in the
primates and rodents are observed in the bats, less frequently in Wahlberg’s epauletted fruit
bat. The wide and bilaminar character of layer III in the bats is consistent with descriptions
made in related fields in primates and rodents. In the Straw-coloured fruit bat and primates,
layer IV is not visible in the ER field (Amaral et al. 1987). Similar observations have been
made in a corresponding field, Pr2 in the rhesus monkey (van Hoesen and Pandya 1975a).
Layer IV is present in the mouse (van Groen 2001) and infrequently observed in Wahlberg’s
epauletted fruit bat and the rat (Insausti et al. 1997). Layer Va is wide and has 3-4 rows of
pyramids, more so in the Straw-coloured fruit bat, resonating with descriptions made in
primates (Amaral et al. 1987; Blaizot et al. 2004). The pyramids associated with this layer
appear incidentally in rodents (Insausti et al. 1997; Mulders et al. 1997). All 3 sublayers of
layer V are only observed in primates. Layers Vb and VI narrow rostrally in the Straw-
coloured fruit bat. In the rat, only layer VI narrows (Insausti et al. 1997). In Wahlberg’s
epauletted fruit bat, mouse (van Groen et al. 2003) and primates (Amaral et al. 1987), layer
VI is well developed.
Timm-staining reveals the laminar organisation of the EC, which assisted in delineating the
EC from adjacent structures, and distinguished between medial and lateral subdivisions and
their layers. In the two species, dark staining is observed in layers Ib and II in all fields, and
part of layer III in the ER field. Layer Va stains moderately and layers IV, Vb and VI are pale.
The staining pattern corresponds to the presence of zinc in the boutons of the terminals of
telencephalic afferents to the EC (Perez-Clausell and Danscher 1985; Slomianka 1992),
where zinc may serve as a neuromodulator of excitatory transmission (Frederickson et al.
2005; Paoletti et al. 2009). The distribution of zinc-containing boutons in bats is comparable
to observations made in the guinea pig, dog, and pig (Geneser-Jensen et al. 1974; Holm and
Geneser 1989; Wóznicka et al. 2006). In the rat and mouse, the staining is moderate in layers
Ib and II of MEA and increases gradually across layer III to become very intense at the layer
III/IV boundary (Slomianka 1992; Slomianka and Geneser 1991).
Parvalbumin-like immunostaining varies along a medio-lateral gradient, and is comparable in
the two fruit bats. Staining mainly occurs in the ER, EL and medial fields, and an intermediate
reactivity in the EI field. Parvalbumin is present in GABAergic interneurons of the EC in
layers II, III, V and VI and predominantly expressed by chandelier and basket cells (DeFelipe
et al. 1989; Fonseca et al. 1993; Hendry et al. 1989). Observations in the fruit bats are
consistent with reported distributions of parvalbumin in rat (Wouterlood et al. 1995), guinea
pig (Uva et al. 2004), macaque (Pitkänen and Amaral 1993) and human EC (Beall and Lewis
1992; Tuñón et al. 1992). Thus, there does not seem to be much difference in the role of this
calcium binding protein and the part of the inhibitory circuitry identified by it in the EC
across species (Hof and Sherwood 2005).
SMI-32 recognises a high molecular weight (200kD) non-phosphorylated neurofilament
protein H (Sternberger and Sternberger 1983) in neuronal soma, dendrites and some large
calibre axons of specific subpopulations of neurons resulting in a distinct cellular and laminar
staining pattern. This protein also occurs in a subset of cortical neurons selectively lost in
Alzheimer’s disease (Hof and Morrison 1990; Morrison et al. 1987). In Wahlberg’s
epauletted fruit bat, immunoreactivity in particular defines layer III of the EC. Most of the
staining is observed in the ER and medial fields. Rostrally, the staining in layers II, III and V
concurs with observations made in the EL field of the macaque and human where a rostral-
caudal gradient was noted (Beall and Lewis 1992; Saleem et al. 2007). The prevalent staining
of layer III is in contrast to the macaque, where it mainly involves layers II and V of LEA
(Saleem et al. 2007). A comparative analysis in the visual cortex shows similarities in
regional staining patterns between closely related species; these patterns are shown to be
predictive of evolutionary relationships (Hof et al. 2000). This may imply that the 2 bat
species may not be as phylogenetically close as we presume. It could also be that the EC is a
less adept model for this hypothesis, and a lack of immunoreactivity in the EC of Straw-
coloured fruit bat, may indicate that the function subserved by SMI-32 immunoreactive
cytoskeletal components may either not be required or is assumed by another protein
(Campbell and Morrison 1989; Goldstein et al. 1983). There are similar reports of a lack of
SMI-32 immunoreactivity in the EC that include an Australian echidna (Tachyglossus
aculeatus) and the Tamar wallaby (Macropus eugenii) (Ashwell et al. 2005; Hassiotis et al.
2004; Hassiotis et al. 2005).
Overall, the organisation of the EC cannot be categorized as either primate- or rodent-like,
rather the EC shows a mosaic of characters. The two bat species share, e.g., a well-developed
layer Va throughout the EC with primates, while they share with rodents the absence of
distinct layer II islands in most EC fields. Species-specific characteristics are largely
restricted to layer II, which is better differentiated in the MEA of Wahlberg’s epauletted fruit
bat, while it, in comparison to the Straw-coloured fruit bat, is poorly differentiated in the
LEA. Buhl and Dann (Buhl and Dann 1991) already noted a primate-like dispersion of
hippocampal pyramidal cells, which we also observed in the species investigated here, and
which in other species provide hippocampal afferents to the EC layer Va (Swanson and
Cowan 1977). The EC layer Va, in turn, provides afferents to neocortical areas (Swanson and
Fruit bats share with primates high indices for encephalization and neocorticalization as well
as high indices for the size of the schizocortex, which contains the entorhinal cortex as the
major contributing area (Table 4). Also, the relation between indices for the hippocampus and
schizocortex is very similar between bats and primates, but differ from that of the rat. It is
therefore not surprising that structural similarities exist in layer V of bats and primates. The
cortical and sub-cortical inputs into layer V, and intrinsic associational connections suggest
that it may play a more significant role in memory operations (Burwell and Amaral 1998;
Hamam et al. 2000; Hamam et al. 2002; Kerr et al. 2007; Suzuki and Amaral 1994). At least
for CA-entorhinal-cortical interactions bats may serve as a primate-like model.
If the morphology of layer II, which appears more rodent-like in structure, can be used as a
guideline, this is not true for cortical-entorhinal-dentate interactions. However, EC layer II
also differs between the two bats, and both bats apparently differ in the cellular composition
and relative sizes of MEA layer II from primates and rodents (see below). We previously
suggested that changes in the structure and connectivity of the phylogenetically rather pliable
dentate gyrus may subserve species-specific demands on hippocampal function (Slomianka
and Geneser 1991; Slomianka and West 1989). Species- and/or group specific changes in the
cortical region providing input to the dentate gyrus, as observed in the EC layer II of bats,
should therefore be expected.
MEA layer II neuronal phenotypes and proportions
Cytoarchitectonic descriptions of the MEA in the rat (Insausti et al. 1997), mouse (Slomianka
and Geneser 1991), dog (Wóznicka et al. 2006), macaque (Amaral et al. 1987), baboon
(Blaizot et al. 2004) and human (Insausti et al. 1995) indicate the stellate cell is the dominant
neuronal phenotype. Quantitative studies estimated that stellate cells constitute 67-81% of the
neuronal population and pyramidal cells between 16-32% in the rat and human MEA layer II
(Mikkonen et al. 2000; Schwartz and Coleman 1981). We found that this layer contains an
unusually large proportion of small cells (24 and 35%) that are neither stellate nor pyramidal.
The stellate cell in the bats, as the single dominant phenotype at 36 and 41% is well below
reported values of over 60%. However, estimates of the pyramidal cell at ~18% are rather
close to those of other species. Intracellular neuronal staining (Mikkonen et al. 2000) may
have been biased by the selection of large cells in restricted EC areas, while Schwartz and
Coleman (Schwartz and Coleman 1981) estimated proportions within only hippocampally
projecting layer II cells. The number-weighted selection of cells for classification using
disector probes at a uniform random systematic set of sampling sites throughout the EC
should be unbiased by cell size and distribution. However, methodological differences cannot
easily explain why one set of numbers is so different while another set is very close, and it is
likely that bats differ from rats and humans in the cellular composition of MEA layer II. To
pinpoint the nature of differences, a design-based stereological assessment of the cellular
composition in the other species is needed.
Neuron number in the rat was estimated to 6.6 × 104 in MEA layer II (Mulders et al. 1997).
This is almost twice the count in Wahlberg’s epauletted fruit bat, which has approximately
the same brain weight (2g) as a rat, and although comparable to the Straw-coloured fruit bat,
the brain weight (4g) is about twice that of the rat. It would be of interest to establish if the
input from layer II is shared by the dentate gyrus and CA2/3 region of the hippocampus in the
ratio (~17.7) reported in the primates and rat (Mulders et al. 1997). In the dentate gyrus, the
Straw-coloured fruit bat has an estimated mean total granule cell count of ~1,200,000 and
that of Wahlberg’s epauletted fruit bat is ~900,000 (C. Gatome, unpublished observations).
The ratios granule cells:MEA layer II of ~19 (Straw-coloured fruit bat) or ~29 (Wahlberg’s
epauletted fruit bat) show that large differences in the divergence of the projection from the
MEA layer II to the dentate gyrus can occur in closely related species. The estimated ratio in
the rat of about ~18 (Mulders et al. 1997; West et al. 1991) distinctly differs from Wahlberg’s
epauletted fruit bat. This difference in divergence may well explain the increase in the size of
some neuronal phenotypes likely to be hippocampal projecting in Wahlberg’s epauletted fruit
bat, which is the smaller species.
Stellate cells are, in the entorhinal cortex as elsewhere, considered modified pyramidal cells
(Germroth et al. 1989b; Germroth et al. 1991; Klink and Alonso 1997a; Peters and Jones
1984) and share with them a number of electrophysiological properties (Alonso and Klink
1993; Alonso and Llinas 1989; van der Linden and Lopes da Silva 1998). However, the two
cell types differ from their morphological complements in layer III (Erchova et al. 2004; van
der Linden and Lopes da Silva 1998), and, for example, in their modulation by cholinergic
inputs also from each other (Klink and Alonso 1997b). This may imply two parallel channels
that act differently on the hippocampus (Alonso and Klink 1993; Klink and Alonso 1997a).
The physiological properties of non-stellate or non-pyramidal neurons are not reported,
although it is known that some phenotypes, such as the horizontal bipolar and tripolar cells
also contribute to the perforant pathway (Germroth et al. 1989a).
The medial EC contains grid cells that fire selectively in specific locations in the environment
(Brun et al. 2008; Fyhn et al. 2004; Hafting et al. 2005). Although correlational evidence
based on dynamic membrane properties points towards the stellate cell as the anatomical
equivalent of the grid cell (Garden et al. 2008; Giocomo and Hasselmo 2008), other
morphological phenotypes cannot be ruled out. The current knowledge on grid cells supports
a 2-dimensional representation of the environment (Hafting et al. 2005). If and how grid cells
represent dimensions that not only reflect movement across a surface but also above a
surface, is a question that, in mammals, can most easily be addressed in bats. It is tempting to
speculate that the relative proportions of cell types within layer II are associated with the
representation of a 3-dimensional environment. Alternatively, the proportions observed in
bats may be a trait reflecting the phylogenetic history of the Chiroptera, which must
accommodate but is not necessarily specific to a 3-dimensional representation.
We thank Prof. Menno Witter for a critical reading of the manuscript. We are grateful for the
help of Mr. Ben Agwanda (National Museums of Kenya, Nairobi), and Dr. Robert Kityo
(Makerere University, Kampala) for guidance on the species biology and ecology and
logistical support, Mr. Francis Muchemi (National Museums of Kenya, Nairobi), and Dr.
Joseph M. Bukenya (Rubaga Hospital, Kampala) for assistance in the capture. Dr. Urs
Ziegler (ZMB, Zürich) kindly introduced us to the 3D modelling software.
Alonso A, Klink R (1993) Differential electroresponsiveness of stellate and pyramidal-like
cells of medial entorhinal cortex layer II. J Neurophysiol 70:128-143.
Alonso A, Llinas RR (1989) Subthreshold Na+-dependent theta-like rhythmicity in stellate
cells of entorhinal cortex layer II. Nature 342:175-177.
Amaral DG, Insausti R, Cowan WM (1987) The entorhinal cortex of the monkey: I.
Cytoarchitectonic organization. J Comp Neurol 264:326-355.
Ashwell KW, Zhang LL, Marotte LR (2005) Cyto- and chemoarchitecture of the cortex of the
tammar wallaby (Macropus eugenii): areal organization. Brain Behav Evol 66:114-
Baron G, Stephan H, Frahm HD (1996a) Atlases of a Megachiroptera brain. In G. Baron, H.
Stephan, H. D. Frahm (eds): Comparative neurobiology of Chiroptera:
Macromorphology, brain structures, tables and atlases (Volume1). Birkhäuser Verlag,
Basel, Switzerland. pp. 433-529.
Baron G, Stephan H, Frahm HD (1996b) Comparative brain characteristics. In G. Baron, H.
Stephan, H. D. Frahm (eds): Comparative neurobiology of Chiroptera:
Macromorphology, brain structures, tables and atlases (Volume1). Birkhäuser Verlag,
Basel, Switzerland. 529 pp.
Beall MJ, Lewis DA (1992) Heterogeneity of layer II neurons in human entorhinal cortex. J
Comp Neurol 321:241-266.
Blaizot X, Martinez-Marcos A, Arroyo-Jimenez MdM, Marcos P, Artacho-Perula E, Munoz
M, Chavoix C, Insausti R (2004) The parahippocampal gyrus in the Baboon:
Anatomical, cytoarchitectonic and magnetic resonance imaging (MRI) Studies. Cereb
Brodmann K (1909) Vergleichende Lokalisationslehre der Grosshirnrinde in ihren Prinzipien
dargestellt auf Grund des Zellenbaues. Barth, Leipzig, Germany.
Brodmann K (1925) Vergleichende Lokalisationslehre der Grosshirnrinde. C. G. Leipzig:
Röder GmbH, Germany. pp. 177-183.
Brun VH, Solstad T, Kjelstrup KB, Fyhn M, Witter MP, Moser EI, Moser MB (2008)
Progressive increase in grid scale from dorsal to ventral medial entorhinal cortex.
Buhl EH, Dann JF (1991) Cytoarchitecture, neuronal composition, and entorhinal afferents of
the flying fox hippocampus. Hippocampus 1:131-152.
Burwell RD, Amaral DG (1998) Perirhinal and postrhinal cortices of the rat:
interconnectivity and connections with the entorhinal cortex. J Comp Neurol 391:293-
Campbell MJ, Morrison JH (1989) Monoclonal antibody to neurofilament protein (SMI-32)
labels a subpopulation of pyramidal neurons in the human and monkey neocortex. J
Comp Neurol 282:191-205.
DeFelipe J, Hendry SH, Jones EG (1989) Visualization of chandelier cell axons by
parvalbumin immunoreactivity in monkey cerebral cortex. Proc Natl Acad Sci U S A
Dorph-Petersen KA, Nyengaard JR, Gundersen HJG (2001) Tissue shrinkage and unbiased
stereological estimation of particle number and size. J Microsc 204:232-246.
Erchova I, Kreck G, Heinemann U, Herz AV (2004) Dynamics of rat entorhinal cortex layer
II and III cells: characteristics of membrane potential resonance at rest predict
oscillation properties near threshold. J Physiol 560:89-110.
Fonseca M, Soriano E, Ferrer I, Martinez A, Tunon T (1993) Chandelier cell axons identified
by parvalbumin-immunoreactivity in the normal human temporal cortex and in
Alzheimer's disease. Neuroscience 55:1107-1116.
Frederickson CJ, Koh JY, Bush AI (2005) The neurobiology of zinc in health and disease.
Nat Rev Neurosci 6:449-462.
Fyhn M, Hafting T, Witter MP, Moser EI, Moser M-B (2008) Grid cells in mice.
Fyhn M, Molden S, Witter MP, Moser EI, Moser M-B (2004) Spatial Representation in the
entorhinal cortex. Science 305:1258-1264.
Garden DL, Dodson PD, O'Donnell C, White MD, Nolan MF (2008) Tuning of synaptic
integration in the medial entorhinal cortex to the organization of grid cell firing fields.
Geneser-Jensen FA, Haug FM, Danscher G (1974) Distribution of heavy metals in the
hippocampal region of the guinea pig. A light microscope study with Timm's sulfide
silver method. Z Zellforsch Mikrosk Anat 147:441-478.
Germroth P, Schwerdtfeger WK, Buhl EH (1989a) GABAergic neurons in the entorhinal
cortex project to the hippocampus. Brain Res 494:187-192.
Germroth P, Schwerdtfeger WK, Buhl EH (1989b) Morphology of identified entorhinal
neurons projecting to the hippocampus. A light microscopical study combining
retrograde tracing and intracellular injection. Neuroscience 30:683-691.
Germroth P, Schwerdtfeger WK, Buhl EH (1991) Ultrastructure and aspects of functional
organization of pyramidal and nonpyramidal entorhinal projection neurons
contributing to the perforant path. J Comp Neurol 305:215-231.
Giocomo LM, Hasselmo ME (2008) Time constants of h current in layer ii stellate cells differ
along the dorsal to ventral axis of medial entorhinal cortex. J Neurosci 28:9414-9425.
Goldstein ME, Sternberger LA, Sternberger NH (1983) Microheterogeneity ("neurotypy") of
neurofilament proteins. Proc Natl Acad Sci U S A 80:3101-3105.
Gundersen HJG, Jensen EB, Kiêu K, Nielsen J (1999) The efficiency of systematic sampling
in stereology--reconsidered. J Microsc 193:199-211.
Hafting T, Fyhn M, Molden S, Moser MB, Moser EI (2005) Microstructure of a spatial map
in the entorhinal cortex. Nature 436:801-806.
Hamam BN, Amaral DG, Alonso AA (2000) Morphological and electrophysiological
characteristics of layer V neurons of the rat medial entorhinal cortex. The Journal of
Comparative Neurology 418:457-472.
Hamam BN, Amaral DG, Alonso AA (2002) Morphological and electrophysiological
characteristics of layer V neurons of the rat lateral entorhinal cortex. J Comp Neurol
Hassiotis M, Paxinos G, Ashwell KW (2004) Cyto- and chemoarchitecture of the cerebral
cortex of the Australian echidna (Tachyglossus aculeatus). I. Areal organization. J
Comp Neurol 475:493-517.
Hassiotis M, Paxinos G, Ashwell KW (2005) Cyto- and chemoarchitecture of the cerebral
cortex of an echidna (Tachyglossus aculeatus). II. Laminar organization and synaptic
density. J Comp Neurol 482:94-122.
Hendry SH, Jones EG, Emson PC, Lawson DE, Heizmann CW, Streit P (1989) Two classes
of cortical GABA neurons defined by differential calcium binding protein
immunoreactivities. Exp Brain Res 76:467-472.
Hof P, Sherwood C (2005) Morphomolecular neuronal phenotypes in the neocortex reflect
phylogenetic relationships among certain mammalian orders. The Anatomical Record
Part A: Discoveries in Molecular, Cellular, and Evolutionary Biology 287A:1153-
Hof PR, Glezer, II, Nimchinsky EA, Erwin JM (2000) Neurochemical and cellular
specializations in the mammalian neocortex reflect phylogenetic relationships:
evidence from primates, cetaceans, and artiodactyls. Brain Behav Evol 55:300-310.
Hof PR, Morrison JH (1990) Quantitative analysis of a vulnerable subset of pyramidal
neurons in Alzheimer's disease: II. Primary and secondary visual cortex. J Comp
Holm IE, Geneser FA (1989) Histochemical demonstration of zinc in the hippocampal region
of the domestic pig: I. Entorhinal area, parasubiculum, and presubiculum. J Comp
Iñiguez C, Gayoso MJ, Carreres J (1985) A versatile and simple method for staining nervous
tissue using Giemsa dye. Journal of Neuroscience Methods 13:77-86.
Insausti R, Herrero MT, Witter MP (1997) Entorhinal cortex of the rat: cytoarchitectonic
subdivisions and the origin and distribution of cortical efferents. Hippocampus 7:146-
Insausti R, Tuñón T, Sobreviela T, Insausti AM, Gonzalo LM (1995) The human entorhinal
cortex: a cytoarchitectonic analysis. J Comp Neurol 355:171-198.
Jeffery KJ (2007) Integration of the sensory inputs to place cells: what, where, why, and
how? Hippocampus 17:775-785.
Johnson JI, Kirsch JA, Reep RL, Switzer RC, 3rd (1994) Phylogeny through brain traits:
more characters for the analysis of mammalian evolution. Brain Behav Evol 43:319-
Jones G, Teeling EC (2006) The evolution of echolocation in bats. Trends in Ecology &
Kerr KM, Agster KL, Furtak SC, Burwell RD (2007) Functional neuroanatomy of the
parahippocampal region: the lateral and medial entorhinal areas. Hippocampus
Kingdon J (1984) Bats: Fruit Bats. In J. Kingdon (ed.): East African mammals: An atlas of
evolution in Africa. Volume 2. Part A (Insectivores and Bats). The University of
Chicago Press. BAS Printers Limited, Over Wallop, Hampshire, Great Britain. pp.
Klink R, Alonso A (1997a) Morphological characteristics of layer II projection neurons in the
rat medial entorhinal cortex. Hippocampus 7:571-583.
Klink R, Alonso A (1997b) Muscarinic modulation of the oscillatory and repetitive firing
properties of entorhinal cortex layer II neurons. J Neurophysiol 77:1813-1828.
Kruska D (1975) [Comparative quantitative study on brains of wild and laboratory rats. II.
Comparison of size of allocortical brain centers] J. Hirnforsch. 16: 485-496.
Lapointe F, Baron G, Legendre P (1999) Encephalization, adaptation and evolution of
chiroptera: A statistical analysis with further evidence for bat monophyly. Brain
Behav Evol 54:119-126.
Lorente de Nó R (1933) Studies on the structure of the cerebral cortex. Journal für
psychologie und neurologie 45:381-438.
Maseko BC, Bourne JA, Manger PR (2007) Distribution and morphology of cholinergic,
putative catecholaminergic and serotonergic neurons in the brain of the Egyptian
rousette flying fox, Rousettus aegyptiacus. J Chem Neuroanat 34:108-127.
Maseko BC, Manger PR (2007) Distribution and morphology of cholinergic,
catecholaminergic and serotonergic neurons in the brain of Schreiber's long-fingered
bat, Miniopterus schreibersii. J Chem Neuroanat 34:80-94.
Mikkonen M, Pitkänen A, Soininen H, Alafuzoff I, Miettinen R (2000) Morphology of spiny
neurons in the human entorhinal cortex: intracellular filling with lucifer yellow.
Morrison JH, Lewis DA, Campbell MJ, Huntley GW, Benson DL, Bouras C (1987) A
monoclonal antibody to non-phosphorylated neurofilament protein marks the
vulnerable cortical neurons in Alzheimer's disease. Brain Res 416:331-336.
Moser EI, Kropff E, Moser MB (2008) Place cells, grid cells, and the brain's spatial
representation system. Annu Rev Neurosci 31:69-89.
Mulders WH, West MJ, Slomianka L (1997) Neuron numbers in the presubiculum,
parasubiculum, and entorhinal area of the rat. J Comp Neurol 385:83-94.
O'Keefe J, Burgess N (2005) Dual phase and rate coding in hippocampal place cells:
Theoretical significance and relationship to entorhinal grid cells. Hippocampus
Paoletti P, Vergnano AM, Barbour B, Casado M (2009) Zinc at glutamatergic synapses.
Perez-Clausell J, Danscher G (1985) Intravesicular localization of zinc in rat telencephalic
boutons. A histochemical study. Brain Res 337:91-98.
Peters A, Jones EG (1984) Classification of cortical neurons. Classification of cortical
neurons In Cerebral Cortex, E G Jones and A Peters (eds): Volume 1 Plenum Press,
New York and London pp107-121.
Pettigrew JD, Jamieson BG, Robson SK, Hall LS, McAnally KI, Cooper HM (1989)
Phylogenetic relations between microbats, megabats and primates (Mammalia:
Chiroptera and Primates). Philos Trans R Soc Lond B Biol Sci 325:489-559.
Pettigrew JD, Maseko BC, Manger PR (2008) Primate-like retinotectal decussation in an
echolocating megabat, Rousettus aegyptiacus. Neuroscience 153:226-231.
Pitkänen A, Amaral DG (1993) Distribution of parvalbumin-immunoreactive cells and fibers
in the monkey temporal lobe: the hippocampal formation. J Comp Neurol 331:37-74.
Ramón y Cajal S (1988) On a special ganglion of the spheno-occipital cortex. In J. DeFelipe
and E. G. Jones (eds): Cajal on the cerebral cortex. An annoted translation of the
complete writings. Oxford University Press Inc, New York. pp. 363-376.
Rose M (1912) Histologische lokalisation der Grosshirnrinde bei Kleinen Saugetierein
(Rodentia, Insectivora, Chiroptera). J für Psychologie und Neurologie 19:119-207.
Rose M (1926) Der Allocortex bei Tier und Mensch. I. Teil. J für Psychology und Neurologie
Rosene DL, Van Hoesen GW (1987) The hippocampal formation of the primate brain. A
review of some comparative aspects of cytoarchitecture and connections. In E. G.
Jones and A. Peters (eds): Cerebral Cortex, Volume 6. Plenum New York. pp. 345-
Saleem KS, Price JL, Hashikawa T (2007) Cytoarchitectonic and chemoarchitectonic
subdivisions of the perirhinal and parahippocampal cortices in macaque monkeys. J
Comp Neurol 500:973-1006.
Schwartz SP, Coleman PD (1981) Neurons of origin of the perforant path. Exp Neurol
Simmons NB, Seymour KL, Habersetzer J, Gunnell GF (2008) Primitive early Eocene bat
from Wyoming and the evolution of flight and echolocation. Nature 451:818-821.
Slomianka L (1992) Neurons of origin of zinc-containing pathways and the distribution of
zinc-containing boutons in the hippocampal region of the rat. Neuroscience 48:325-
Slomianka L, Geneser FA (1991) Distribution of acetylcholinesterase in the hippocampal
region of the mouse: I. Entorhinal area, parasubiculum, retrosplenial area, and
presubiculum. J Comp Neurol 303:339-354.
Slomianka L, West MJ (1989) Comparative quantitative study of the hippocampal region of
two closely related species of wild mice: interspecific and intraspecific variations in
volumes of hippocampal components. J Comp Neurol 280:544-552.
Slomianka L, West MJ (2005) Estimators of the precision of stereological estimates: An
example based on the CA1 pyramidal cell layer of rats. Neuroscience 136:757-767.
Sternberger LA, Sternberger NH (1983) Monoclonal antibodies distinguish phosphorylated
and nonphosphorylated forms of neurofilaments in situ. Proc Natl Acad Sci U S A
Stephan H, Baron G, Frahm HD, Stephan M (1986) [Comparison of the size of brains and
brain structures of mammals.] Z Mikrosk Anat Forsch 100:189-212.
Suzuki W, Amaral DG (1994) Topographic organization of the reciprocal connections
between the monkey entorhinal cortex and the perirhinal and parahippocampal
cortices. J Neurosci 14:1856-1877.
Swanson LW, Cowan WM (1977) An autoradiographic study of the organization of the
efferent connections of the hippocampal formation in the rat. J Comp Neurol 172:49-
Swanson LW, Köhler C (1986) Anatomical evidence for direct projections from the
entorhinal area to the entire cortical mantle in the rat. J Neurosci 6:3010-3023.
Tandrup T (1993) A method for unbiased and efficient estimation of number and mean
volume of specified neuron subtypes in rat dorsal root ganglion. J Comp Neurol
Teeling EC, Scally M, Kao DJ, Romagnoli ML, Springer MS, Stanhope MJ (2000) Molecular
evidence regarding the origin of echolocation and flight in bats. Nature 403:188-192.
Tuñón T, Insausti R, Ferrer I, Sobreviela T, Soriano E (1992) Parvalbumin and calbindin D-
28K in the human entorhinal cortex. An immunohistochemical study. Brain Research
Ulanovsky N, Moss CF (2007) Hippocampal cellular and network activity in freely moving
echolocating bats. Nat Neurosci 10:224-233.
Uva L, Grüschke S, Biella G, De Curtis M, Witter MP (2004) Cytoarchitectonic
characterization of the parahippocampal region of the guinea pig. J Comp Neurol
van der Linden S, Lopes da Silva FH (1998) Comparison of the electrophysiology and
morphology of layers III and II neurons of the rat medial entorhinal cortex in vitro.
Eur J Neurosci 10:1479-1489.
van Groen T (2001) Entorhinal cortex of the mouse: cytoarchitectonical organization.
van Groen T, Miettinen P, Kadish I (2003) The entorhinal cortex of the mouse: Organization
of the projection to the hippocampal formation. Hippocampus 13:133-149.
van Hoesen G, Pandya DN (1975a) Some connections of the entorhinal (area 28) and
perirhinal (area 35) cortices of the rhesus monkey. I. Temporal lobe afferents. Brain
West MJ, Slomianka L, Gundersen HJG (1991) Unbiased stereological estimation of the total
number of neurons in thesubdivisions of the rat hippocampus using the optical
fractionator. Anat Rec 231:482-497.
Wouterlood FG, Hartig W, Bruckner G, Witter MP (1995) Parvalbumin-immunoreactive
neurons in the entorhinal cortex of the rat: localization, morphology, connectivity and
ultrastructure. J Neurocytol 24:135-153.
Wóznicka A, Malinowska M, Kosmal A (2006) Cytoarchitectonic organization of the
entorhinal cortex of the canine brain. Brain Research Reviews 52:346-367.
MEA layer II neuronal phenotypes, profile areas and proportions
Cell type Species N Mean Area (µm2) SD (µm2) Proportion (%)
352 162 44 35.8
E. wahlbergi 193 164 46 40.8
48 193 51 4.9
E. wahlbergi 61 212 60 12.9
181 196 51 18.4
E. wahlbergi 83 232 61 17.5
Oblique pyramidal E. helvum
62 192 43 6.3
E. wahlbergi 23 212 66 4.9
339 115 39 34.5
E. wahlbergi 113 98 26 23.9
All cells measured E. helvum
982 156 54 100
E. wahlbergi 473 168 67 100
Total number of neurons in MEA layer II
Species Animal Estimated total cell
1 56067 0.06
2 61632 0.08
3 56265 0.09
4 74123 0.08
E. wahlbergi 1 31712 0.09
2 27344 0.1
3 33147 0.08
4 30497 0.1
Summary of cytoarchitectural comparison between bats, primates and rodents.
ECL / CE
EC / ME
EI / VIE
layers II and III mostly similar
poorly developed layer IV in primates
layer Va moderately-developed in rodents, well developed in bats and best
developed in primates
layer II well-differentiated in rodents and Wahlberg’s epauletted fruit bat,
but islands only in primates
layer III bilaminar only in rodents
deep layers as in the ECL/CE
layer II moderately-differentiated in bats and rodents; distinct islands only
well developed layers III and IV in rodents, primates and bats
well-defined Va only in primates and bats
EL / DLE
ER / DIE
layer II well-differentiated in the Straw-coloured fruit bat and rodents, but
distinct islands only in primates; poorly differentiated layer II in
Wahlberg’s epauletted fruit bat
wide layer III in bats and primates; narrow in rodents
layer IV absent in primates
layer Va well developed in primates and bats; indistinct Va in rodents
indistinct islands in layer II of the Straw-coloured fruit bat and rodents;
wide cell islands in primates
bilaminar wide layer III in bats, rodents and primates
layer IV well-developed in mouse, poorly developed in Wahlberg’s
epauletted fruit bat and rat; absent in Straw-coloured fruit bat and primates
Layer Va poorly developed in rodents; well developed in bats; all sublayers
best developed in primates
well-developed layer VI in mouse, primates, and Wahlberg’s epauletted
CE, ME, VIE, DIE, DLE regions in rodents.
Indices for encephalisation (EI), neocorticalization (NEO) and the sizes of the hippocampus
(HIP) and schizocortex (SCH) for the bats investigated in this study, rat and the major
primate groups. For E. wahlbergi only the encehalization index is available. Epomophorus
labiatus, which is equal in body weight to E. wahlbergi, was included to present values for
NEO, HIP and SCH
Species EI NEO HIP SCH
E. helvum a 311 1088 283 373
E. wahlbergi a 302
E. labiatus a 292 999 281 328
Rattus norwegicus b 155 475 132 122
prosimian primates d 415 2100 ~290 ~360
simian primates d 801 5100 ~280 ~320
human d 3012 20000 ~500 ~680
Data from a Baron et al. 1996b, b calculated from Baron et al. 1996b and Kruska 1975 and c
Stephan et al. 1986.
Schematic drawing of the brain of Pteropus reproduced from Brodmann, 1909. Lateral (a)
and medial (b) view showing the location of Brodmann area 28. Three-dimensional
representation of the entorhinal fields in the Straw-coloured fruit bat (c, e) and Wahlberg’s
epauletted fruit bat (d, f) showing the approximate level of Figures 2, 3 (i), 4 (ii), 6(iii), 7, 8
(iv) and 9 (v). c, d: Lateral view. e, f: Caudal view showing the approximate level of Figures
4 (iv) and 9 (v). Brain of Wahlberg’s epauletted fruit bat showing a schematic drawing of the
EC fields in the Piriform lobe, and in relation to the Perirhinal cortex (PRh), Parasubiculum
(PaS), Postrhinal cortex (POR), Pre-piriform cortex (PPC), and rhinal sulcus. g: Ventral view.
h: Lateral view. Scale bars 200 μm. (g, h).
Horizontal sections at a dorsal level showing rostrocaudal extent of fields ECL, EC, and EI,
indicated by arrows, in the Straw-coloured fruit bat (a, c, e, g) and Wahlberg’s epauletted
fruit bat (b, d, f, h). c, d: ECL layer II is well-delineated from layer I, layer IV is prominent
and layers V and VI are radial and columnar. e, f: EC has an indistinct layer II and stratified
Va in (e) and distinct layer II in (f). Nissl-stained sections showing different neuronal
phenotypes (indicated by arrows) in MEA layer II in Wahlberg’s epauletted fruit bat (g, i, j,
k, l, m) and the Straw-coloured fruit bat (h, m) . Pial surface is to the top. g: ovoid stellate
cell. h: polygonal stellate cell. i: pyramidal cell. j: oblique pyramidal cell. k: horizontal
tripolar cell. l: bipolar cell. m: fusiform cell. n: small round cell. Scale bar in a, b: 300 μm; c
- f: 250 μm; g-n: 50 μm.
Timm-stained horizontal sections showing the rostrocaudal extent of ECL and EC. Deep layer
I and II stain darkly, layer III is moderately staining and is separated from a dark sublayer Va
by a narrow pale layer IV. Deeper layers are pale staining. a: Straw-coloured fruit bat. A
staining artefact was removed (indicated by star). b: Wahlberg’s epauletted fruit bat. Scale
bar: 300 μm.
Coronal sections at a caudal level showing the mediolateral extent of the fields, indicated by
arrows, in the Straw-coloured fruit bat (a, c, e) and Wahlberg’s epauletted fruit bat (b, d, f).
a, b: Nissl-stained. c, d: Parvalbumin-like reactivity of neurons and fibre plexus involving
layers II and III. Most reactivity is observed in ECL, EL and ER. e: Lack of SMI 32-like
staining in the Straw-coloured fruit bat. f: SMI 32-like staining in layers II and III of ECL, EL
and ER of Wahlberg’s epauletted fruit bat. Scale bar: 300 μm.
Parvalbumin-like stained sections in Wahlberg’s epauletted fruit bat (a-h, j, k) and the Straw-
coloured fruit bat (i). a: Fibre plexus and several neurons and processes in layers I-III. b:
Multipolar neuron in layer II with processes that extend into layer I. c: Layer III is well
delineated from the weakly stained layer IV. Large spherical multipolar neuron with an
ascending thick and descending thin processes, next to a horizontally oriented neuron
(arrow). d: Medium-sized conical neurons with thick ascending processes in mid layer III. e:
Large inverted multipolar neuron (arrow) with a thick descending process next to a spherical
multipolar neuron in layer III. f: Large multipolar neuron (arrow) in layer III sends a process
into the weakly stained border between ECL and parasubiculum g: Unipolar neuron (arrow) in
layer IV and few spherical neurons in layers V. h: Survey of layers II to VI. i: Multipolar
neuron in layer V with radial processes. j: Large conical neuron (arrow) with long smooth
ascending and descending processes in layer V k: Small unipolar cell in layer VI. SMI 32-
like staining in Wahlberg’s epauletted fruit bat (l, m). l: Pyramidal neuron in layer III. m:
Multipolar neuron in layer III. n: SMI 32-like stained pyramidal neurons in the
somatosensory cortex in the Straw-coloured fruit bat. Scale bar: 100 μm.
Horizontal sections at mid dorsoventral level showing the extent of fields EI, EL and ER,
indicated by arrows, in the Straw-coloured fruit bat (a, c, e) and Wahlberg’s epauletted fruit
bat (b, d, f). The Presubiculum (PrS) and Subiculum (S), and hippocampal regions CA1 and
CA3 are indicated. c, d: EI field has a layer II with dispersed neurons and deep layers have a
radial columnar organisation. Layer VI is not well delineated from the white matter in (c). e:
Small dark staining neurons in layer II. f: Multiple neuronal phenotypes in layer II. Scale bar
in a, b: 300 μm; c, d: 250 μm; e, f: 100 μm
Horizontal sections at a ventral level showing extent of the lateral fields, EI, EL and ER,
indicated by arrows, in the Straw-coloured fruit bat (a, c, e, g) and Wahlberg’s epauletted
fruit bat (b, d, f, h). c, d: EL has a layer II with clustered neurons better visible in (c), patchy
layer III, and wide Va. e, f: ER has clustered layer II neurons, more distinct in (e). g, h: Layer
II clusters. Scale bar in a, b: 300 μm; c - f: 250 μm; g, h: 100 μm.
Timm-stained horizontal sections at a ventral level showing the rostrocaudal extent of fields
EI, EL and ER, indicated by arrows. Deep layer I, II and III stain darkly, Va stains moderately
and Vb and layer VI are pale staining. a: Straw-coloured fruit bat. b: Wahlberg’s epauletted
fruit bat. Scale bar: 300 μm.
Coronal sections at a rostral level showing the mediolateral extent of the fields, indicated by
arrows, in the Straw-coloured fruit bat (a, c, e) and Wahlberg’s epauletted fruit bat (b, d, f).
a, b: Nissl-stained. c, d: Parvalbumin-like reactivity of neurons and fibre plexus in field ER.
e: Lack of SMI 32-like staining. f: SMI 32-like staining in layer III in field ER. Scale bar: 300
Click here to download high resolution image